Mitsunori Fukuda

Exosome biogenesis is poorly understood. The small GTPases Rab27a and Rab27b and their effectors, Slp4 and Slac2b, control exosome secretion at different steps by regulating the peripheral localization, retention and docking of exosomal precursors, the multivesicular endosomes. Exosomes are secreted membrane vesicles that share structural and biochemical characteristics with intraluminal vesicles of multivesicular endosomes (MVEs). Exosomes could be involved in intercellular communication and in the pathogenesis of infectious and degenerative diseases. The molecular mechanisms of exosome biogenesis and secretion are, however, poorly understood. Using an RNA interference (RNAi) screen, we identified five Rab GTPases that promote exosome secretion in HeLa cells. Among these, Rab27a and Rab27b were found to function in MVE docking at the plasma membrane. The size of MVEs was strongly increased by Rab27a silencing, whereas MVEs were redistributed towards the perinuclear region upon Rab27b silencing. Thus, the two Rab27 isoforms have different roles in the exosomal pathway. In addition, silencing two known Rab27 effectors, Slp4 (also known as SYTL4, synaptotagmin-like 4) and Slac2b (also known as EXPH5, exophilin 5), inhibited exosome secretion and phenocopied silencing of Rab27a and Rab27b, respectively. Our results therefore strengthen the link between MVEs and exosomes, and introduce ways of manipulating exosome secretion in vivo.

Two ubiquitin-like molecules, Atg12 and LC3/Atg8, are involved in autophagosome biogenesis. Atg12 is conjugated to Atg5 and forms an approximately 800-kDa protein complex with Atg16L (referred to as Atg16L complex). LC3/Atg8 is conjugated to phosphatidylethanolamine and is associated with autophagosome formation, perhaps by enabling membrane elongation. Although the Atg16L complex is required for efficient LC3 lipidation, its role is unknown. Here, we show that overexpression of Atg12 or Atg16L inhibits autophagosome formation. Mechanistically, the site of LC3 lipidation is determined by the membrane localization of the Atg16L complex as well as the interaction of Atg12 with Atg3, the E2 enzyme for the LC3 lipidation process. Forced localization of Atg16L to the plasma membrane enabled ectopic LC3 lipidation at that site. We propose that the Atg16L complex is a new type of E3-like enzyme that functions as a scaffold for LC3 lipidation by dynamically localizing to the putative source membranes for autophagosome formation.

Recently, we described a 160 kDa protein (designated AS160, for Akt substrate of 160 kDa) with a predicted Rab GAP (GTPase-activating protein) domain that is phosphorylated on multiple sites by the protein kinase Akt. Phosphorylation of AS160 in adipocytes is required for insulin-stimulated translocation of the glucose transporter GLUT4 to the plasma membrane. The aim of the present study was to determine whether AS160 is in fact a GAP for Rabs, and, if so, what its specificity is. We first identified a group of 16 Rabs in a preparation of intracellular vesicles containing GLUT4 by MS. We then prepared the recombinant GAP domain of AS160 and examined its activity against many of these Rabs, as well as several others. The GAP domain was active against Rabs 2A, 8A, 10 and 14. There was no significant activity against 14 other Rabs. GAP activity was further validated by the finding that the recombinant GAP domain with the predicted catalytic arginine residue replaced by lysine was inactive. Finally, it was found by immunoblotting that Rabs 2A, 8A and 14 are present in GLUT4 vesicles. These results indicate that AS160 is a Rab GAP, and suggest novel Rabs that may participate in GLUT4 translocation.

Myosin Va is a member of the unconventional class V myosin family, and a mutation in the myosin Va gene causes pigment granule transport defects in human Griscelli syndrome and dilute mice. How myosin Va recognizes its cargo (i.e. melanosomes), however, has re- mained undetermined over the past decade. In this study, we discovered Slac2-a/melanophilin to be the “missing link” between myosin Va and GTP-Rab27A present in the melanosome. Deletion analysis and site-directed mutagenesis showed that the N-terminal Slp (synaptotagmin-like protein) homology domain of Slac2-a specifically binds Rab27A/B isoforms and that the C-terminal half directly binds the globular tail of myosin Va. The tripartite protein complex (Rab27A· Slac2-a·myosin Va) in melanoma cells was further confirmed by immunoprecipitation. The discovery that myosin Va indirectly recognizes its cargo through Slac2-a, a novel Rab27A/B effector, should shed light on molecular recognition of its specific cargo by class V myosin. Myosin Va is a member of the unconventional class V myosin family, and a mutation in the myosin Va gene causes pigment granule transport defects in human Griscelli syndrome and dilute mice. How myosin Va recognizes its cargo (i.e. melanosomes), however, has re- mained undetermined over the past decade. In this study, we discovered Slac2-a/melanophilin to be the “missing link” between myosin Va and GTP-Rab27A present in the melanosome. Deletion analysis and site-directed mutagenesis showed that the N-terminal Slp (synaptotagmin-like protein) homology domain of Slac2-a specifically binds Rab27A/B isoforms and that the C-terminal half directly binds the globular tail of myosin Va. The tripartite protein complex (Rab27A· Slac2-a·myosin Va) in melanoma cells was further confirmed by immunoprecipitation. The discovery that myosin Va indirectly recognizes its cargo through Slac2-a, a novel Rab27A/B effector, should shed light on molecular recognition of its specific cargo by class V myosin. The synaptotagmin-like protein (Slp) 1The abbreviations used are: Slpsynaptotagmin-like proteinGSTglutathione S-transferaseGTglobular tailHRPhorseradish peroxidaseSHDSlp homology domainSlac2Slp homologue lacking C2 domains family is classified as a subfamily of C-terminal-type tandem C2 proteins (1.Marquèze B. Berton F. Seagar M. Biochimie (Paris). 2000; 82: 409-420Crossref PubMed Scopus (88) Google Scholar, 2.Duncan R.R. Shipston M.J. Chow R.H. Biochimie (Paris). 2000; 82: 421-426Crossref PubMed Scopus (40) Google Scholar, 3.Fukuda M. Saegusa C. Kanno E. Mikoshiba K. J. Biol. Chem. 2001; 276: 24441-24444Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 4.Fukuda M. Mikoshiba K. Biochem. Biophys. Res. Commun. 2001; 281: 1226-1233Crossref PubMed Scopus (80) Google Scholar, 5.Fukuda M. Saegusa C. Mikoshiba K. Biochem. Biophys. Res. Commun. 2001; 283: 513-519Crossref PubMed Scopus (68) Google Scholar, 6.Fukuda M. Mikoshiba K. FEBS Lett. 2001; 503: 217-218Crossref PubMed Scopus (16) Google Scholar, 7.Fukuda M. Mikoshiba K. Biochem. J. 2001; 360: 441-448Crossref PubMed Google Scholar) and was originally defined as an N-terminal Slp homology domain (SHD) (5.Fukuda M. Saegusa C. Mikoshiba K. Biochem. Biophys. Res. Commun. 2001; 283: 513-519Crossref PubMed Scopus (68) Google Scholar) and C-terminal tandem C2 domains, putative Ca2+ binding motifs (known as the C2A domain and C2B domain) (8.Nalefski E.A. Falke J.J. Protein Sci. 1996; 5: 2375-2390Crossref PubMed Scopus (691) Google Scholar). To date, four members of the Slp family (Slp1/Jfc1, Slp2-a, Slp3-a, and Slp4/granuphilin-a) have been described in the mouse and human (4.Fukuda M. Mikoshiba K. Biochem. Biophys. Res. Commun. 2001; 281: 1226-1233Crossref PubMed Scopus (80) Google Scholar, 5.Fukuda M. Saegusa C. Mikoshiba K. Biochem. Biophys. Res. Commun. 2001; 283: 513-519Crossref PubMed Scopus (68) Google Scholar,9.McAdara Berkowitz J.K. Catz S.D. Johnson J.L. Ruedi J.M. Thon V. Babior B.M. J. Biol. Chem. 2001; 276: 18855-18862Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 10.Wang J. Takeuchi T. Yokota H. Izumi T. J. Biol. Chem. 1999; 274: 28542-28548Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar), and several alternatively splicing isoforms have been identified in Slp2, Slp3, and Slp4 (5.Fukuda M. Saegusa C. Mikoshiba K. Biochem. Biophys. Res. Commun. 2001; 283: 513-519Crossref PubMed Scopus (68) Google Scholar, 10.Wang J. Takeuchi T. Yokota H. Izumi T. J. Biol. Chem. 1999; 274: 28542-28548Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). An additional slp gene (slp5) has been found on chromosome X (Xp21.1) in the human genome (5.Fukuda M. Saegusa C. Mikoshiba K. Biochem. Biophys. Res. Commun. 2001; 283: 513-519Crossref PubMed Scopus (68) Google Scholar). synaptotagmin-like protein glutathione S-transferase globular tail horseradish peroxidase Slp homology domain Slp homologue lacking C2 domains The Slp family contains two conserved domains at the N terminus, referred to as SHD1 and SHD2 (5.Fukuda M. Saegusa C. Mikoshiba K. Biochem. Biophys. Res. Commun. 2001; 283: 513-519Crossref PubMed Scopus (68) Google Scholar). The SHD1 and SHD2 of Slp3-a and Slp4 are separated by a sequence containing two zinc-finger motifs, whereas Slp1 and Slp2-a lack such zinc-finger motifs, and their SHD1 and SHD2 are linked together (5.Fukuda M. Saegusa C. Mikoshiba K. Biochem. Biophys. Res. Commun. 2001; 283: 513-519Crossref PubMed Scopus (68) Google Scholar). The SHD has also been found in other proteins, including Slac2-a (Slp homologue lackingC2 domains-a) and Slac2-b/KIAA0624 (11.Ishikawa K. Nagase T. Suyama M. Miyajima N. Tanaka A. Kotani H. Nomura N. Ohara O. DNA Res. 1998; 5: 169-176Crossref PubMed Scopus (170) Google Scholar), suggesting a general role of the SHD in cellular signaling. Two very recent important discoveries have been made concerning the functional relationship between the SHD and Rab27A, one of the small GTP-binding proteins believed to be essential for membrane trafficking in eukaryotic cells (12.Zerial M. McBride H. Nat. Rev. Mol. Cell. Biol. 2001; 2: 107-117Crossref PubMed Scopus (2730) Google Scholar). The first was our discovery that the SHD of the Slp family and Slac2-a directly interact with the GTP-bound form of Rab27A both in vitro and in intact cells (13.Kuroda T.S. Fukuda M. Ariga H. Mikoshiba K. J. Biol. Chem. 2002; 277: 9212-9218Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Since mutations in the rab27A gene cause hemophagocytic syndrome (Griscelli syndrome), an uncontrolled T lymphocyte and macrophage activation syndrome in humans (14.Menasche G. Pastural E. Feldmann J. Certain S. Ersoy F. Dupuis S. Wulffraat N. Bianchi D. Fischer A. Le Deist F. de Saint Basile G. Nat. Genet. 2000; 25: 173-176Crossref PubMed Scopus (745) Google Scholar, 15.Bahadoran P. Aberdam E. Mantoux F. Busca R. Bille K. Yalman N. de Saint-Basile G. Casaroli-Marano R. Ortonne J.P. Ballotti R. J. Cell Biol. 2001; 152: 843-850Crossref PubMed Scopus (174) Google Scholar), and defects in granule exocytosis in cytotoxic T lymphocytes and melanosome transport in ashen mice (16.Wilson S.M. Yip R. Swing D.A. O'Sullivan T.N. Zhang Y. Novak E.K. Swank R.T. Russell L.B. Copeland N.G. Jenkins N.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7933-7938Crossref PubMed Scopus (339) Google Scholar, 17.Stinchcombe J.C. Barral D.C. Mules E.H. Booth S. Hume A.N. Machesky L.M. Seabra M.C. Griffiths G.M. J. Cell Biol. 2001; 152: 825-834Crossref PubMed Scopus (328) Google Scholar, 18.Haddad E.K. Wu X. Hammer III, J.A. Henkart P.A. J. Cell Biol. 2001; 152: 835-842Crossref PubMed Scopus (208) Google Scholar, 19.Hume A.N. Collinson L.M. Rapak A. Gomes A.Q. Hopkins C.R. Seabra M.C. J. Cell Biol. 2001; 152: 795-808Crossref PubMed Scopus (274) Google Scholar), we hypothesized that the Slp family and Slac2 are involved in such membrane trafficking (13.Kuroda T.S. Fukuda M. Ariga H. Mikoshiba K. J. Biol. Chem. 2002; 277: 9212-9218Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). The second very recent important discovery is the identification of Slac2-a as melanophilin and that a mutation in the mlph gene causes defects in melanosome transport in leaden mice (20.Matesic L.E. Yip R. Reuss A.E. Swing D.A. O'Sullivan T.N. Fletcher C.F. Copeland N.G. Jenkins N.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10238-10243Crossref PubMed Scopus (206) Google Scholar). Interestingly ashen mice, which carry a mutation in the rab27A gene, and dilute mice, which carry a mutation in the myosin Va gene, which encodes one of the actin-based motor proteins (21.Reck-Peterson S.L. Provance Jr., D.W. Mooseker M.S. Mercer J.A. Biochim. Biophys. Acta. 2000; 1496: 36-51Crossref PubMed Scopus (244) Google Scholar, 22.Sellers J.R. Biochim. Biophys. Acta. 2000; 1496: 3-25Crossref PubMed Scopus (622) Google Scholar), have shown similar defects in pigment granule transport (i.e. clumping of melanosomes in the perinuclear region), and as a result ashen, leaden, and dilute mice all exhibit a similarly lighter coat color (14.Menasche G. Pastural E. Feldmann J. Certain S. Ersoy F. Dupuis S. Wulffraat N. Bianchi D. Fischer A. Le Deist F. de Saint Basile G. Nat. Genet. 2000; 25: 173-176Crossref PubMed Scopus (745) Google Scholar, 16.Wilson S.M. Yip R. Swing D.A. O'Sullivan T.N. Zhang Y. Novak E.K. Swank R.T. Russell L.B. Copeland N.G. Jenkins N.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7933-7938Crossref PubMed Scopus (339) Google Scholar, 19.Hume A.N. Collinson L.M. Rapak A. Gomes A.Q. Hopkins C.R. Seabra M.C. J. Cell Biol. 2001; 152: 795-808Crossref PubMed Scopus (274) Google Scholar, 23.Moore K.J. Seperack P.K. Strobel M.C. Swing D.A. Copeland N.G. Jenkins N.A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8131-8135Crossref PubMed Scopus (28) Google Scholar, 24.Mercer J.A. Seperack P.K. Strobel M.C. Copeland N.G. Jenkins N.A. Nature. 1991; 349: 709-713Crossref PubMed Scopus (457) Google Scholar, 25.Marks M.S. Seabra M.C. Nat. Rev. Mol. Cell. Biol. 2001; 2: 738-748Crossref PubMed Scopus (354) Google Scholar). In addition, genetic analysis has shown that these three proteins function in the same or overlapping transport pathways (20.Matesic L.E. Yip R. Reuss A.E. Swing D.A. O'Sullivan T.N. Fletcher C.F. Copeland N.G. Jenkins N.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10238-10243Crossref PubMed Scopus (206) Google Scholar), but the functional relationships between these three molecules, Rab27A, Slac2-a/melanophilin, and myosin Va, in melanosome transport remained to be clarified (25.Marks M.S. Seabra M.C. Nat. Rev. Mol. Cell. Biol. 2001; 2: 738-748Crossref PubMed Scopus (354) Google Scholar). In this study, we report on two domain structures of Slac2-a, the N-terminal SHD, which specifically interacts with the Rab27A and Rab27B isoforms, and the C-terminal domain, which directly interacts with the globular tail of myosin Va, and we discuss the role of the tripartite protein complex in melanosome transport based on our findings. cDNA encoding a full open reading frame of mouse Rab27B, Rab34, and myosin Va was amplified from Marathon-Ready adult brain cDNA (CLONTECH) by reverse transcriptase PCR as described previously (26.Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). The purified PCR products were directly inserted into the pGEM-T Easy vector (Promega, Madison, WI). Both strands were completely sequenced using a Hitachi SQ-5500 DNA sequencer. We identified one deletion as a result of alternative splicing (deletion of amino acids 1387–1411) and several amino acid differences (R695A, D904E, and V905L) compared with the reported myosin Va sequences (24.Mercer J.A. Seperack P.K. Strobel M.C. Copeland N.G. Jenkins N.A. Nature. 1991; 349: 709-713Crossref PubMed Scopus (457) Google Scholar), and they were unlikely to be PCR-induced errors because we found the same differences in at least two independent clones. Addition of the FLAG tag (or T7 tag) to the N terminus of Rab34 (pEF-FLAG-Rab34) or to the N terminus of myosin Va (pEF-T7- or -FLAG-myosin Va) and construction of the expression vector were performed as described previously (26.Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 27.Mizushima S. Nagata S. Nucleic Acids Res. 1990; 18: 5332Crossref Scopus (1499) Google Scholar, 28.Fukuda M. Aruga J. Niinobe M. Aimoto S. Mikoshiba K. J. Biol. Chem. 1994; 269: 29206-29211Abstract Full Text PDF PubMed Google Scholar, 29.Fukuda M. Mikoshiba K. J. Biol. Chem. 2000; 275: 28180-28185Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). pEF-T7-Slac2-a, -FLAG-Slac2-a, and other -FLAG-Rabs were prepared as described previously (13.Kuroda T.S. Fukuda M. Ariga H. Mikoshiba K. J. Biol. Chem. 2002; 277: 9212-9218Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Deletion mutants of Slac2-a (pEF-T7-Slac2-a-SHD and -Δ146) and of myosin Va (pEF-FLAG-myosin Va-GT (globular tail)) were essentially constructed by conventional PCR as described previously (28.Fukuda M. Aruga J. Niinobe M. Aimoto S. Mikoshiba K. J. Biol. Chem. 1994; 269: 29206-29211Abstract Full Text PDF PubMed Google Scholar, 30.Fukuda M. Kanno E. Ogata Y. Mikoshiba K. J. Biol. Chem. 2001; 276: 40319-40325Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) using the following oligonucleotides with restriction enzyme sites (underlined) or stop codons (in bold): 5′-CGGATCCGGTGGAGGTGGATCTGAGCC-3′ (Slac2-a-Δ146 primer; sense), 5′-GCGAATTC ATGGCTCAGATCCACCTCCAC-3′ (Slac2-a-SHD 3′-primer), and 5′-CGGATCCGAAAAGCAGGATAAAACTGT-3′ (myosin Va-Δ1417 primer; sense). pEF-T7-Slac2-a-(A4) carrying a SLEWY-to-ALEAA substitution (Fig. 1A) was constructed by two-step PCR techniques as described previously using the following mutagenic oligonucleotides (31.Fukuda M. Kojima T. Aruga J. Niinobe M. Mikoshiba K. J. Biol. Chem. 1995; 270: 26523-26527Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar): 5′-GGCGGCCGCCAGAGCACCGATCTTCAC-3′ (A4 primer 1; antisense) and 5′-GCGGCCGCCTACCAGCACGTGAGGGCT-3′ (A4 primer 2; sense). pEF-T7-GST-Slac2-a-Δ146, -T7-GST-Slac2-a, -T7-Rab27A, -T7-GST-FLAG-myosin Va-GT, and pEF-T7-GST vectors were constructed by PCR essentially as described elsewhere. 2M. Fukuda, manuscript in preparation. COS-7 cells (5 × 105 cells (the day before transfection)/10-cm dish) were transfected with 4 μg of plasmids as described previously (30.Fukuda M. Kanno E. Ogata Y. Mikoshiba K. J. Biol. Chem. 2001; 276: 40319-40325Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Three days after transfection, cells were harvested and homogenized in 1 ml of a buffer containing 50 mm HEPES-KOH, pH 7.2, 250 mm NaCl, 0.1 mmphenylmethylsulfonyl fluoride, 10 μm leupeptin, and 10 μm pepstatin A in a glass-Teflon Potter homogenizer with 10 strokes at 900–1000 rpm. After solubilization with 1% Triton X-100, insoluble material was removed by centrifugation at 15,000 rpm for 10 min. Expressed GST fusion proteins were affinity-purified on glutathione-Sepharose beads (wet volume, 20 μl; Amersham Biosciences) according to the manufacturer's recommendations. After extensively washing the beads with 10 mm HEPES-KOH, pH 7.2, 100 mm NaCl, and 0.2% Triton X-100, thrombin (1 unit, Sigma) digestion was performed on the same column at 25 °C for 1 h. The eluate containing FLAG-myosin Va-GT protein (or Rab27A) was then incubated with benzamidine-Sepharose 6B (wet volume, 20 μl; Amersham Biosciences) to remove the thrombin. Protein concentrations were estimated by 10% SDS-PAGE or determined with a Bio-Rad protein assay kit using bovine serum albumin as a reference. The purified FLAG-myosin Va-GT protein and Rab27A were incubated with glutathione-Sepharose beads (wet volume, 20 μl) either coupled with T7-GST-Slac2-a or T7-GST alone in 50 mm HEPES-KOH, pH 7.2, 100 mm NaCl, 1 mm MgCl2, and 0.2% Triton X-100 for 1 h at 4 °C. After washing three times with 1 ml of the binding buffer without recombinant proteins, proteins trapped with the beads were analyzed by 10% SDS-PAGE followed by immunoblotting with horseradish (HRP)-conjugated anti-FLAG tag antibody (Sigma) and anti-Rab27 mouse monoclonal antibody (Transduction Laboratories, Lexington, KY) as described previously (26.Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 30.Fukuda M. Kanno E. Ogata Y. Mikoshiba K. J. Biol. Chem. 2001; 276: 40319-40325Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). cDNA encoding the C terminus of Slac2-a (amino acids 401–590) was amplified by conventional PCR and subcloned into the pGEX-4T-3 vector (named pGEX-4T-3-Slac2-aΔ400) (Amersham Biosciences) as described previously (28.Fukuda M. Aruga J. Niinobe M. Aimoto S. Mikoshiba K. J. Biol. Chem. 1994; 269: 29206-29211Abstract Full Text PDF PubMed Google Scholar). GST fusion proteins were expressed and purified on glutathione-Sepharose beads by the standard method (32.Smith D.B. Johnson K.S. Gene (Amst.). 1988; 67: 31-40Crossref PubMed Scopus (5047) Google Scholar). New Zealand White rabbits were immunized with purified GST-Slac2-aΔ400 (or FLAG-myosin Va-GT), and anti-Slac2-a antibody was affinity-purified by exposure to antigen-bound Affi-Gel 10 beads (Bio-Rad) as described previously (33.Fukuda M. Mikoshiba K. J. Biol. Chem. 1999; 274: 31428-31434Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Specificity of the antibody was checked by immunoblotting using recombinant T7-tagged Slac2-a expressed in COS-7 cells (34.Fukuda M. Martin T.F.J. Kowalchyk J.A. Zhang X. Mikoshiba K. J. Biol. Chem. 2002; 277: 4601-4604Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Melanoma cell line B16-F1 derived from the wild-type mouse was cultured as described previously (13.Kuroda T.S. Fukuda M. Ariga H. Mikoshiba K. J. Biol. Chem. 2002; 277: 9212-9218Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Cells (10-cm dish) were homogenized in a buffer containing 1 ml of 50 mm HEPES-KOH, pH 7.2, 150 mm NaCl, and protease inhibitors, and proteins were solubilized with 1% Triton X-100 at 4 °C for 1 h. After removal of insoluble material by centrifugation, the supernatant was incubated with either anti-Slac2-a IgG (10 μg/ml) or normal rabbit IgG for 1 h at 4 °C followed by incubation with protein A-Sepharose beads (Amersham Biosciences) for 1 h at 4 °C. After washing the beads five times with 50 mm HEPES-KOH, pH 7.2, 150 mm NaCl, 0.2% Triton X-100, and protease inhibitors, the immunoprecipitates were subjected to 10 or 7.5% SDS-PAGE followed by immunoblotting with anti-Slac2-a (1:250 dilution), anti-myosin Va (1:100 dilution), or anti-Rab27 monoclonal antibody (1:250 dilution) as described previously (26.Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 33.Fukuda M. Mikoshiba K. J. Biol. Chem. 1999; 274: 31428-31434Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Cotransfection of pEF-T7-Slac2-a, pEF-FLAG-Rab27A/B, and/or pEF-T7-myosin Va into COS-7 cells (5 × 105 cells (the day before transfection)/10-cm dish) was performed as described previously (30.Fukuda M. Kanno E. Ogata Y. Mikoshiba K. J. Biol. Chem. 2001; 276: 40319-40325Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Proteins were solubilized with a buffer containing 1% Triton X-100, 250 mm NaCl, 1 mm MgCl2, 50 mm HEPES-KOH, pH 7.2, and protease inhibitors at 4 °C for 1 h. T7-Slac2-a and FLAG-Rab27A/B were immunoprecipitated with anti-T7 tag antibody-conjugated agarose (Novagen, Madison, WI) and anti-FLAG M2 antibody-conjugated agarose (Sigma), respectively, as described previously (26.Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 29.Fukuda M. Mikoshiba K. J. Biol. Chem. 2000; 275: 28180-28185Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 35.Fukuda M. Mikoshiba K. J. Biol. Chem. 2001; 276: 27670-27676Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). SDS-PAGE and immunoblotting analyses with HRP-conjugated anti-FLAG and anti-T7 tag antibodies (Novagen) were also performed as described previously (26.Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 30.Fukuda M. Kanno E. Ogata Y. Mikoshiba K. J. Biol. Chem. 2001; 276: 40319-40325Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The blots and gels shown in this paper are representative of at least two or three independent experiments. We recently discovered that the SHD of Slac2-a/b directly binds the GTP-bound form of Rab27A in vitro and in intact cells (13.Kuroda T.S. Fukuda M. Ariga H. Mikoshiba K. J. Biol. Chem. 2002; 277: 9212-9218Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). However, it remained undetermined whether the full-length Slac2-a protein specifically recognizes the Rab27A molecule and whether Rab27A interacts with the SHD alone or also with the large Slac2-a C-terminal domain with unknown function. To address these issues, we first investigated the specific interaction of full-length Slac2-a with various Rab proteins (Rab1, Rab2, Rab3A, Rab4A, Rab5A, Rab6A, Rab7, Rab8, Rab9, Rab10, Rab11A, Rab17, Rab18, Rab20, Rab22, Rab23, Rab25, Rab27A, Rab27B, Rab28, Rab34, or Rab37) in intact cells by co-expression assay (30.Fukuda M. Kanno E. Ogata Y. Mikoshiba K. J. Biol. Chem. 2001; 276: 40319-40325Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 35.Fukuda M. Mikoshiba K. J. Biol. Chem. 2001; 276: 27670-27676Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). In brief, T7-tagged Slac2-a and each of the FLAG-tagged Rabs were co-expressed in COS-7 cells, and T7-Slac2-a protein was immunoprecipitated with the anti-T7 antibody-conjugated agarose (26.Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 30.Fukuda M. Kanno E. Ogata Y. Mikoshiba K. J. Biol. Chem. 2001; 276: 40319-40325Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). As expected, T7-Slac2-a protein specifically co-immunoprecipitated with the FLAG-Rab 27A and -Rab27B isoforms but not with other Rabs (Fig. 1, B, lane 18, and D, lane 1). The faint signal observed in Fig. 1B, lane 20, was probably attributable to nonspecific interaction of Rab34 with the agarose beads. Next we investigated the possible involvement of the C terminus of Slac2-a in Rab27A/B binding in the same co-transfection assay. The SHD domain alone efficiently co-immunoprecipitated with both the Rab27A and Rab27B isoforms (Fig. 1, C and D, lane 2), but the C-terminal domain lacking the SHD (Δ146) did not (Fig. 1, C and D, lane 3). In addition, the Slac2-a mutant carrying the SLEWY-to-ALEAA substitutions in SHD2 (referred to as Slac2-a(A4), see Fig. 1A) dramatically reduced the Rab27A/B binding activity (Fig. 1, C and D, lane 4). Consistent with this, crystallographic analysis has shown the corresponding sequence (SGAWFF) in rabphilin-3 directly interacts with Rab3A (36.Ostermeier C. Brunger A.T. Cell. 1999; 96: 363-374Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). These findings indicated that the SHD of Slac2-a specifically binds Ra27A/B isoforms but that the large C-terminal domain is not involved in the recognition of Rab27A/B molecules and might have different functions. The results of a genetic analysis comparing dilute, ashen, and leaden mice have indicated that myosin Va, Rab27A, and Slac2-a function in the same or overlapping transport pathways in melanosome transport (20.Matesic L.E. Yip R. Reuss A.E. Swing D.A. O'Sullivan T.N. Fletcher C.F. Copeland N.G. Jenkins N.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10238-10243Crossref PubMed Scopus (206) Google Scholar). Consistent with this, myosin Va in extracts from melanocytes has been shown to co-immunoprecipitate with Rab27A (19.Hume A.N. Collinson L.M. Rapak A. Gomes A.Q. Hopkins C.R. Seabra M.C. J. Cell Biol. 2001; 152: 795-808Crossref PubMed Scopus (274) Google Scholar). However, when FLAG-Rab27A and T7-myosin Va were co-expressed in COS-7 cells, no Rab27A-myosin Va complex was detected in the cell lysates (Fig. 2A, lane 8), indicating that an additional protein must link Rab27A and myosin Va. Since the SHD of Slac2-a specifically binds Rab27A (Fig. 1), we hypothesized that Slac2-a is the missing link between Rab27A and myosin Va in melanosome transport. To test this hypothesis, three proteins (FLAG-Rab27A, T7-Slac2-a, and T7-myosin Va) were co-expressed in COS-7 cells, and their associations were analyzed by immunoprecipitation as described above (26.Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 35.Fukuda M. Mikoshiba K. J. Biol. Chem. 2001; 276: 27670-27676Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). As expected, in the presence of full-length T7-Slac2-a, T7-myosin Va co-immunoprecipitated with FLAG-Rab27A (Fig. 2A, lane 5), whereas in the absence of T7-Slac2-a, T7-myosin Va was undetectable in the anti-FLAG antibody immunoprecipitants (Fig. 2A, lane 8). Interestingly the SHD alone (Slac2-a-SHD) or the C-terminal half alone (Slac2-a-Δ146) failed to mediate co-immunoprecipitation of T7-myosin Va with FLAG-Rab27A (Fig. 2A, lanes 6 and 7), suggesting that different domains of Slac2-a may be involved in Rab27A binding and myosin Va binding. Similar results were obtained when FLAG-Rab27B was used instead of FLAG-Rab27A (data not shown). We next sought to identify the myosin Va-binding site in Slac2-a by dual tag (T7 and FLAG) co-expression assay. When T7-Slac2-a deletion mutants and FLAG-myosin Va were co-expressed in COS-7 cells, T7-Slac2-a-Δ146, but not T7-Slac2-a-SHD, co-immunoprecipitated with FLAG-myosin Va (Fig. 2B), indicating the two domain structures of Slac2-a: the N-terminal SHD involved in the GTP-bound form of Rab27A binding and the C-terminal domain involved in myosin Va binding. Since the globular tail of myosin Va was thought to be essential for cargo recognition (21.Reck-Peterson S.L. Provance Jr., D.W. Mooseker M.S. Mercer J.A. Biochim. Biophys. Acta. 2000; 1496: 36-51Crossref PubMed Scopus (244) Google Scholar) (Fig. 3A), we investigated the interaction between Slac2-a and the myosin Va globular tail by using the same dual tag (T7 and FLAG) co-expression assay, and as shown in Fig. 3B, Slac2-a-Δ146 interacted with the globular tail of myosin Va in intact cells. We used purified recombinant proteins (GST-Slac2-a (or -Δ146), Rab27A, and FLAG-myosin Va-GT) for an in vitro binding assay to further confirm the direct interaction between the C terminus of Slac2-a and the globular tail of myosin Va as well as the in vitro formation of a tripartite protein complex from purified components. As expected, FLAG-myosin Va-GT bound GST-Slac2-a-Δ146 but not GST alone (Fig. 3C, lane 2, arrow), and full-length Slac2-a bound both FLAG-myosin Va-GT and Rab27A (Fig. 3D, arrows). Lastly, immunoprecipitation analysis was performed to investigate whether the tripartite protein complex (Rab27A·Slac2-a·myosin Va) is formed at physiological conditions. As shown in Fig. 3F, both myosin Va and Rab27A were co-immunoprecipitated with anti-Slac2-a-specific antibody (Fig. 3E), but not control IgG, from melanoma cell lysates. Thus, the tripartite protein complex (Rab27A·Slac2-a·myosin Va) demonstrated by in vitro binding experiments should be physiologically relevant. The results of a recent biochemical analysis have suggested that the tail domain of myosin Va (or Vb) recognizes its cargo by directly binding to the proteins present in the cargo (e.g. Rab11, Rab25, and synaptobrevin·synaptophysin complex) (37.Prekeris R. Terrian D.M. J. Cell Biol. 1997; 137: 1589-1601Crossref PubMed Scopus (218) Google Scholar, 38.Cheney R.E. Rodriguez O.C. Science. 2001; 293: 1263-1264Crossref PubMed Scopus (7) Google Scholar, 39.Lapierre L.A. Kumar R. Hales C.M. Navarre J. Bhartur S.G. Burnette J.O. Provance Jr., D.W. Mercer J.A. Bahler M. Goldenring J.R. Mol. Biol. Cell. 2001; 12: 1843-1857Crossref PubMed Scopus (347) Google Scholar). However, since myosin Va did not directly recognize Rab27A in the melanosomes, an additional linker protein was proposed to assist melanosome recognition in melanosome transport (25.Marks M.S. Seabra M.C. Nat. Rev. Mol. Cell. Biol. 2001; 2: 738-748Crossref PubMed Scopus (354) Google Scholar). In the present study, we discovered that Slac2-a is a missing link between Rab27A and myosin Va in melanosome transport and demonstrated how myosin Va recognizes its specific cargo (i.e. melanosomes) by its globular tail domain. The possible role of the tripartite protein complex (Slac2-a, Rab27A, and myosin Va) in melanosome capture in actin-rich cell periphery is summarized in Fig. 4. The SHD of Slac2-a specifically binds the GTP-Rab27A in the melanosomes, and the C terminus of Slac2-a binds the globular tail of myosin Va, which binds actin filament via the head domain. Since Slac2-a is expressed in various tissues, including the brain (20.Matesic L.E. Yip R. Reuss A.E. Swing D.A. O'Sullivan T.N. Fletcher C.F. Copeland N.G. Jenkins N.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10238-10243Crossref PubMed Scopus (206) Google Scholar), the Slac2-a·Rab27·myosin Va complex may be involved in other types of membrane trafficking. For instance, Slac2-a·Rab27B·myosin Va may be involved in endoplasmic reticulum transport to dendrites in neurons because the inositol 1,4,5-trisphosphate receptor on the endoplasmic reticulum does not migrate to the postsynaptic spines in dilute mice (40.Takagishi Y. Oda S. Hayasaka S. Dekker-Ohno K. Shikata T. Inouye M. Yamamura H. Neurosci. Lett. 1996; 215: 169-172Crossref PubMed Scopus (140) Google Scholar, 41.Miyata M. Finch E.A. Khiroug L. Hashimoto K. Hayasaka S. Oda S.I. Inouye M. Takagishi Y. Augustine G.J. Kano M. Neuron. 2000; 28: 233-244Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). Further work is necessary to define the universality and/or specialty of the tripartite protein complex, Slac2-a, Rab27A/B, and myosin Va, in membrane trafficking.

GLUT4 trafficking to the plasma membrane of muscle and fat cells is regulated by insulin. An important component of insulin-regulated GLUT4 distribution is the Akt substrate AS160 rab GTPase-activating protein. Here we show that Rab10 functions as a downstream target of AS160 in the insulin-signaling pathway that regulates GLUT4 translocation in adipocytes. Overexpression of a mutant of Rab10 defective for GTP hydrolysis increased GLUT4 on the surface of basal adipocytes. Rab10 knockdown resulted in an attenuation of insulin-induced GLUT4 redistribution to the plasma membrane and a concomitant 2-fold decrease in GLUT4 exocytosis rate. Re-expression of a wild-type Rab10 restored normal GLUT4 translocation. The basal increase in plasma-membrane GLUT4 due to AS160 knockdown was partially blocked by knocking down Rab10 in the same cells, further indicating that Rab10 is a target of AS160 and a positive regulator of GLUT4 trafficking to the cell surface upon insulin stimulation.

The Rab family of small GTPases regulates intracellular membrane trafficking by orchestrating the biogenesis, transport, tethering, and fusion of membrane‐bound organelles and vesicles. Like other small GTPases, Rabs cycle between two states, an active (GTP‐loaded) state and an inactive (GDP‐loaded) state, and their cycling is catalyzed by guanine nucleotide exchange factors (GEFs) and GTPase‐activating proteins (GAPs). Because an active form of each Rab localizes on a specific organelle (or vesicle) and recruits various effector proteins to facilitate each step of membrane trafficking, knowing when and where Rabs are activated and what effectors Rabs recruit is crucial to understand their functions. Since the discovery of Rabs, they have been regarded as one of the central hubs for membrane trafficking, and numerous biochemical and genetic studies have revealed the mechanisms of Rab functions in recent years. The results of these studies have included the identification and characterization of novel GEFs, GAPs, and effectors, as well as post‐translational modifications, for example, phosphorylation, of Rabs. Rab functions beyond the simple effector‐recruiting model are also emerging. Furthermore, the recently developed CRISPR/Cas technology has enabled acceleration of knockout analyses in both animals and cultured cells and revealed previously unknown physiological roles of many Rabs. In this review article, we provide the most up‐to‐date and comprehensive lists of GEFs, GAPs, effectors, and knockout phenotypes of mammalian Rabs and discuss recent findings in regard to their regulation and functions.

Although ubiquitin is thought to be important for the autophagic sequestration of invading bacteria (also called xenophagy), its precise role remains largely enigmatic. Here we determined how ubiquitin is involved in this process. After invasion, ubiquitin is conjugated to host cellular proteins in endosomes that contain Salmonella or transfection reagent-coated latex (polystyrene) beads, which mimic invading bacteria. Ubiquitin is recognized by the autophagic machinery independently of the LC3-ubiquitin interaction through adaptor proteins, including a direct interaction between ubiquitin and Atg16L1. To ensure that invading pathogens are captured and degraded, Atg16L1 targeting is secured by two backup systems that anchor Atg16L1 to ubiquitin-decorated endosomes. Thus, we reveal that ubiquitin is a pivotal molecule that connects bacteria-containing endosomes with the autophagic machinery upstream of LC3.

Leucine-rich repeat kinase 2 (LRRK2) has been associated with a variety of human diseases, including Parkinson's disease and Crohn's disease, whereas LRRK2 deficiency leads to accumulation of abnormal lysosomes in aged animals. However, the cellular roles and mechanisms of LRRK2-mediated lysosomal regulation have remained elusive. Here, we reveal a mechanism of stress-induced lysosomal response by LRRK2 and its target Rab GTPases. Lysosomal overload stress induced the recruitment of endogenous LRRK2 onto lysosomal membranes and activated LRRK2. An upstream adaptor Rab7L1 (Rab29) promoted the lysosomal recruitment of LRRK2. Subsequent family-wide screening of Rab GTPases that may act downstream of LRRK2 translocation revealed that Rab8a and Rab10 were specifically accumulated on overloaded lysosomes dependent on their phosphorylation by LRRK2. Rab7L1-mediated lysosomal targeting of LRRK2 attenuated the stress-induced lysosomal enlargement and promoted lysosomal secretion, whereas Rab8 stabilized by LRRK2 on stressed lysosomes suppressed lysosomal enlargement and Rab10 promoted lysosomal secretion, respectively. These effects were mediated by the recruitment of Rab8/10 effectors EHBP1 and EHBP1L1. LRRK2 deficiency augmented the chloroquine-induced lysosomal vacuolation of renal tubules in vivo. These results implicate the stress-responsive machinery composed of Rab7L1, LRRK2, phosphorylated Rab8/10, and their downstream effectors in the maintenance of lysosomal homeostasis.

Rab27, a member of the small GTPase Rab family, is widely conserved in metazoan, and two Rab27 isoforms, Rab27A and Rab27B, are present in vertebrates. Rab27A was the first Rab protein whose dysfunction was found to cause a human hereditary disease, type 2 Griscelli syndrome, which is characterized by silvery hair and immunodeficiency. The discovery in the 21st century of three distinct types of mammalian Rab27A effectors [synaptotagmin-like protein (Slp), Slp homologue lacking C2 domains (Slac2), and Munc13-4] that specifically bind active Rab27A has greatly accelerated our understanding not only of the molecular mechanisms of Rab27A-mediated membrane traffic (e.g. melanosome transport and regulated secretion) but of the symptoms of Griscelli syndrome patients at the molecular level. Because Rab27B is widely expressed in various tissues together with Rab27A and has been found to have the ability to bind all of the Rab27A effectors that have been tested, Rab27A and Rab27B were initially thought to function redundantly by sharing common Rab27 effectors. However, recent evidence has indicated that by interacting with different Rab27 effectors Rab27A and Rab27B play different roles in special types of secretion (e.g. exosome secretion and mast cell secretion) even within the same cell type. In this review article, I describe the current state of our understanding of the functions of Rab27 effectors in secretory pathways.

IP4BPISynaptotagmin I1 is an inositol-1,3,4,5-tetrakisphosphate (IP,) or inositol polyphosphate-binding protein, which is accumulated at nerve terminals.Here we report a novel function of the C2B domain, which was originally thought to be responsible for Ca2+-dependent binding to phospholipid membranes.A study of deletion mutants showed that about 30 amino acids of the central region of the C2B domain of mouse IP4BP/ synaptotagmin I1 (315 IHLMQNGKRLKKKKTTVKKK-TLNPYFNESFSF 346) are essential for inositol polyphosphate binding.This binding domain includes a sequence corresponding to the squid Pep20 peptide, which is also known to be essential for neurotransmitter release (Bommert, K., Charlton, M. P., DeBello, W. M., Chin, G. J., Betz, H., and Augustine, G. J. (1993) Nature 363, 163-165), suggesting that inositol polyphosphate has some effect on neurotransmitter release.Rabphilin 3A, another neuronal protein containing C2 domains, cannot bind IP,, indicating that the IP, binding property is specific to the C2B domain of synaptotagmin.Phospholipid and IP, binding experiments clearly indicated that the C2A and C2B domains have different functions.The C2A domain binds phospholipid in a Ca2+dependent manner, but the C2B domain binds inositol polyphosphate and phospholipid irrespective of the presence of Ca".Our data suggest that the C2B domain of synaptotagmin is the inositol polyphosphate sensor at the synaptic vesicle and may be involved in synaptic function.Inositol-1,3,4,5-tetrakisphosphate UP,)' has been suggested to be a potent second messenger regulating the intracellular Ca2+ concentration (1-8), but the intracellular site of action of IP, is still unknown because specific IP, receptor proteins have

Macroautophagy is a mechanism of degradation of cytoplasmic components in all eukaryotic cells. In macroautophagy, cytoplasmic components are wrapped by double-membrane structures called autophagosomes, whose formation involves unique membrane dynamics, i.e., de novo formation of a double-membrane sac called the isolation membrane and its elongation. However, the precise regulatory mechanism of isolation membrane formation and elongation remains unknown. In this study, we showed that Golgi-resident small GTPase Rab33B (and Rab33A) specifically interacts with Atg16L, an essential factor in isolation membrane formation, in a guanosine triphosphate-dependent manner. Expression of a GTPase-deficient mutant Rab33B (Rab33B-Q92L) induced the lipidation of LC3, which is an essential process in autophagosome formation, even under nutrient-rich conditions, and attenuated macroautophagy, as judged by the degradation of p62/sequestosome 1. In addition, overexpression of the Rab33B binding domain of Atg16L suppressed autophagosome formation. Our findings suggest that Rab33 modulates autophagosome formation through interaction with Atg16L.

Synaptotagmin is a proposed Ca2+ sensor on the vesicle for regulated exocytosis and exhibits Ca2+-dependent binding to phospholipids, syntaxin, and SNAP-25 in vitro, but the mechanism by which Ca2+ triggers membrane fusion is uncertain. Previous studies suggested that SNAP-25 plays a role in the Ca2+ regulation of secretion. We found that synaptotagmins I and IX associate with SNAP-25 during Ca2+-dependent exocytosis in PC12 cells, and we identified C-terminal amino acids in SNAP-25 (Asp179, Asp186, Asp193) that are required for Ca2+-dependent synaptotagmin binding. Replacement of SNAP-25 in PC12 cells with SNAP-25 containing C-terminal Asp mutations led to a loss-of-function in regulated exocytosis at the Ca2+-dependent fusion step. These results indicate that the Ca2+-dependent interaction of synaptotagmin with SNAP-25 is essential for the Ca2+-dependent triggering of membrane fusion.

Small GTPase Rab is generally thought to control intracellular membrane trafficking through interaction with specific effector molecules.Because of the large number of Rab isoforms in mammals, however, the effectors of most of the mammalian Rabs have never been identified, and the Rab binding specificity of the Rab effectors previously reported has never been thoroughly investigated.In this study we systematically screened for novel Rab effectors by a yeast two-hybrid assay with 28 different mouse or human Rabs (Rab1-30) as bait and identified 27 Rab-binding proteins, including 19 novel ones.We further investigated their Rab binding specificity by a yeast twohybrid assay with a panel of 60 different GTP-locked mouse or human Rabs.Unexpectedly most (17 of 27) of the Rab-binding proteins we identified exhibited broad Rab binding specificity and bound multiple Rab isoforms.As an example, inositol-polyphosphate 5-phosphatase OCRL (oculocerebrorenal syndrome of Lowe) bound the greatest number of Rabs (i.e.16 distinct Rabs).Others, however, specifically recognized only a single Rab isoform or only two closely related Rab isoforms.The interaction of eight of the novel Rab-binding proteins identified (e.g.INPP5E and Cog4) with a specific Rab isoform was confirmed by co-immunoprecipitation assay and/or colocalization analysis in mammalian cell cultures, and the novel Rab2B-binding domain of Golgi-associated Rab2B interactor (GARI) and GARI-like proteins was identified by deletion and homology search analyses.The findings suggest that most Rab effectors (or Rab-binding proteins) regulate intracellular membrane trafficking through interaction with several Rab isoforms rather than through a single Rab isoform.

Platelets store self-agonists such as ADP and serotonin in dense core granules. Although exocytosis of these granules is crucial for hemostasis and thrombosis, the underlying mechanism is not fully understood. Here, we show that incubation of permeabilized platelets with unprenylated active mutant Rab27A-Q78L, wild type Rab27A, and Rab27B inhibited the secretion, whereas inactive mutant Rab27A-T23N and other GTPases had no effects. Furthermore, we affinity-purified a GTP-Rab27A-binding protein in platelets and identified it as Munc13-4, a homologue of Munc13-1 known as a priming factor for neurotransmitter release. Recombinant Munc13-4 directly bound to GTP-Rab27A and -Rab27B <i>in vitro</i>, but not other GTPases, and enhanced secretion in an <i>in vitro</i> assay. The inhibition of secretion by unprenylated Rab27A was rescued by the addition of Munc13-4, suggesting that Munc13-4 mediates the function of GTP-Rab27. Thus, Rab27 regulates the dense core granule secretion in platelets by employing its binding protein, Munc13-4.

<i>rab27A</i>, which encodes a small GTP-binding protein, was recently identified as a gene in which mutations caused human hemophagocytic syndrome (Griscelli syndrome) and<i>ashen</i> mice, which exhibit defects in melanosome transport as well as in regulated granule exocytosis in cytotoxic T lymphocytes. However, little is known about the molecular mechanism of Rab27A-dependent membrane trafficking or the specific effector molecules of Rab27A. In this study, we discovered that the Slp (synaptotagmin-like protein) homology domain (SHD) of Slp1–3 and Slac2-a/b specifically and directly binds the GTP-bound form of Rab27A both <i>in vitro</i> and in intact cells but not of the other Rabs tested (Rab1, Rab2, Rab3A, Rab4, Rab5A, Rab6A, Rab7, Rab8, Rab9, Rab10, Rab11A, Rab17, Rab18, Rab20, Rab22, Rab23, Rab25, Rab28, and Rab37). Immunocytochemical analysis revealed that Slp2 (or Slp1) colocalized with Rab27A in the melanosomes of melanoma cells. Slp2 and Rab27A were distributed to the periphery of the cells (especially at the dendritic tips) in the wild-type melanoma cells, whereas they accumulated in the perinuclear region in the melanosome transport-defective cells (S91/Cloudman). These results strongly indicated that the SHD of Slp1–3 and Slac2 functions as an <i>in vivo</i> Rab27A binding domain.

Bruton's tyrosine kinase (Btk), a cytoplasmic protein-tyrosine kinase, plays a pivotal role in B cell activation and development. Mutations in the pleckstrin homology (PH) domain of the Btk gene cause human X-linked agammaglobulinemia (XLA) and murine X-linked immunodeficiency (Xid). In this paper, we report that the PH domain of Btk functions as an inositol 1,3,4,5-tetrakisphosphate (IP4), inositol 1,3,4,5,6-pentakisphosphate, and inositol 1,2,3,4,5,6-hexakisphosphate (IP6) binding domain (Kd of approximately 40 nM for IP4), and that all of the XLA (Phe replaced by Ser at position 25 (F25S), R28H, T33P, V64F, and V113D) and Xid mutations (R28C) found in the PH domain result in a dramatic reduction of IP4 binding activity. Furthermore, the rare alternative splicing variant, with 33 amino acids deleted in the PH domain, corresponding to exon 3 of the Btk gene, also impaired IP4 binding capacity. In contrast, a gain-of-function mutant called Btk*, which carries a E41K mutation in the PH domain, binds IP6 with two times higher affinity than the wild type. Our data suggest that B cell differentiation is closely correlated with the IP4 binding capacity of the PH domain of Btk. Bruton's tyrosine kinase (Btk), a cytoplasmic protein-tyrosine kinase, plays a pivotal role in B cell activation and development. Mutations in the pleckstrin homology (PH) domain of the Btk gene cause human X-linked agammaglobulinemia (XLA) and murine X-linked immunodeficiency (Xid). In this paper, we report that the PH domain of Btk functions as an inositol 1,3,4,5-tetrakisphosphate (IP4), inositol 1,3,4,5,6-pentakisphosphate, and inositol 1,2,3,4,5,6-hexakisphosphate (IP6) binding domain (Kd of approximately 40 nM for IP4), and that all of the XLA (Phe replaced by Ser at position 25 (F25S), R28H, T33P, V64F, and V113D) and Xid mutations (R28C) found in the PH domain result in a dramatic reduction of IP4 binding activity. Furthermore, the rare alternative splicing variant, with 33 amino acids deleted in the PH domain, corresponding to exon 3 of the Btk gene, also impaired IP4 binding capacity. In contrast, a gain-of-function mutant called Btk*, which carries a E41K mutation in the PH domain, binds IP6 with two times higher affinity than the wild type. Our data suggest that B cell differentiation is closely correlated with the IP4 binding capacity of the PH domain of Btk.

Calcium-binding synaptotagmins (Syts) are membrane proteins that are conserved from nematode to human. Fifteen Syts (Syts I–XV) have been identified in mammalian species. Syt I has been well studied and is a candidate for the Ca 2+ -sensor that triggers evoked exocytosis underlying fast synaptic transmission. Whereas the functions of the other Syts are unclear, Syt IV is of particular interest because it is rapidly up-regulated after chronic depolarization or seizures, and because null mutations exhibit deficits in fine motor coordination and hippocampus-dependent memory. Screening Syts I–XIII, which are enriched in brain, we find that Syt IV is located in processes of astroglia in situ . Reduction of Syt IV in astrocytes by RNA interference decreases Ca 2+ -dependent glutamate release, a gliotransmission pathway that regulates synaptic transmission. Mutants of the C2B domain, the only putative Ca 2+ -binding domain in Syt IV, act in a dominant-negative fashion over Ca 2+ -regulated glial glutamate release, but not gliotransmission induced by changes in osmolarity. Because we find that Syt IV is expressed predominantly by astrocytes and is not in the presynaptic terminals of the hippocampus, and because Syt IV knockout mice exhibit hippocampal-based memory deficits, our data raise the intriguing possibility that Syt IV-mediated gliotransmission contributes to hippocampal-based memory.

The neurotrophin receptors TrkA, TrkB, and TrkC are localized at the surface of the axon terminus and transmit key signals from brain-derived neurotrophic factor (BDNF) for diverse effects on neuronal survival, differentiation, and axon formation. Trk receptors are sorted into axons via the anterograde transport of vesicles and are then inserted into axonal plasma membranes. However, the transport mechanism remains largely unknown. Here, we show that the Slp1/Rab27B/CRMP-2 complex directly links TrkB to Kinesin-1, and that this association is required for the anterograde transport of TrkB-containing vesicles. The cytoplasmic tail of TrkB binds to Slp1 in a Rab27B-dependent manner, and CRMP-2 connects Slp1 to Kinesin-1. Knockdown of these molecules by siRNA reduces the anterograde transport and membrane targeting of TrkB, thereby inhibiting BDNF-induced ERK1/2 phosphorylation in axons. Our data reveal a molecular mechanism for the selective anterograde transport of TrkB in axons and show how the transport is coupled to BDNF signaling.

Rabphilin, Rim, and Noc2 have generally been believed to be the Rab3 isoform (Rab3A/B/C/D)-specific effectors that regulate secretory vesicle exocytosis in neurons and in some endocrine cells. The results of recent genetic analysis of rabphilin knock-out animals, however, strongly refute this notion, because there are no obvious genetic interactions between Rab3 and rabphilin in nematoda (Staunton, J., Ganetzky, B., and Nonet, M. L. (2001) J. Neurosci. 21, 9255-9264), suggesting that Rab3 is not a major ligand of rabphilin in vivo. In this study, I tested the interaction of rabphilin, Rim1, Rim2, and Noc2 with 42 different Rab proteins by cotransfection assay and found differences in rabphilin, Rim1, Rim2, and Noc2 binding to several Rab proteins that belong to the Rab functional group III (Rab3A/B/C/D, Rab26, Rab27A/B, and Rab37) and/or VIII (Rab8A and Rab10). Rim1 interacts with Rab3A/B/C/D, Rab10, Rab26, and Rab37; Rim2 interacts with Rab3A/B/C/D and Rab8A; and rabphilin and Noc2 interact with Rab3A/B/C/D, Rab8A, and Rab27A/B. By contrast, the synaptotagmin-like protein homology domain of Slp homologue lacking C2 domains-a (Slac2-a)/melanophilin specifically recognizes Rab27A/B but not other Rabs. I also found that alternative splicing events in the first alpha-helical region (alpha(1)) of the Rab binding domain of Rim1 alter the Rab binding specificity of Rim1. Site-directed mutagenesis and chimeric analyses of Rim2 and Slac2-a indicate that the acidic cluster (Glu-50, Glu-51, and Glu-52) in the alpha(1) region of the Rab binding domain of Rim2, which is not conserved in the synaptotagmin-like pro tein homology domain of Slac2-a, is a critical determinant of Rab3A recognition. Based on these results, I propose that Rim, rabphilin, and Noc2 function differently in concert with functional group III and/or VIII Rab proteins, including Rab3 isoforms.

The most common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in individuals with cystic fibrosis, ΔF508, causes retention of ΔF508-CFTR in the endoplasmic reticulum and leads to the absence of CFTR Cl^- channels in the apical plasma membrane. Rescue of ΔF508-CFTR by reduced temperature or chemical means reveals that the ΔF508 mutation reduces the half-life of ΔF508-CFTR in the apical plasma membrane. Because ΔF508-CFTR retains some Cl^- channel activity, increased expression of ΔF508-CFTR in the apical membrane could serve as a potential therapeutic approach for cystic fibrosis. However, little is known about the mechanisms responsible for the short apical membrane half-life of ΔF508-CFTR in polarized human airway epithelial cells. Accordingly, the goal of this study was to determine the cellular defects in the trafficking of rescued ΔF508-CFTR that lead to the decreased apical membrane half-life of ΔF508-CFTR in polarized human airway epithelial cells. We report that in polarized human airway epithelial cells (CFBE41o-) the ΔF508 mutation increased endocytosis of CFTR from the apical membrane without causing a global endocytic defect or affecting the endocytic recycling of CFTR in the Rab11a-specific apical recycling compartment.

Recent studies have suggested that two small GTPases, Rab3A and Rab27A, play a key role in the late steps of dense-core vesicle exocytosis in endocrine cells; however, neither the precise mechanisms by which these two GTPases regulate dense-core vesicle exocytosis nor the functional relationship between them is clear. In this study, we expressed a number of different Rab proteins, from Rab1 to Rab41 in PC12 cells and systematically screened them for those that are specifically localized on dense-core vesicles. We found that four Rabs (Rab3A, Rab27A, Rab33A, Rab37) are predominantly targeted to dense-core vesicles in PC12 cells, and that three of them (Rab3A, Rab27A, Rab33A) are endogenously expressed on dense-core vesicles. We further investigated the effect of silencing each Rab with specific small interfering RNA on vesicle dynamics by total internal reflection fluorescence microscopy in a single PC12 cell. Silencing either Rab3A or Rab27A in PC12 cells significantly decreased the number of dense-core vesicles docked to the plasma membrane without altering the kinetics of individual exocytotic events, whereas silencing of Rab33A had no effect at all. Simultaneous silencing of Rab3A and Rab27A caused a significantly greater decrease in number of vesicles docked to the plasma membrane. Our findings indicate that Rab3A and Rab27A cooperatively regulate docking step(s) of dense-core vesicles to the plasma membrane.

Synaptotagmins I and II are inositol high polyphosphate series (inositol 1,3,4,5-tetrakisphosphate (IP4), inositol 1,3,4,5,6-pentakisphosphate, and inositol 1,2,3,4,5,6-hexakisphosphate) binding proteins, which are thought to be essential for Ca2+-regulated exocytosis of neurosecretory vesicles. In this study, we analyzed the inositol high polyphosphate series binding site in the C2B domain by site-directed mutagenesis and compared the IP4 binding properties of the C2B domains of multiple synaptotagmins (II-IV). The IP4 binding domain of synaptotagmin II is characterized by a cluster of highly conserved, positively charged amino acids (321 GKRLKKKKTTVKKK 324). Among these, three lysine residues, at positions 327, 328, and 332 in the middle of the C2B domain, which is not conserved in the C2A domain, were found to be essential for IP4 binding in synaptotagmin II. When these lysine residues were altered to glutamine, the IP4 binding ability was completely abolished. The primary structures of the IP4 binding sites are highly conserved among synaptotagmins I through IV. However, synaptotagmin III did not show significant binding ability, which may be due to steric hindrance by the C-terminal flanking region. These functional diversities of C2B domains suggest that not all synaptotagmins function as inositol high polyphosphate sensors at the synaptic vesicle. Synaptotagmins I and II are inositol high polyphosphate series (inositol 1,3,4,5-tetrakisphosphate (IP4), inositol 1,3,4,5,6-pentakisphosphate, and inositol 1,2,3,4,5,6-hexakisphosphate) binding proteins, which are thought to be essential for Ca2+-regulated exocytosis of neurosecretory vesicles. In this study, we analyzed the inositol high polyphosphate series binding site in the C2B domain by site-directed mutagenesis and compared the IP4 binding properties of the C2B domains of multiple synaptotagmins (II-IV). The IP4 binding domain of synaptotagmin II is characterized by a cluster of highly conserved, positively charged amino acids (321 GKRLKKKKTTVKKK 324). Among these, three lysine residues, at positions 327, 328, and 332 in the middle of the C2B domain, which is not conserved in the C2A domain, were found to be essential for IP4 binding in synaptotagmin II. When these lysine residues were altered to glutamine, the IP4 binding ability was completely abolished. The primary structures of the IP4 binding sites are highly conserved among synaptotagmins I through IV. However, synaptotagmin III did not show significant binding ability, which may be due to steric hindrance by the C-terminal flanking region. These functional diversities of C2B domains suggest that not all synaptotagmins function as inositol high polyphosphate sensors at the synaptic vesicle.

The TBC (Tre-2/Bub2/Cdc16) domain was originally identified as a conserved domain among the tre-2 oncogene product and the yeast cell cycle regulators Bub2 and Cdc16, and it is now widely recognized as a conserved protein motif that consists of approx. 200 amino acids in all eukaryotes. Since the TBC domain of yeast Gyps [GAP (GTPase-activating protein) for Ypt proteins] has been shown to function as a GAP domain for small GTPase Ypt/Rab, TBC domain-containing proteins (TBC proteins) in other species are also expected to function as a certain Rab-GAP. More than 40 different TBC proteins are present in humans and mice, and recent accumulating evidence has indicated that certain mammalian TBC proteins actually function as a specific Rab-GAP. Some mammalian TBC proteins {e.g. TBC1D1 [TBC (Tre-2/Bub2/Cdc16) domain family, member 1] and TBC1D4/AS160 (Akt substrate of 160 kDa)} play an important role in homoeostasis in mammals, and defects in them are directly associated with mouse and human diseases (e.g. leanness in mice and insulin resistance in humans). The present study reviews the structure and function of mammalian TBC proteins, especially in relation to Rab small GTPases.

Slac2-a (synaptotagmin-like protein (Slp) homologue lacking C2domains-a)/melanophilin is a melanosome-associated protein that links Rab27A on melanosomes with myosin Va, an actin-based motor protein, and formation of the tripartite protein complex (Rab27A·Slac2-a·myosin Va) has been suggested to regulate melanosome transport (Fukuda, M., Kuroda, T. S., and Mikoshiba, K. (2002) J. Biol. Chem. 277, 12432–12436). Here we report the structure of a novel form of Slac2, named Slac2-c, that is homologous to Slac2-a. Slac2-a and Slac2-c exhibit the same overall structure, consisting of a highly conserved N-terminal Slp homology domain (about 50% identity) and a less conserved C-terminal myosin Va-binding domain (about 20% identity). As with other Slac2 members and the Slp family, the Slp homology domain of Slac2-c was found to interact specifically with the GTP-bound form of Rab27A/B bothin vitro and in intact cells, and the C-terminal domain of Slac2-c interacted with myosin Va and myosin VIIa. In addition, we discovered that the most C-terminal conserved region of Slac2-a (amino acids 400–590) and Slac2-c (amino acids 670–856), which is not essential for myosin Va binding, directly binds actin and that expression of these regions in PC12 cells and melanoma cells colocalized with actin filaments at the cell periphery, suggesting a novel role of Slac2-a/c in capture of Rab27-containing organelles in the actin-enriched cell periphery. Slac2-a (synaptotagmin-like protein (Slp) homologue lacking C2domains-a)/melanophilin is a melanosome-associated protein that links Rab27A on melanosomes with myosin Va, an actin-based motor protein, and formation of the tripartite protein complex (Rab27A·Slac2-a·myosin Va) has been suggested to regulate melanosome transport (Fukuda, M., Kuroda, T. S., and Mikoshiba, K. (2002) J. Biol. Chem. 277, 12432–12436). Here we report the structure of a novel form of Slac2, named Slac2-c, that is homologous to Slac2-a. Slac2-a and Slac2-c exhibit the same overall structure, consisting of a highly conserved N-terminal Slp homology domain (about 50% identity) and a less conserved C-terminal myosin Va-binding domain (about 20% identity). As with other Slac2 members and the Slp family, the Slp homology domain of Slac2-c was found to interact specifically with the GTP-bound form of Rab27A/B bothin vitro and in intact cells, and the C-terminal domain of Slac2-c interacted with myosin Va and myosin VIIa. In addition, we discovered that the most C-terminal conserved region of Slac2-a (amino acids 400–590) and Slac2-c (amino acids 670–856), which is not essential for myosin Va binding, directly binds actin and that expression of these regions in PC12 cells and melanoma cells colocalized with actin filaments at the cell periphery, suggesting a novel role of Slac2-a/c in capture of Rab27-containing organelles in the actin-enriched cell periphery. synaptotagmin-like protein brain glutathioneS-transferase globular tail horseradish peroxidase melanocyte 5′-rapid amplification of cDNA ends reverse transcriptase Slp homology domain expressed sequence tag green fluorescent protein The Rab family, one of the small GTP-binding protein subfamilies (1Pereira-Leal J.B. Seabra M.C. J. Mol. Biol. 2000; 301: 1077-1087Crossref PubMed Scopus (374) Google Scholar), is thought to control intracellular membrane trafficking in eukaryotic cells (reviewed in Refs. 2Zerial M. McBride H. Nat. Rev. Mol. Cell Biol. 2001; 2: 107-117Crossref PubMed Scopus (2700) Google Scholar, 3Segev N. Curr. Opin. Cell Biol. 2001; 13: 500-511Crossref PubMed Scopus (247) Google Scholar, 4Pfeffer S.R. Trends Cell Biol. 2001; 11: 487-491Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar). More than 60 distinct Rab proteins have been identified in humans (5Bock J.B. Matern H.T. Peden A.A. Scheller R.H. Nature. 2001; 409: 839-841Crossref PubMed Scopus (521) Google Scholar, 6Pereira-Leal J.B. Seabra M.C. J. Mol. Biol. 2001; 313: 889-901Crossref PubMed Scopus (611) Google Scholar), and they seem to regulate various steps of membrane trafficking (e.g. vesicle formation, docking, tethering, and fusion) (2Zerial M. McBride H. Nat. Rev. Mol. Cell Biol. 2001; 2: 107-117Crossref PubMed Scopus (2700) Google Scholar, 3Segev N. Curr. Opin. Cell Biol. 2001; 13: 500-511Crossref PubMed Scopus (247) Google Scholar, 4Pfeffer S.R. Trends Cell Biol. 2001; 11: 487-491Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar). Although Rab proteins are generally believed to act with specific effector molecule(s) that preferentially bind the GTP-bound activated form of Rab, only a limited number of effector molecules have been identified to date (2Zerial M. McBride H. Nat. Rev. Mol. Cell Biol. 2001; 2: 107-117Crossref PubMed Scopus (2700) Google Scholar, 3Segev N. Curr. Opin. Cell Biol. 2001; 13: 500-511Crossref PubMed Scopus (247) Google Scholar, 4Pfeffer S.R. Trends Cell Biol. 2001; 11: 487-491Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar), and the exact roles of most of the Rab proteins remain to be elucidated. Recent studies have suggested that several Rabs (i.e. Rab6, Rab11, and Rab25) are involved in the movement of transport vesicles from their site of formation to their site of fusion, because these Rabs have been found to interact directly with specific microtubule- or actin-based motor proteins (see Refs. 7Echard A. Jollivet F. Martinez O. Lacapere J.J. Rousselet A. Janoueix-Lerosey I. Goud B. Science. 1998; 279: 580-585Crossref PubMed Scopus (411) Google Scholar, 8Lapierre L.A. Kumar R. Hales C.M. Navarre J. Bhartur S.G. Burnette J.O. Provance Jr., D.W. Mercer J.A. Bahler M. Goldenring J.R. Mol. Biol. Cell. 2001; 12: 1843-1857Crossref PubMed Scopus (342) Google Scholar, 9Schott D., Ho, J. Pruyne D. Bretscher A. J. Cell Biol. 1999; 147: 791-808Crossref PubMed Scopus (199) Google Scholar, and reviewed in Ref. 10Hammer III., J.A. Wu X.S. Curr. Opin. Cell Biol. 2002; 14: 69-75Crossref PubMed Scopus (124) Google Scholar). For instance, Rab6 interacts with the C-terminal domain of Rabkinesin-6 (7Echard A. Jollivet F. Martinez O. Lacapere J.J. Rousselet A. Janoueix-Lerosey I. Goud B. Science. 1998; 279: 580-585Crossref PubMed Scopus (411) Google Scholar), whereas Rab11 and Rab25 interact with the C-terminal domain of the myosin Vb tail (8Lapierre L.A. Kumar R. Hales C.M. Navarre J. Bhartur S.G. Burnette J.O. Provance Jr., D.W. Mercer J.A. Bahler M. Goldenring J.R. Mol. Biol. Cell. 2001; 12: 1843-1857Crossref PubMed Scopus (342) Google Scholar). Very recently, another type of Rab-motor protein interaction has been discovered in melanosome transport (11Fukuda M. Kuroda T.S. Mikoshiba K. J. Biol. Chem. 2002; 277: 12432-12436Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, 12Wu X.S. Rao K. Zhang H. Wang F. Sellers J.R. Matesic L.E. Copeland N.G. Jenkins N.A. Hammer III., J.A. Nat. Cell Biol. 2002; 4: 271-278Crossref PubMed Scopus (380) Google Scholar, 13Hume A.N. Collinson L.M. Hopkins C.R. Strom M. Barral D.C. Bossi G. Griffiths G.M. Seabra M.C. Traffic. 2002; 3: 193-202Crossref PubMed Scopus (132) Google Scholar, 14Provance Jr., D.W. James T.L. Mercer J.A. Traffic. 2002; 3: 124-132Crossref PubMed Scopus (111) Google Scholar) and plasma membrane recycling systems (15Hales C.M. Griner R. Hobdy-Henderson K.C. Dorn M.C. Hardy D. Kumar R. Navarre J. Chan E.K. Lapierre L.A. Goldenring J.R. J. Biol. Chem. 2001; 276: 39067-39075Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar); myosin Va indirectly recognizes Rab27A on melanosomes via Slac2-a (synaptotagmin-like protein (Slp)1 homologuelacking C2 domains-a) (also called melanophilin), a linker protein that interacts specifically and directly with the GTP-bound form of Rab27A and myosin Va (11Fukuda M. Kuroda T.S. Mikoshiba K. J. Biol. Chem. 2002; 277: 12432-12436Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, 12Wu X.S. Rao K. Zhang H. Wang F. Sellers J.R. Matesic L.E. Copeland N.G. Jenkins N.A. Hammer III., J.A. Nat. Cell Biol. 2002; 4: 271-278Crossref PubMed Scopus (380) Google Scholar, 16Kuroda T.S. Fukuda M. Ariga H. Mikoshiba K. J. Biol. Chem. 2002; 277: 9212-9218Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar,17Fukuda M. J. Biol. Chem. 2002; 277: 40118-40124Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), whereas myosin Vb interacts with Rab11 (and Rab25), as well as Rab11-FIP2 (15Hales C.M. Griner R. Hobdy-Henderson K.C. Dorn M.C. Hardy D. Kumar R. Navarre J. Chan E.K. Lapierre L.A. Goldenring J.R. J. Biol. Chem. 2001; 276: 39067-39075Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). Although five members of the Rab11-FIP family have been described to date (15Hales C.M. Griner R. Hobdy-Henderson K.C. Dorn M.C. Hardy D. Kumar R. Navarre J. Chan E.K. Lapierre L.A. Goldenring J.R. J. Biol. Chem. 2001; 276: 39067-39075Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, 18Prekeris R. Davies J.M. Scheller R.H. J. Biol. Chem. 2001; 276: 38966-38970Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 19Lindsay A.J. Hendrick A.G. Cantalupo G. Senic-Matuglia F. Goud B. Bucci C. McCaffrey M.W. J. Biol. Chem. 2002; 277: 12190-12199Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar), no information is available for the existence of a Slac2-a homologue (or Slac2 family). Because Rab27A and myosin Va are known to be expressed in tissues other than melanocytes, we hypothesized that other linker protein(s) must exist in the body. In this paper, we report on a novel Slac2-a homologue (named Slac2-c) that interacts specifically with Rab27A/B and myosin Va/VIIa by means of EST database searches and biochemical experiments. We also discovered that the conserved most C-terminal region of Slac2 functions as a novel actin-binding site. Based on our findings, we discuss the role of Slac2 in Rab27A-dependent membrane trafficking. cDNAs encoding the N-terminal region of Slac2-c were amplified from Marathon-Ready adult mouse brain cDNA (Clontech Laboratories, Inc.; Palo Alto, CA) by 5′-rapid amplification of cDNA ends (RACE) as described previously (20Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). First 5′-RACE reactions were carried out using the adapter primer 1 (5′-CCATCCTAATACGACTCACTATAGGGC-3′) and Slac2-c-stop primer (5′-TTAGTACATCACAGCTGACT-3′; termination codon is shown in boldface letters) designed on the basis of rat and mouse EST sequences (GenBankTM accession numbers BF287121 and BG869374). Second RACE reactions were carried out using the internal adapter primer 2 (5′-ACTCACTATAGGGCTCGAGCGGC-3′) and Slac2-c C1 primer (5′-GTGCTGGACCGGGAATTCTG-3′). The purified PCR products were inserted directly into the pGEM-T Easy vector (Promega; Madison, WI), and both strands were sequenced completely with a Hitachi SQ-5500 DNA sequencer as described previously (20Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). cDNAs encoding the open reading frame of the mouse Slac2-c were amplified by reverse transcriptase (RT)-PCR from Marathon-Ready adult mouse brain cDNA with Slac2-c Met (5′-GGATCCATGGGGAGGAAGCTGGACCT-3′;BamHI site is underlined) and Slac2-c-stop primers. The purified PCR products were subcloned into the pGEM-T Easy vector (named pGEM-T-Slac2-c) and verified by DNA sequencing. Addition of the T7 tag to the N terminus of Slac2-c and construction of the mammalian cell expression vector (named pEF-T7-Slac2-c) were performed as described previously (20Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 21Mizushima S. Nagata S. Nucleic Acids Res. 1990; 18: 5332Crossref Scopus (1499) Google Scholar, 22Fukuda M. Aruga J. Niinobe M. Aimoto S. Mikoshiba K. J. Biol. Chem. 1994; 269: 29206-29211Abstract Full Text PDF PubMed Google Scholar). The human Slac2-c cDNA was determined by database searching (standard BLAST search) using the mouse Slac2-c as a query. cDNA encoding a tail domain of mouse myosin VIIa (23Gibson F. Walsh J. Mburu P. Varela A. Brown K.A. Antonio M. Beisel K.W. Steel K.P. Brown S.D. Nature. 1995; 374: 62-64Crossref PubMed Scopus (545) Google Scholar) or MC-type myosin Va (− exon B, + exon D, and + exon F) (24Seperack P.K. Mercer J.A. Strobel M.C. Copeland N.G. Jenkins N.A. EMBO J. 1995; 14: 2326-2332Crossref PubMed Scopus (114) Google Scholar, 25Huang J.D. Mermall V. Strobel M.C. Russell L.B. Mooseker M.S. Copeland N.G. Jenkins N.A. Genetics. 1998; 148: 1963-1972Crossref PubMed Google Scholar) was amplified from Marathon-Ready adult brain cDNA or spleen cDNA from mouse MTC Panel I (Clontech Laboratories, Inc.), respectively, by RT-PCR as described previously (11Fukuda M. Kuroda T.S. Mikoshiba K. J. Biol. Chem. 2002; 277: 12432-12436Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, 16Kuroda T.S. Fukuda M. Ariga H. Mikoshiba K. J. Biol. Chem. 2002; 277: 9212-9218Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 20Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). The following oligonucleotides were used for amplification of the mouse myosin VIIa cDNA: 5′-CGGATCCCGGGTTGAGTACCAGCGGCG-3′ (myosin VIIa-N1 primer, sense), 5′-GATGTGGTCAATTATGCCCG-3′ (myosin VIIa- N2 primer, sense), 5′-CTCAAGTACATGGGCGACTA-3′ (myosin VIIa-N3 primer, sense), 5′-GCCTGAGAATTTGTAAGCTT-3′ (myosin VIIa-C1 primer, antisense), 5′-CTCAAAGATCTGGTCAGTGA-3′ (myosin VIIa-C2 primer, antisense), and 5′-TCACCTCCCGCTCCTGGAGT-3′ (myosin VIIa-stop primer, antisense). The purified PCR products were inserted directly into the pGEM-T Easy vector and were sequenced completely. We identified one amino acid difference (L1156F) compared with the reported myosin VIIa sequences (GenBankTM accession number NM_008663) (23Gibson F. Walsh J. Mburu P. Varela A. Brown K.A. Antonio M. Beisel K.W. Steel K.P. Brown S.D. Nature. 1995; 374: 62-64Crossref PubMed Scopus (545) Google Scholar), and it is unlikely to be a PCR-induced error, because we found the same difference in at least two independent clones. Addition of the FLAG tag to the N terminus of myosin VIIa tail or MC-type myosin Va tail and construction of the expression vector (pEF-FLAG-myosin VIIa-tail and pEF-FLAG-MC-myosin Va-tail) were performed as described previously (20Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 21Mizushima S. Nagata S. Nucleic Acids Res. 1990; 18: 5332Crossref Scopus (1499) Google Scholar, 22Fukuda M. Aruga J. Niinobe M. Aimoto S. Mikoshiba K. J. Biol. Chem. 1994; 269: 29206-29211Abstract Full Text PDF PubMed Google Scholar). pEF-T7-GST-FLAG-MC-myosin Va-F-GT (MC-specific exon F + globular tail) and pEF-FLAG-MC-myosin Va-F-GT were constructed by PCR essentially as described previously (11Fukuda M. Kuroda T.S. Mikoshiba K. J. Biol. Chem. 2002; 277: 12432-12436Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). Brain (BR)-type myosin Va cDNA (+ exon B, − exon D, and − exon F) (pEF-FLAG-BR-myosin Va) was prepared as described previously (11Fukuda M. Kuroda T.S. Mikoshiba K. J. Biol. Chem. 2002; 277: 12432-12436Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). Deletion mutants of Slac2-a (pEF-T7-Slac2-a-Δ146/481, -Δ146/321, -Δ400, and -Δ240) and of Slac2-c (pEF-T7-Slac2-c-SHD, -ΔSHD, -Δ146/494, -495/856, and pEF-T7-GST-Slac2-c-ΔSHD) were constructed essentially by conventional PCR as described previously (11Fukuda M. Kuroda T.S. Mikoshiba K. J. Biol. Chem. 2002; 277: 12432-12436Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, 26Fukuda M. Kanno E. Ogata Y. Mikoshiba K. J. Biol. Chem. 2001; 276: 40319-40325Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 27Fukuda M. Mikoshiba K. J. Biol. Chem. 2001; 276: 27670-27676Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar) using the following oligonucleotides with restriction enzyme sites (underlined) or stop codons (in bold): 5′-CGGATCCGAGGGCCTAGAGGAGACTGG-3′ (Slac2-a-Δ240 primer; sense), 5′-CGGATCCTCATCAGAAGATGAGACCAA-3′ (Slac2-a-Δ400 primer; sense), 5′-GCACTAGT CAAGCTGCGATCCTGGACTGGA-3′ (Slac2-a-Δ481 primer; antisense), 5′-GCACTAGT CAACCCTGGATACTGTCTTCAT-3′ (Slac2-a-Δ321 primer; antisense), 5′-GCACTAGT CAGGGCTCCAGAGAGGTGGTGT-3′ (Slac2-a-Δ241 primer; antisense), 5′-CGGATCCCGTCTGGAGAGCGGTGCCTG-3′ (Slac2-c-ΔSHD primer; sense), 5′-CGGATCCTTCAACCCTCAGGCAGCCGG-3′ (Slac2-c-Δ494 primer; sense), 5′-TCAGTGTTTTCTGTACAGGTTCT-3′ (Slac2-c-Δ146 primer, antisense), and 5′-TCAATTGACGTCCAGTTCTCCCT-3′ (Slac2-c-Δ495 primer, antisense). pEGFP-C1-Slac2-a, -Slac2-a-SHD, and -Slac2-a-Δ400 (Clontech Laboratories, Inc.) were constructed similarly by PCR. Other expression constructs (pEF-FLAG-Rab1, -Rab2, -Rab3A, -Rab4A, -Rab5A, -Rab6A, -Rab7, -Rab8, -Rab9, -Rab10, -Rab11A, -Rab17, -Rab18, -Rab20, -Rab22, -Rab23, -Rab25, -Rab27A, -Rab27B, -Rab28, -Rab34, -Rab37, and pEF-T7-GST-FLAG-myosin Va-GT) were prepared as described previously (11Fukuda M. Kuroda T.S. Mikoshiba K. J. Biol. Chem. 2002; 277: 12432-12436Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, 16Kuroda T.S. Fukuda M. Ariga H. Mikoshiba K. J. Biol. Chem. 2002; 277: 9212-9218Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). Mutant Rab27A plasmids carrying a Thr to Asn substitution at amino acid position 23 (T23N) (dominant negative form) or a Q78L substitution (dominant active form) were produced by PCR using the following mutagenic oligonucleotides and SP6 primer: 5′-CCTTGGGAGACTCTGGGGTAGGGAAGAACAGT-3′ (T23N primer) or 5′-CTGCAGTTATGGGACACGGCGGGGCTGGAG-3′ (Q78L primer). pGEM-T-Rab27A was used as a template (16Kuroda T.S. Fukuda M. Ariga H. Mikoshiba K. J. Biol. Chem. 2002; 277: 9212-9218Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). The mutant Rab27A fragments were subcloned into the pEF-FLAG-tag vector (20Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar) using appropriate restriction enzyme sites (underlined above). Mouse first-strand cDNAs prepared from various tissues and developmental stages were obtained from Clontech Laboratories, Inc. (mouse MTC Panel I) (28Fukuda M. Mikoshiba K. Biochem. Biophys. Res. Commun. 2001; 281: 1226-1233Crossref PubMed Scopus (80) Google Scholar, 29Fukuda M. Saegusa C. Mikoshiba K. Biochem. Biophys. Res. Commun. 2001; 283: 513-519Crossref PubMed Scopus (68) Google Scholar). PCRs were carried out in the presence of Perfect Match PCR enhancer (Stratagene; La Jolla, CA) for 30 cycles (for G3PDH), 35 cycles (for Slac2-a), or 40 cycles (for Slac2-c), each consisting of denaturation at 94 °C for 1 min, annealing at 55 °C for 2 min, and extension at 72 °C for 2 min. Slac2-a-Δ146 primer (16Kuroda T.S. Fukuda M. Ariga H. Mikoshiba K. J. Biol. Chem. 2002; 277: 9212-9218Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar) and Slac2-a-Δ321 primer and Slac2-c-N1 primer (5′-AGAGACTGACATCAGCAACG-3′) and Slac2-c-stop primer were used for amplification. The PCR products were analyzed by 1% agarose gel electrophoresis followed by ethidium bromide staining. The authenticity of the products was verified by subcloning into a pGEM-T Easy vector and DNA sequencing as described above. GST-Slac2-a-Δ400 and T7-GST-Slac2-a-Δ146 were prepared as described previously (11Fukuda M. Kuroda T.S. Mikoshiba K. J. Biol. Chem. 2002; 277: 12432-12436Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). G-actin and F-actin (Sigma) were prepared as described by Perelroizen et al. (30Perelroizen I. Didry D. Christensen H. Chua N.-H. Carlier M.-F. J. Biol. Chem. 1996; 271: 12302-12309Abstract Full Text PDF PubMed Scopus (123) Google Scholar) and were immobilized with anti-actin mouse monoclonal antibody (C-2)-conjugated agarose (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Approximately 1 μg of GST-Slac2-a-Δ400 (or GST alone) were incubated with the G-actin (or F-actin) beads in G-buffer (5 mm Tris-HCl, pH 7.5, 1 mm dithiothreitol, 0.2 mm ATP, and 0.1 mm CaCl2) or F-buffer (5 mmTris-HCl, pH 7.5, 1 mm dithiothreitol, 0.2 mmATP, 0.1 mm CaCl2, 2 mmMgCl2, and 0.1 m KCl) at 4 °C for 1 h and then washed five times with 1 ml of the binding buffer without actin. Proteins that bound the beads were analyzed by 10% SDS-PAGE and then immunoblotted with anti-actin goat polyclonal antibody (1/200 dilution) and horseradish peroxidase (HRP)-conjugated anti-GST antibody (1/2000 dilution; Santa Cruz Biotechnology, Inc.) as described previously (16Kuroda T.S. Fukuda M. Ariga H. Mikoshiba K. J. Biol. Chem. 2002; 277: 9212-9218Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 20Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). New Zealand White rabbits were immunized with GST-Slac2-a-SHD (amino acids 1–153) (11Fukuda M. Kuroda T.S. Mikoshiba K. J. Biol. Chem. 2002; 277: 12432-12436Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, 16Kuroda T.S. Fukuda M. Ariga H. Mikoshiba K. J. Biol. Chem. 2002; 277: 9212-9218Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar), and anti-Slac2-a-SHD antibody was affinity-purified by exposure to antigen-bound Affi-Gel 10 beads (Bio-Rad Laboratories, Hercules, CA) as described previously (31Fukuda M. Mikoshiba K. J. Biol. Chem. 1999; 274: 31428-31434Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Specificity of the antibody was checked by immunoblotting with recombinant T7-tagged Slac2-a and Slac2-c expressed in COS-7 cells (32Ibata K. Fukuda M. Hamada T. Kabayama H. Mikoshiba K. J. Neurochem. 2000; 74: 518-526Crossref PubMed Scopus (65) Google Scholar, 33Fukuda M. Kowalchyk J.A. Zhang X. Martin T.F.J. Mikoshiba K. J. Biol. Chem. 2002; 277: 4601-4604Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 34Zhang X. Kim-Miller M.J. Fukuda M. Kowalchyk J.A. Martin T.F.J. Neuron. 2002; 34: 599-611Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). Melanoma cells (B16-F1; 10-cm dish) were homogenized in a buffer containing 1 ml of the F-buffer and protease inhibitors, and proteins were solubilized with 1% Triton X-100 at 4 °C for 1 h. After removal of insoluble material by centrifugation, the supernatant was incubated with either anti-Slac2-a-SHD IgG (10 μg/ml) or control rabbit IgG for 1 h at 4 °C followed by incubation with protein A-Sepharose beads (Amersham Biosciences) for 1 h at 4 °C. After washing the beads five times with the F-buffer containing 0.2% Triton X-100 and protease inhibitors, the immunoprecipitates were subjected to 10% SDS-PAGE followed by immunoblotting with anti-actin (1/200 dilution) and anti-myosin Va goat antibodies (1/100 dilution; Santa Cruz Biotechnology, Inc.) as described previously (11Fukuda M. Kuroda T.S. Mikoshiba K. J. Biol. Chem. 2002; 277: 12432-12436Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, 20Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). PC12 cells were cultured on glass-bottom dishes (35-mm dish; MatTek Corp., Ashland, MA) as described previously (31Fukuda M. Mikoshiba K. J. Biol. Chem. 1999; 274: 31428-31434Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). A 4-μg sample of pEF-T7-Slac2-c-495/856, pEGFP-C1-Slac2-a-SHD, or -Slac2-a-Δ400 was transfected into PC12 cells by using LipofectAMINE 2000 reagent (Invitrogen) according to the manufacturer's instructions. Exposure to nerve growth factor (Merck KGaA, Darmstadt, Germany) was performed as described previously (32Ibata K. Fukuda M. Hamada T. Kabayama H. Mikoshiba K. J. Neurochem. 2000; 74: 518-526Crossref PubMed Scopus (65) Google Scholar). Melanoma cell line B16-F1 was cultured as described previously (16Kuroda T.S. Fukuda M. Ariga H. Mikoshiba K. J. Biol. Chem. 2002; 277: 9212-9218Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). pEGFP-C1-Slac2-a was transfected into melanoma cells by using FuGENE 6 (Roche Molecular Biochemicals) according to the manufacturer's instructions. Three days after transfection, cells were fixed in 4% paraformaldehyde, permeabilized with 0.3% Triton X-100 for 2 min, incubated for 1 h at room temperature with anti-T7 tag (1/4000 dilution; Novagen, Madison, WI) or anti-Rab27A mouse monoclonal antibody (1/250 dilution; Transduction Laboratories; Lexinton, KY), and visualized by a second antibody (1/10,000 dilution; anti-mouse Alexa 488 antibodies; Molecular Probes, Inc.; Eugene, OR) or Texas Red-conjugated phalloidin (1/200 or 1/500 dilution; Molecular Probes, Inc.) as described previously (31Fukuda M. Mikoshiba K. J. Biol. Chem. 1999; 274: 31428-31434Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 32Ibata K. Fukuda M. Hamada T. Kabayama H. Mikoshiba K. J. Neurochem. 2000; 74: 518-526Crossref PubMed Scopus (65) Google Scholar). The cells were then examined with a confocal fluorescence microscope (Fluoview; Olympus, Tokyo, Japan). Images were pseudo-colored and superimposed with Adobe Photoshop software (version 5.0). Cotransfection of pEF-T7-Slac2-c and pEF-FLAG-Rabs (or -FLAG-myosin Va) into COS-7 cells (7.5 × 105 cells, the day before transfection/10-cm dish) was performed as described previously (26Fukuda M. Kanno E. Ogata Y. Mikoshiba K. J. Biol. Chem. 2001; 276: 40319-40325Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Proteins were solubilized with a buffer containing 1% Triton X-100, 250 mm NaCl, 1 mm MgCl2, 50 mm HEPES-KOH, pH 7.2, 0.1 mm phenylmethylsulfonyl fluoride, 10 μmleupeptin, and 10 μm pepstatin A at 4 °C for 1 h. T7-Slac2-c was immunoprecipitated with anti-T7 tag antibody-conjugated agarose (Novagen) as described previously (20Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 27Fukuda M. Mikoshiba K. J. Biol. Chem. 2001; 276: 27670-27676Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Direct interaction of myosin Va-GT with Slac2-c-ΔSHD and in vitro formation of the tripartite protein complex (Slac2-c·Rab27A·myosin Va-F-GT) were also performed as described previously (11Fukuda M. Kuroda T.S. Mikoshiba K. J. Biol. Chem. 2002; 277: 12432-12436Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). SDS-PAGE and immunoblotting analyses with HRP-conjugated anti-FLAG tag (Sigma) and anti-T7 tag antibodies (Novagen) were also performed as described previously (20Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 26Fukuda M. Kanno E. Ogata Y. Mikoshiba K. J. Biol. Chem. 2001; 276: 40319-40325Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The blots and gels shown are representative of at least two or three independent experiments. Slac2 was identified originally as a protein that contains an N-terminal SHD without tandem C2 domains (29Fukuda M. Saegusa C. Mikoshiba K. Biochem. Biophys. Res. Commun. 2001; 283: 513-519Crossref PubMed Scopus (68) Google Scholar, 35McAdara Berkowitz J.K. Catz S.D. Johnson J.L. Ruedi J.M. Thon V. Babior B.M. J. Biol. Chem. 2001; 276: 18855-18862Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 36Wang J. Takeuchi T. Yokota H. Izumi T. J. Biol. Chem. 1999; 274: 28542-28548Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 37Ishikawa K. Nagase T. Suyama M. Miyajima N. Tanaka A. Kotani H. Nomura N. Ohara O. DNA Res. 1998; 5: 169-176Crossref PubMed Scopus (170) Google Scholar). Two forms of Slac2 (Slac2-a and Slac2-b/KIAA0624) have been reported in humans, but they do not show any significant homology except for the SHD (16Kuroda T.S. Fukuda M. Ariga H. Mikoshiba K. J. Biol. Chem. 2002; 277: 9212-9218Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). Recently, Slac2-a was identified as melanophilin, and a mutation in themlph gene was discovered to cause defects in melanosome transport in leaden mice exhibiting a lighter coat color (i.e. accumulation of melanosomes in the perinuclear region) (38Matesic L.E. Yip R. Reuss A.E. Swing D.A. O'Sullivan T.N. Fletcher C.F. Copeland N.G. Jenkins N.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10238-10243Crossref PubMed Scopus (201) Google Scholar). Slac2-a/melanophilin has been shown to function as a linker protein that bridges between Rab27A on melanosomes (39Menasche G. Pastural E. Feldmann J. Certain S. Ersoy F. Dupuis S. Wulffraat N. Bianchi D. Fischer A., Le Deist F. de Saint Basile G. Nat. 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Rab small GTPases are involved in the transport of vesicles between different membranous organelles. RAB-3 is an exocytic Rab that plays a modulatory role in synaptic transmission. Unexpectedly, mutations in the Caenorhabditis elegans RAB-3 exchange factor homologue, aex-3, cause a more severe synaptic transmission defect as well as a defecation defect not seen in rab-3 mutants. We hypothesized that AEX-3 may regulate a second Rab that regulates these processes with RAB-3. We found that AEX-3 regulates another exocytic Rab, RAB-27. Here, we show that C. elegans RAB-27 is localized to synapse-rich regions pan-neuronally and is also expressed in intestinal cells. We identify aex-6 alleles as containing mutations in rab-27. Interestingly, aex-6 mutants exhibit the same defecation defect as aex-3 mutants. aex-6; rab-3 double mutants have behavioral and pharmacological defects similar to aex-3 mutants. In addition, we demonstrate that RBF-1 (rabphilin) is an effector of RAB-27. Therefore, our work demonstrates that AEX-3 regulates both RAB-3 and RAB-27, that both RAB-3 and RAB-27 regulate synaptic transmission, and that RAB-27 potentially acts through its effector RBF-1 to promote soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) function.

Membrane and secretory trafficking are essential for proper neuronal development. However, the molecular mechanisms that organize secretory trafficking are poorly understood. Here, we identify Bicaudal-D-related protein 1 (BICDR-1) as an effector of the small GTPase Rab6 and key component of the molecular machinery that controls secretory vesicle transport in developing neurons. BICDR-1 interacts with kinesin motor Kif1C, the dynein/dynactin retrograde motor complex, regulates the pericentrosomal localization of Rab6-positive secretory vesicles and is required for neural development in zebrafish. BICDR-1 expression is high during early neuronal development and strongly declines during neurite outgrowth. In young neurons, BICDR-1 accumulates Rab6 secretory vesicles around the centrosome, restricts anterograde secretory transport and inhibits neuritogenesis. Later during development, BICDR-1 expression is strongly reduced, which permits anterograde secretory transport required for neurite outgrowth. These results indicate an important role for BICDR-1 as temporal regulator of secretory trafficking during the early phase of neuronal differentiation.

Macroautophagy is a bulk degradation system conserved in all eukaryotic cells. A ubiquitin-like protein, Atg8, and its homologues are essential for autophagosome formation and act as a landmark for selective autophagy of aggregated proteins and damaged organelles. In this study, we report evidence demonstrating that OATL1, a putative Rab guanosine triphosphatase–activating protein (GAP), is a novel binding partner of Atg8 homologues in mammalian cells. OATL1 is recruited to isolation membranes and autophagosomes through direct interaction with Atg8 homologues and is involved in the fusion between autophagosomes and lysosomes through its GAP activity. We further provide evidence that Rab33B, an Atg16L1-binding protein, is a target substrate of OATL1 and is involved in the fusion between autophagosomes and lysosomes, the same as OATL1. Because both its GAP activity and its Atg8 homologue–binding activity are required for OATL1 to function, we propose a model that OATL1 uses Atg8 homologues as a scaffold to exert its GAP activity and to regulate autophagosomal maturation.

Cytokinesis bridge instability leads to binucleated cells that can promote tumorigenesis in vivo [1Fujiwara T. Bandi M. Nitta M. Ivanova E.V. Bronson R.T. Pellman D. Cytokinesis failure generating tetraploids promotes tumorigenesis in p53-null cells.Nature. 2005; 437: 1043-1047Crossref PubMed Scopus (732) Google Scholar]. Membrane trafficking is crucial for animal cell cytokinesis [2Glotzer M. Animal cell cytokinesis.Annu. Rev. Cell Dev. Biol. 2001; 17: 351-386Crossref PubMed Scopus (281) Google Scholar, 3Eggert U.S. Mitchison T.J. Field C.M. Animal cytokinesis: from parts list to mechanisms.Annu. Rev. Biochem. 2006; 75: 543-566Crossref PubMed Scopus (331) Google Scholar, 4Montagnac G. Echard A. Chavrier P. Endocytic traffic in animal cell cytokinesis.Curr. Opin. Cell Biol. 2008; 20: 454-461Crossref PubMed Scopus (101) Google Scholar, 5Prekeris R. Gould G.W. Breaking up is hard to do—membrane traffic in cytokinesis.J. 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Cell. 2005; 16: 849-860Crossref PubMed Scopus (225) Google Scholar, 10Fielding A.B. Schonteich E. Matheson J. Wilson G. Yu X. Hickson G.R. Srivastava S. Baldwin S.A. Prekeris R. Gould G.W. Rab11-FIP3 and FIP4 interact with Arf6 and the exocyst to control membrane traffic in cytokinesis.EMBO J. 2005; 24: 3389-3399Crossref PubMed Scopus (233) Google Scholar, 11Pellinen T. Tuomi S. Arjonen A. Wolf M. Edgren H. Meyer H. Grosse R. Kitzing T. Rantala J.K. Kallioniemi O. et al.Integrin trafficking regulated by Rab21 is necessary for cytokinesis.Dev. Cell. 2008; 15: 371-385Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 12Kouranti I. Sachse M. Arouche N. Goud B. Echard A. Rab35 regulates an endocytic recycling pathway essential for the terminal steps of cytokinesis.Curr. Biol. 2006; 16: 1719-1725Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 13Dambournet D. Machicoane M. Chesneau L. Sachse M. Rocancourt M. El Marjou A. Formstecher E. Salomon R. Goud B. Echard A. Rab35 GTPase and OCRL phosphatase remodel lipids and F-actin for successful cytokinesis.Nat. Cell Biol. 2011; 13: 981-988Crossref PubMed Scopus (184) Google Scholar, 14Schweitzer J.K. D'Souza-Schorey C. Localization and activation of the ARF6 GTPase during cleavage furrow ingression and cytokinesis.J. Biol. Chem. 2002; 277: 27210-27216Crossref PubMed Scopus (85) Google Scholar, 15Montagnac G. Sibarita J.B. Loubéry S. Daviet L. Romao M. Raposo G. Chavrier P. ARF6 Interacts with JIP4 to control a motor switch mechanism regulating endosome traffic in cytokinesis.Curr. Biol. 2009; 19: 184-195Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 16Cascone I. Selimoglu R. Ozdemir C. Del Nery E. Yeaman C. White M. Camonis J. Distinct roles of RalA and RalB in the progression of cytokinesis are supported by distinct RalGEFs.EMBO J. 2008; 27: 2375-2387Crossref PubMed Scopus (88) Google Scholar] contribute to the postfurrowing steps of cytokinesis. However, little is known about how these pathways are coordinated for successful cytokinesis. The Rab35 GTPase controls a fast endocytic recycling pathway and must be activated for SEPTIN cytoskeleton localization at the intercellular bridge, and thus for completion of cytokinesis [12Kouranti I. Sachse M. Arouche N. Goud B. Echard A. Rab35 regulates an endocytic recycling pathway essential for the terminal steps of cytokinesis.Curr. Biol. 2006; 16: 1719-1725Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar]. Here, we report that the ARF6 GTPase [17D'Souza-Schorey C. Chavrier P. ARF proteins: roles in membrane traffic and beyond.Nat. Rev. Mol. Cell Biol. 2006; 7: 347-358Crossref PubMed Scopus (987) Google Scholar, 18Grant B.D. Donaldson J.G. Pathways and mechanisms of endocytic recycling.Nat. Rev. Mol. Cell Biol. 2009; 10: 597-608Crossref PubMed Scopus (892) Google Scholar] negatively regulates Rab35 activation and hence the Rab35 pathway. Human cells expressing a constitutively activated, GTP-bound ARF6 mutant display identical endocytic recycling and cytokinesis defects as those observed upon overexpression of the inactivated, GDP-bound Rab35 mutant. As a molecular mechanism, we identified the Rab35 GAP EPI64B as an effector of ARF6 in negatively regulating Rab35 activation. Unexpectedly, this regulation takes place at clathrin-coated pits, and activated ARF6 reduces Rab35 loading into the endocytic pathway. Thus, an effector of an ARF protein is a GAP for a downstream Rab protein, and we propose that this hierarchical ARF/Rab GTPase cascade controls the proper activation of a common endocytic pathway essential for cytokinesis.

Insulin secretory granules (ISGs) are cytoplasmic organelles of pancreatic β-cells. They are responsible for the storage and secretion of insulin. To date, only about 30 different proteins have been clearly described to be associated with these organelles. However, data from two-dimensional gel electrophoresis analyses suggested that almost 150 different polypeptides might be present within ISGs. The elucidation of the identity and function of the ISG proteins by proteomics strategies would be of considerable help to further understand some of the underlying mechanisms implicated in ISG biogenesis and trafficking. Furthermore it should give the bases to the comprehension of impaired insulin secretion observed during diabetes. A proteomics analysis of an enriched insulin granule fraction from the rat insulin-secreting cell line INS-1E was performed. The efficacy of the fractionation procedure was assessed by Western blot and electron microscopy. Proteins of the ISG fraction were separated by SDS-PAGE, excised from consecutive gel slices, and tryptically digested. Peptides were analyzed by nano-LC-ESI-MS/MS. This strategy identified 130 different proteins that were classified into four structural groups including intravesicular proteins, membrane proteins, novel proteins, and other proteins. Confocal microscopy analysis demonstrated the association of Rab37 and VAMP8 with ISGs in INS-1E cells. In conclusion, the present study identified 130 proteins from which 110 are new proteins associated with ISGs. The elucidation of their role will further help in the understanding of the mechanisms governing impaired insulin secretion during diabetes.

Upon antigen recognition, T-cell receptor (TCR/CD3) and other signaling molecules become enriched in a specialized contact site between the T cell and antigen-presenting cell, i.e. the immunological synapse (IS). Enrichment occurs via mechanisms that include polarized secretion from recycling endosomes, but the Rabs and RabGAPs that regulate this are unknown. EPI64C (TBC1D10C) is an uncharacterized candidate RabGAP we identified by mass spectrometry as abundant in human peripheral blood T cells that is preferentially expressed in hematopoietic cells. EPI64C is a Rab35-GAP based both on in vitro Rab35-specific GAP activity and findings in transfection assays. EPI64C and Rab35 dominant negative (DN) constructs each impaired transferrin export from a recycling pathway in Jurkat T-cells and induced large vacuoles marked by transferrin receptor, TCR, and SNAREs implicated in TCR-polarized secretion. Rab35 localized to the plasma membrane and to intracellular vesicles where it substantially colocalized with TfR and with TCR. Rab35 was strongly recruited to the IS. Conjugate formation was impaired by transfection with Rab35-DN or EPI64C and by EPI64C knock down. TCR enrichment at the IS was impaired by Rab35-DN. Thus, EPI64C and Rab35 regulate a recycling pathway in T cells and contribute to IS formation, most likely by participating in TCR transport to the IS.

The formation of epithelial tissues requires both the generation of apical–basal polarity and the coordination of this polarity between neighbouring cells to form a central lumen. During de novo lumen formation, vectorial membrane transport contributes to the formation of a singular apical membrane, resulting in the contribution of each cell to only a single lumen. Here, from a functional screen for genes required for three-dimensional epithelial architecture, we identify key roles for synaptotagmin-like proteins 2-a and 4-a (Slp2-a/4-a) in the generation of a single apical surface per cell. Slp2-a localizes to the luminal membrane in a PtdIns(4,5)P2-dependent manner, where it targets Rab27-loaded vesicles to initiate a single lumen. Vesicle tethering and fusion is controlled by Slp4-a, in conjunction with Rab27/Rab3/Rab8 and the SNARE syntaxin-3. Together, Slp2-a/4-a coordinate the spatiotemporal organization of vectorial apical transport to ensure that only a single apical surface, and thus the formation of a single lumen, occurs per cell. By performing a screen for genes that regulate epithelial architecture, Martín–Belmonte and colleagues identify key roles for the synaptotagmin-like proteins Slp2-a and Slp4-a in restricting lumen generation. They find that Slp2-a targets Rab27a/b-positive vesicles to PtdIns(4,5)P2-enriched apical membranes, whereas Slp4-a controls subsequent vesicle tethering and fusion. Their coordinated activities ensure the creation of a single lumen per cell.

A non-coding hexanucleotide repeat expansion in intron 1 of the C9orf72 gene is the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD), however, the precise molecular mechanism by which the C9orf72 hexanucleotide repeat expansion directs C9ALS/FTD pathogenesis remains unclear. Here, we report a novel disease mechanism arising due to the interaction of C9ORF72 with the RAB7L1 GTPase to regulate vesicle trafficking. Endogenous interaction between C9ORF72 and RAB7L1 was confirmed in human SH-SY5Y neuroblastoma cells. The C9orf72 hexanucleotide repeat expansion led to haploinsufficiency resulting in severely defective intracellular and extracellular vesicle trafficking and a dysfunctional trans-Golgi network phenotype in patient-derived fibroblasts and induced pluripotent stem cell-derived motor neurons. Genetic ablation of RAB7L1or C9orf72 in SH-SY5Y cells recapitulated the findings in C9ALS/FTD fibroblasts and induced pluripotent stem cell neurons. When C9ORF72 was overexpressed or antisense oligonucleotides were targeted to the C9orf72 hexanucleotide repeat expansion to upregulate normal variant 1 transcript levels, the defective vesicle trafficking and dysfunctional trans-Golgi network phenotypes were reversed, suggesting that both loss- and gain-of-function mechanisms play a role in disease pathogenesis. In conclusion, we have identified a novel mechanism for C9ALS/FTD pathogenesis highlighting the molecular regulation of intracellular and extracellular vesicle trafficking as an important pathway in C9ALS/FTD pathogenesis.

We recently showed that the exocytosis regulator Synaptotagmin (Syt) V is recruited to the nascent phagosome and remains associated throughout the maturation process. In this study, we investigated the possibility that Syt V plays a role in regulating interactions between the phagosome and the endocytic organelles. Silencing of Syt V by RNA interference revealed that Syt V contributes to phagolysosome biogenesis by regulating the acquisition of cathepsin D and the vesicular proton-ATPase. In contrast, recruitment of cathepsin B, the early endosomal marker EEA1 and the lysosomal marker LAMP1 to phagosomes was normal in the absence of Syt V. As Leishmania donovani promastigotes inhibit phagosome maturation, we investigated their potential impact on the phagosomal association of Syt V. This inhibition of phagolysosome biogenesis is mediated by the virulence glycolipid lipophosphoglycan, a polymer of the repeating Galbeta1,4Manalpha1-PO(4) units attached to the promastigote surface via an unusual glycosylphosphatidylinositol anchor. Our results showed that insertion of lipophosphoglycan into ganglioside GM1-containing microdomains excluded or caused dissociation of Syt V from phagosome membranes. As a consequence, L. donovani promatigotes established infection in a phagosome from which the vesicular proton-ATPase was excluded and which failed to acidify. Collectively, these results reveal a novel function for Syt V in phagolysosome biogenesis and provide novel insight into the mechanism of vesicular proton-ATPase recruitment to maturing phagosomes. We also provide novel findings into the mechanism of Leishmania pathogenesis, whereby targeting of Syt V is part of the strategy used by L. donovani promastigotes to prevent phagosome acidification.

Summary In yeast, C-tail-anchored mitochondrial outer membrane protein Fis1 recruits the mitochondrial-fission-regulating GTPase Dnm1 to mitochondrial fission sites. However, the function of its mammalian homologue remains enigmatic because it has been reported to be dispensable for the mitochondrial recruitment of Drp1, a mammalian homologue of Dnm1. We identified TBC1D15 as a Fis1-binding protein in HeLa cell extracts. Immunoprecipitation revealed that Fis1 efficiently interacts with TBC1D15 but not with Drp1. Bacterially expressed Fis1 and TBC1D15 formed a direct and stable complex. Exogenously expressed TBC1D15 localized mainly in cytoplasm in HeLa cells, but when coexpressed with Fis1 it localized to mitochondria. Knockdown of TBC1D15 induced highly developed mitochondrial network structures similar to the effect of Fis1 knockdown, suggesting that the TBC1D15 and Fis1 are associated with the regulation of mitochondrial morphology independently of Drp1. These data suggest that Fis1 acts as a mitochondrial receptor in the recruitment of mitochondrial morphology protein in mammalian cells.

Two small GTPases, Rab and Arf, are well-known molecular switches that function in diverse membrane-trafficking routes in a coordinated manner; however, very little is known about the direct crosstalk between Rab and Arf. Although Rab35 and Arf6 were independently reported to regulate the same cellular events, including endocytic recycling, phagocytosis, cytokinesis and neurite outgrowth, the molecular basis that links them remains largely unknown. Here we show that centaurin-β2 (also known as ACAP2) functions both as a Rab35 effector and as an Arf6-GTPase-activating protein (GAP) during neurite outgrowth of PC12 cells. We found that Rab35 accumulates at Arf6-positive endosomes in response to nerve growth factor (NGF) stimulation and that centaurin-β2 is recruited to the same compartment in a Rab35-dependent manner. We further showed by knockdown and rescue experiments that after the Rab35-dependent recruitment of centaurin-β2, the Arf6-GAP activity of centaurin-β2 at the Arf6-positive endosomes was indispensable for NGF-induced neurite outgrowth. These findings suggest a novel mode of crosstalk between Rab and Arf: a Rab effector and Arf-GAP coupling mechanism, in which Arf-GAP is recruited to a specific membrane compartment by its Rab effector function.

Transverse (T)-tubules make-up a specialized network of tubulated muscle cell membranes involved in excitation-contraction coupling for power of contraction. Little is known about how T-tubules maintain highly organized structures and contacts throughout the contractile system despite the ongoing muscle remodeling that occurs with muscle atrophy, damage and aging. We uncovered an essential role for autophagy in T-tubule remodeling with genetic screens of a developmentally regulated remodeling program in Drosophila abdominal muscles. Here, we show that autophagy is both upregulated with and required for progression through T-tubule disassembly stages. Along with known mediators of autophagosome-lysosome fusion, our screens uncovered an unexpected shared role for Rab2 with a broadly conserved function in autophagic clearance. Rab2 localizes to autophagosomes and binds to HOPS complex members, suggesting a direct role in autophagosome tethering/fusion. Together, the high membrane flux with muscle remodeling permits unprecedented analysis both of T-tubule dynamics and fundamental trafficking mechanisms.

MDCK II cells, a widely used model of polarized epithelia, develop into different structures depending on culture conditions: two-dimensional (2D) monolayers when grown on synthetic supports or three-dimensional (3D) cysts when surrounded by an extracellular matrix. The establishment of epithelial polarity is accompanied by transcytosis of the apical marker podocalyxin from the outer plasma membrane to the newly formed apical domain, but its exact route and regulation remain poorly understood. Here, through comprehensive colocalization and knockdown screenings, we identified the Rab GTPases mediating podocalyxin transcytosis and showed that different sets of Rabs coordinate its transport during cell polarization in 2D and 3D structures. Moreover, we demonstrated that different Rab35 effectors regulate podocalyxin trafficking in 2D and 3D environments; trafficking is mediated by OCRL in 2D monolayers and ACAP2 in 3D cysts. Our results give substantial insight into regulation of the transcytosis of this apical marker and highlight differences between trafficking mechanisms in 2D and 3D cell cultures.

Retromer is a protein assembly that orchestrates the sorting of transmembrane cargo proteins into endosome-to-Golgi and endosome-to-plasma-membrane transport pathways. Here, we have employed quantitative proteomics to define the interactome of human VPS35, the core retromer component. This has identified a number of new interacting proteins, including ankyrin-repeat domain 50 (ANKRD50), seriologically defined colon cancer antigen 3 (SDCCAG3) and VPS9-ankyrin-repeat protein (VARP, also known as ANKRD27). Depletion of these proteins resulted in trafficking defects of retromer-dependent cargo, but differential and cargo-specific effects suggested a surprising degree of functional heterogeneity in retromer-mediated endosome-to-plasma-membrane sorting. Extending this, suppression of the retromer-associated WASH complex did not uniformly affect retromer cargo, thereby confirming cargo-specific functions for retromer-interacting proteins. Further analysis of the retromer-VARP interaction identified a role for retromer in endosome-to-melanosome transport. Suppression of VPS35 led to mistrafficking of the melanogenic enzymes, tyrosinase and tryrosine-related protein 1 (Tyrp1), establishing that retromer acts in concert with VARP in this trafficking pathway. Overall, these data reveal hidden complexities in retromer-mediated sorting and open up new directions in our molecular understanding of this essential sorting complex.

Recycling endosomes are stations that sort endocytic cargoes to their appropriate destinations. Tubular endosomes have been characterized as a recycling endosomal compartment for clathrin-independent cargoes. However, the molecular mechanism by which tubular endosome formation is regulated is poorly understood. In this study, we identified Rab10 as a novel protein localized at tubular endosomes by using a comprehensive localization screen of EGFP-tagged Rab small GTPases. Knockout of Rab10 completely abolished tubular endosomal structures in HeLaM cells. We also identified kinesin motors KIF13A and KIF13B as novel Rab10-interacting proteins by means of in silico screening. The results of this study demonstrated that both the Rab10-binding homology domain and the motor domain of KIF13A are required for Rab10-positive tubular endosome formation. Our findings provide insight into the mechanism by which the Rab10-KIF13A (or KIF13B) complex regulates tubular endosome formation. This article has an associated First Person interview with the first author of the paper.

Rab7 (or Ypt7 in yeast) is one of the well-characterized members of the Rab family small GTPases, which serve as master regulators of membrane trafficking in eukaryotes. It localizes to late endosomes and lysosomes and has multiple functions in the autophagic pathway as well as in the endocytic pathway. Because Rab7/Ypt7 has previously been shown to regulate the autophagosome-lysosome fusion step in yeast and fruit flies (i.e., autophagosome accumulation has been observed in both Ypt7-knockout [KO] yeast and Rab7-knockdown fruit flies), it is widely assumed that Rab7 regulates the autophagosome-lysosome fusion step in mammals. A recent analysis of Rab7-KO mammalian cultured cells, however, has revealed that Rab7 is essential for autolysosome maturation (i.e., autolysosome accumulation has been observed in Rab7-KO cells), but not for autophagosome-lysosome fusion, under nutrient-rich conditions. Thus, although Rab7/Ypt7 itself is essential for the proper progression of autophagy in eukaryotes, the function of Rab7/Ypt7 in autophagy in yeast/fruit flies and mammals must be different. In this review article, we describe novel roles of Rab7 in mammalian autophagy and discuss its functional diversification during evolution.

Exosomes, important players in cell-cell communication, are small extracellular vesicles of endocytic origin. Although single cells are known to release various kinds of exosomes (referred to as exosomal heterogeneity), very little is known about the mechanisms by which they are produced and released. Here, we established methods of studying exosomal heterogeneity by using polarized epithelial cells and showed that distinct types of small extracellular vesicles (more specifically CD9- and CD63-positive, Annexin I-negative small extracellular vesicles, which we refer to as exosomes herein) are differentially secreted from the apical and basolateral sides of polarized epithelial cells. We also identify GPRC5C (G protein-coupled receptor class C group 5 member C) as an apical exosome-specific protein. We further demonstrate that basolateral exosome release depends on ceramide, whereas ALIX, an ESCRT (endosomal sorting complexes required for transport)-related protein, not the ESCRT machinery itself, is required for apical exosome release. Thus, two independent machineries, the ALIX-Syntenin1-Syndecan1 machinery (apical side) and the sphingomyelinase-dependent ceramide production machinery (basolateral side), are likely to be responsible for the polarized exosome release from epithelial cells.

<ns4:p>Melanin pigments are responsible for human skin and hair color, and they protect the body from harmful ultraviolet light. The black and brown melanin pigments are synthesized in specialized lysosome-related organelles called melanosomes in melanocytes. Mature melanosomes are transported within melanocytes and transferred to adjacent keratinocytes, which constitute the principal part of human skin. The melanosomes are then deposited inside the keratinocytes and darken the skin (a process called tanning). Owing to their dark color, melanosomes can be seen easily with an ordinary light microscope, and melanosome research dates back approximately 150 years; since then, biochemical studies aimed at isolating and purifying melanosomes have been conducted. Moreover, in the last two decades, hundreds of molecules involved in regulating melanosomal functions have been identified by analyses of the genes of coat-color mutant animals and patients with genetic diseases characterized by pigment abnormalities, such as hypopigmentation. In recent years, dynamic analyses by more precise microscopic observations have revealed specific functions of a variety of molecules involved in melanogenesis. This review article focuses on the latest findings with regard to the steps (or mechanisms) involved in melanosome formation and transport of mature melanosomes within epidermal melanocytes. Finally, we will touch on current topics in melanosome research, particularly on the "melanosome transfer" and "post-transfer" steps, and discuss future directions in pigment research.</ns4:p>

The synaptotagmins now constitute a large family of membrane proteins characterized by one transmembrane region and two C2 domains. Dimerization of synaptotagmin (Syt) I, a putative low affinity Ca²⁺ sensor for neurotransmitter release, is thought to be important for expression of function during exocytosis of synaptic vesicles. However, little is known about the self-dimerization properties of other isoforms. In this study, we demonstrate that a subclass of synaptotagmins (III, V, VI, and X) (Ibata, K., Fukuda, M., and Mikoshiba, K. (1998) <i>J. Biol. Chem.</i> 273, 12267–12273) forms β-mercaptoethanol-sensitive homodimers and identify three evolutionarily conserved cysteine residues at the N terminus (N-terminal cysteine motif, at amino acids 10, 21, and 33 of mouse Syt III) that are not conserved in other isoforms. Site-directed mutagenesis of these cysteine residues and co-immunoprecipitation experiments clearly indicate that the first cysteine residue is essential for the stable homodimer formation of Syt III, V, or VI, and heterodimer formation between Syts III, V, VI, and X. We also show that native Syt III from mouse brain forms a β-mercaptoethanol-sensitive homodimer. Our results suggest that the cysteine-based heterodimerization between Syt III and Syt V, VI, or X, which have different biochemical properties, may modulate the proposed function of Syt III as a putative high affinity Ca²⁺ sensor for neurotransmitter release.

Rab27a is a GTPase associated with insulin-containing secretory granules of pancreatic beta-cells. Selective reduction of Rab27a expression by RNA interference did not alter granule distribution and basal secretion but impaired exocytosis triggered by insulin secretagogues. Screening for potential effectors of the GTPase revealed that the Rab27a-binding protein Slac2c/MyRIP is associated with secretory granules of beta-cells. Attenuation of Slac2c/MyRIP expression by RNA interference did not modify basal secretion but severely impaired hormone release in response to secretagogues. Although beta-cells express Myosin-Va, a potential partner of Slac2c/MyRIP, no functional link between the two proteins could be demonstrated. In fact, overexpression of the Myosin-Va binding domain of Slac2c/MyRIP did not affect granule localization and hormone exocytosis. In contrast, overexpression of the actin-binding domain of Slac2c/MyRIP led to a potent inhibition of exocytosis without detectable alteration in granule distribution. This effect was prevented by point mutations that abolish actin binding. Taken together our data suggest that Rab27a and Slac2c/MyRIP are part of a complex mediating the interaction of secretory granules with cortical actin cytoskeleton and participate to the regulation of the final steps of insulin exocytosis.

Synaptotagmin (Syt) is an inositol high-polyphosphate series [IHPS inositol 1,3,4,5-tetrakisphosphate (IP4), inositol 1,3,4,5,6-pentakisphosphate, and inositol 1,2,3,4,5,6-hexakisphosphate] binding synaptic vesicle protein. A polyclonal antibody against the C2B domain (anti-Syt-C2B), an IHPS binding site, was produced. The specificity of this antibody to the C2B domain was determined by comparing its ability to inhibit IP4 binding to the C2B domain with that to inhibit the Ca2+/phospholipid binding to the C2A domain. Injection of the anti-Syt-C2B IgG into the squid giant presynapse did not block synaptic release. Coinjection of IP4 and anti-Syt-C2B IgG failed to block transmitter release, while IP4 itself was a powerful synpatic release blocker. Repetitive stimulation to presynaptic fiber injected with anti-Syt-C2B IgG demonstrated a rapid decline of the postsynaptic response amplitude probably due to its block of synaptic vesicle recycling. Electron microscopy of the anti-Syt-C2B-injected presynapse showed a 90% reduction of the numbers of synaptic vesicles. These results, taken together, indicate that the Syt molecule is central, in synaptic vesicle fusion by Ca2+ and its regulation by IHPS, as well as in the recycling of synaptic vesicles.

It has recently been proposed that the TBC (Tre2/Bub2/Cdc16) domain functions as a GAP (GTPase‐activating protein) domain for small GTPase Rab. Because of the large number of Rab proteins in mammals, however, most TBC domains have never been investigated for Rab‐GAP activity. In this study we established panels of the GTP‐fixed form of 60 different Rabs constructed in pGAD‐C1, a yeast two‐hybrid bait vector. We also constructed a yeast two‐hybrid prey vector (pGBDU‐C1) that harbors the cDNA of 40 distinct TBC proteins. Systematic investigation of 2400 combinations of 60 GTP‐fixed Rabs and 40 TBC proteins by yeast two‐hybrid screening revealed that seven TBC proteins specifically and differentially interact with specific Rabs (e.g. OATL1 interacts with Rab2A; FLJ12085 with Rab5A/B/C; and Evi5‐like with Rab10). Measurement of in vitro Rab‐GAP activity revealed that OATL1 and Evi5‐like actually possess significant Rab2A‐ and Rab10‐GAP activity, respectively, but that FLJ12085 do not display Rab5A‐GAP activity at all. These results indicate that specific interaction between TBC protein and Rab would be a useful indicator for screening for the target Rabs of some TBC/Rab‐GAP domains, but that there is little correlation between the Rab‐binding activity and Rab‐GAP activity of other TBC proteins.

Neurotrophins are key modulators of various neuronal functions, including differentiation, survival, and synaptic plasticity, but the molecules that regulate their secretion are poorly understood. We isolated a clone that is predominantly expressed in granule cells of postnatally developing mouse cerebellum, which turned out to be a paralog of CAPS (Ca2+-dependent activator protein for secretion), and named CAPS2. CAPS2 is enriched on vesicular structures of presynaptic parallel fiber terminals of granule cells connecting postsynaptic spines of Purkinje cell dendrites. Vesicle factions affinity-purified by the CAPS2 antibody from mouse cerebella contained significant amounts of neurotrophin-3 (NT-3), brain-derived neurotrophic factor (BDNF), and chromogranin B but not marker proteins for synaptic vesicle synaptophysin and synaptotagmin. In cerebellar primary cultures, punctate CAPS2 immunoreactivities are primarily colocalized with those of NT-3 and BDNF and near those of a postsynaptic marker, postsynaptic density-95, around dendritic arborization of Purkinje cells. Exogenously expressed CAPS2 enhanced release of exogenous NT-3 and BDNF from PC12 cells and endogenous NT-3 from cultured granule cells in a depolarization-dependent manner. Moreover, the overexpression of CAPS2 in granule cells promotes the survival of Purkinje cells in cerebellar cultures. Thus, we suggest that CAPS2 mediates the depolarization-dependent release of NT-3 and BDNF from granule cells, leading to regulation in cell differentiation and survival during cerebellar development.

Synaptotagmins are membrane proteins that possess tandem C2 domains and play an important role in regulated membrane fusion in metazoan organisms. Here we show that both synaptotagmins I and II, the two major neuronal isoforms, can interact with the syntaxin/synaptosomal-associated protein of 25 kDa (SNAP-25) dimer, the immediate precursor of the soluble NSF attachment protein receptor (SNARE) fusion complex. A stretch of basic amino acids highly conserved throughout the animal kingdom is responsible for this calcium-independent interaction. Inositol hexakisphosphate modulates synaptotagmin coupling to the syntaxin/SNAP-25 dimer, which is mirrored by changes in chromaffin cell exocytosis. Our results shed new light on the functional importance of the conserved polybasic synaptotagmin motif, suggesting that synaptotagmin interacts with the t-SNARE dimer to up-regulate the probability of SNARE-mediated membrane fusion.

Melanosomes containing melanin pigments are transported from the cell body of melanocytes to the tips of their dendrites by a combination of microtubule- and actin-dependent machinery. Three proteins, Rab27A, myosin Va, and Slac2-a/melanophilin (a linker protein between Rab27A and myosin Va), are known to be essential for proper actin-based melanosome transport in melanocytes. Although Slac2-a directly interacts with Rab27A and myosin Va via its N-terminal region (amino acids 1 to 146) and the middle region (amino acids 241 to 405), respectively, the functional importance of the putative actin-binding domain of the Slac2-a C terminus (amino acids 401 to 590) in melanosome transport has never been elucidated. In this study we showed that formation of a tripartite protein complex between Rab27A, Slac2-a, and myosin Va alone is insufficient for peripheral distribution of melanosomes in melanocytes and that the C-terminal actin-binding domain of Slac2-a is also required for proper melanosome transport. When a Slac2-a deletion mutant (DeltaABD) or point mutant (KA) that lacks actin-binding ability was expressed in melanocytes, the Slac2-a mutants induced melanosome accumulation in the perinuclear region, possibly by a dominant negative effect, the same as the Rab27A-binding-defective mutant of Slac2-a or the myosin Va-binding-defective mutant. Our findings indicate that Slac2-a organizes actin-based melanosome transport in cooperation with Rab27A, myosin Va, and actin.

Squid synaptotagmin (Syt) cDNA, including its open reading frame, was cloned and polyclonal antibodies were obtained in rabbits immunized with glutathione S-transferase (GST)-Syt-C2A. Binding assays indicated that the antibody, anti-Syt-C2A, recognized squid Syt and inhibited the Ca(2+)-dependent phospholipid binding to the C2A domain. This antibody, when injected into the preterminal at the squid giant synapse, blocked transmitter release in a manner similar to that previously reported for the presynaptic injection of members of the inositol high-polyphosphate series. The block was not accompanied by any change in the presynaptic action potential or the amplitude or voltage dependence of the presynaptic Ca2+ current. The postsynaptic potential was rather insensitive to repetitive presynaptic stimulation, indicating a direct effect of the antibody on the transmitter release system. Following block of transmitter release, confocal microscopical analysis of the preterminal junction injected with rhodamine-conjugated anti-Syt-C2A demonstrated fluorescent spots at the inner surface of the presynaptic plasmalemma next to the active zones. Structural analysis of the same preparations demonstrated an accumulation of synaptic vesicles corresponding in size and distribution to the fluorescent spots demonstrated confocally. Together with the finding that such antibody prevents Ca2+ binding to a specific receptor in the C2A domain, these results indicate that Ca2+ triggers transmitter release by activating the C2A domain of Syt. We conclude that the C2A domain is directly related to the fusion of synaptic vesicles that results in transmitter release.

In fat and muscle cells, insulin stimulates the movement to and fusion of intracellular vesicles containing GLUT4 with the plasma membrane, a process referred to as GLUT4 translocation. Previous studies have indicated that Akt [also known as PKB (protein kinase B)] phosphorylation of AS160, a GAP (GTPase-activating protein) for Rabs, is required for GLUT4 translocation. The results suggest that this phosphorylation suppresses the GAP activity and leads to the elevation of the GTP form of one or more Rabs required for GLUT4 translocation. Based on their presence in GLUT4 vesicles and activity as AS160 GAP substrates, Rabs 8A, 8B, 10 and 14 are candidate Rabs. Here, we provide further evidence that Rab10 participates in GLUT4 translocation in 3T3-L1 adipocytes. Among Rabs 8A, 8B, 10 and 14, only the knockdown of Rab10 inhibited GLUT4 translocation. In addition, we describe the subcellular distribution of Rab10 and estimate the fraction of Rab10 in the active GTP form in vivo. Approx. 5% of the total Rab10 was present in GLUT4 vesicles isolated from the low-density microsomes. In both the basal and the insulin state, 90% of the total Rab10 was in the inactive GDP state. Thus, if insulin increases the GTP form of Rab10, the increase is limited to a small portion of the total Rab10. Finally, we report that the Rab10 mutant considered to be constitutively active (Rab10 Q68L) is a substrate for the AS160 GAP domain and, hence, cannot be used to deduce rigorously the function of Rab10 in its GTP form.

Abstract The Rab family belongs to the Ras-like small GTPase superfamily and is implicated in membrane trafficking through interaction with specific effector molecules. Because of the large number of Rab isoforms in mammals, however, the effectors of most of the mammalian Rabs are yet to be identified. In this study, we systematically screened five different cell or tissue lysates for novel Rab effectors by a combination of glutathione S-transferase (GST) pull-down assay with 60 different mammalian Rabs and mass spectroscopic analysis. Three of the 21 Rab-binding proteins we identified, mKIAA1055/TBC1D2B (Rab22-binding protein), GAPCenA/TBC1D11 (Rab36-binding protein) and centaurin β2/ACAP2 (Rab35-binding protein), are GTPase-activating proteins (GAPs) for Rab or Arf. Although it has recently been proposed that the Rab–GAP (Tre-2 /Bub2/Cdc16) domain physically interacts with its substrate Rab, these three GAPs interacted with specific Rabs via a domain other than a GAP domain, e.g. centaurin β2 binds GTP-Rab35 via the ankyrin repeat (ANKR) domain. Although centaurin β2 did not exhibit any Rab35–GAP activity in vitro, the Rab35-binding ANKR domain of centaurin β2 was found to be required for its plasma membrane localization and regulation of Rab35-dependent neurite outgrowth of PC12 cells through inactivation of Arf6. These findings suggest a novel mode of interaction between Rab and GAP.

Recent findings indicate that mammalian Sonic hedgehog (Shh) signal transduction occurs within primary cilia, although the cell biological mechanisms underlying both Shh signaling and ciliogenesis have not been fully elucidated. We show that an uncharacterized TBC domain-containing protein, Broad-minded (Bromi), is required for high-level Shh responses in the mouse neural tube. We find that Bromi controls ciliary morphology and proper Gli2 localization within the cilium. By use of a zebrafish model, we further show that Bromi is required for proper association between the ciliary membrane and axoneme. Bromi physically interacts with cell cycle-related kinase (CCRK), whose Chlamydomonas homolog regulates flagellar length. Biochemical and genetic interaction data indicate that Bromi promotes CCRK stability and function. We propose that Bromi and CCRK control the structure of the primary cilium by coordinating assembly of the axoneme and ciliary membrane, allowing Gli proteins to be properly activated in response to Shh signaling.

Cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl^- secretion across fluid-transporting epithelia is regulated, in part, by modulating the number of CFTR Cl^- channels in the plasma membrane by adjusting CFTR endocytosis and recycling. However, the mechanisms that regulate CFTR recycling in airway epithelial cells remain unknown, at least in part, because the recycling itineraries of CFTR in these cells are incompletely understood. In a previous study, we demonstrated that CFTR undergoes trafficking in Rab11a-specific apical recycling endosomes in human airway epithelial cells. Myosin Vb is a plus-end-directed, actin-based mechanoenzyme that facilitates protein trafficking in Rab11a-specific recycling vesicles in several cell model systems. There are no published studies examining the role of myosin Vb in airway epithelial cells. Thus, the goal of this study was to determine whether myosin Vb facilitates CFTR recycling in polarized human airway epithelial cells. Endogenous CFTR formed a complex with endogenous myosin Vb and Rab11a. Silencing myosin Vb by RNA-mediated interference decreased the expression of wild-type CFTR and ΔF508-CFTR in the apical membrane and decreased CFTR-mediated Cl^- secretion across polarized human airway epithelial cells. A recombinant tail domain fragment of myosin Vb attenuated the plasma membrane expression of CFTR by arresting CFTR recycling. The dominant-negative effect was dependent on the ability of the myosin Vb tail fragment to interact with Rab11a. Taken together, these data indicate that myosin Vb is required for CFTR recycling in Rab11a-specific apical recycling endosomes in polarized human airway epithelial cells.

Two small GTPase Rabs, Rab32 and Rab38, have recently been proposed to regulate trafficking of melanogenic enzymes to melanosomes in mammalian epidermal melanocytes; however, the exact molecular mechanism of Rab32/38-mediated transport of melanogenic enzymes has never been clarified, because no Rab32/38-specific effector has ever been identified. In this study, we screened for a Rab32/38-specific effector by a yeast two-hybrid assay using a guanosine triphosphate (GTP)-locked Rab32/38 as bait and found that VPS9-ankyrin-repeat protein (Varp)/Ankrd27, characterized previously as a guanine nucleotide exchange factor (GEF) for Rab21, functions as a specific Rab32/38-binding protein in mouse melanocyte cell line melan-a. Deletion analysis showed that the first ankyrin-repeat (ANKR1) domain functions as a GTP-dependent Rab32/38-binding domain, but that the N-terminal VPS9 domain (i.e., Rab21-GEF domain) does not. Small interfering RNA-mediated knockdown of endogenous Varp in melan-a cells caused a dramatic reduction in Tyrp1 (tyrosinase-related protein 1) signals from melanosomes but did not cause any reduction in Pmel17 signals. Furthermore, expression of the ANKR1 domain in melan-a cells also caused a dramatic reduction of Tyrp1 signals, whereas the VPS9 domain had no effect. Based on these findings, we propose that Varp functions as the Rab32/38 effector that controls trafficking of Tyrp1 in melanocytes.

Transferrin receptor (TfR) is a well-characterized plasma membrane protein that travels between the plasma membrane and intracellular membrane compartments. Although TfR itself should undergo degradation, the same as other intracellular proteins, whether a specific TfR degradation pathway exists has never been investigated. In this study, we screened small GTPase Rab proteins, common regulators of membrane traffic in all eukaryotes, for proteins that are specifically involved in TfR degradation. We performed the screening by three sequential methods, i.e. colocalization of Rab with TfR, colocalization with lysosomes, and knockdown of Rab by specific small interfering RNA (siRNA), and succeeded in identifying Rab12, a previously uncharacterized Rab isoform, as a prime candidate among the 60 human or mouse Rabs screened. We showed that expression of a constitutive active mutant of Rab12 reduced the amount of TfR protein, whereas functional ablation of Rab12 by knockdown of either Rab12 itself or its upstream activator Dennd3 increased the amount of TfR protein. Interestingly, however, knockdown of Rab12 had no effect on the degradation of epidermal growth factor receptor (EGFR) protein, i.e. on a conventional degradation pathway. Our findings indicated that TfR is constitutively degraded by a Rab12-dependent pathway (presumably from recycling endosomes to lysosomes), which is independent of the conventional degradation pathway.

Host cell factors can either positively or negatively regulate the assembly and egress of HIV-1 particles from infected cells. Recent reports have identified a previously uncharacterized transmembrane protein, tetherin/CD317/BST-2, as a crucial host restriction factor that acts during a late budding step in HIV-1 replication by inhibiting viral particle release. Although tetherin has been shown to promote the retention of nascent viral particles on the host cell surface, the precise molecular mechanisms that occur during and after these tethering events remain largely unknown. We here report that a RING-type E3 ubiquitin ligase, BCA2 (Breast cancer-associated gene 2; also called Rabring7, ZNF364 or RNF115), is a novel tetherin-interacting host protein that facilitates the restriction of HIV-1 particle production in tetherin-positive cells. The expression of human BCA2 in "tetherin-positive" HeLa, but not in "tetherin-negative" HOS cells, resulted in a strong restriction of HIV-1 particle production. Upon the expression of tetherin in HOS cells, BCA2 was capable of inhibiting viral particle production as in HeLa cells. The targeted depletion of endogenous BCA2 by RNA interference (RNAi) in HeLa cells reduced the intracellular accumulation of viral particles, which were nevertheless retained on the plasma membrane. BCA2 was also found to facilitate the internalization of HIV-1 virions into CD63(+) intracellular vesicles leading to their lysosomal degradation. These results indicate that BCA2 accelerates the internalization and degradation of viral particles following their tethering to the cell surface and is a co-factor or enhancer for the tetherin-dependent restriction of HIV-1 release from infected cells.

A large protein complex consisting of Atg5, Atg12 and Atg16L1 has recently been shown to be essential for the elongation of isolation membranes (also called phagophores) during mammalian autophagy. However, the precise function and regulation of the Atg12–5-16L1 complex has largely remained unknown. In this study we identified a novel isoform of mammalian Atg16L, termed Atg16L2, that consists of the same domain structures as Atg16L1. Biochemical analysis revealed that Atg16L2 interacts with Atg5 and self-oligomerizes to form an ~800-kDa complex, the same as Atg16L1 does. A subcellular distribution analysis indicated that, despite forming the Atg12–5-16L2 complex, Atg16L2 is not recruited to phagophores and is mostly present in the cytosol. The results also showed that Atg16L2 is unable to compensate for the function of Atg16L1 in autophagosome formation, and knockdown of endogenous Atg16L2 did not affect autophagosome formation, indicating that Atg16L2 does not possess the ability to mediate canonical autophagy. Moreover, a chimeric analysis between Atg16L1 and Atg16L2 revealed that their difference in function in regard to autophagy is entirely attributable to the difference between their middle regions that contain a coiled-coil domain. Based on the above findings, we propose that formation of the Atg12–5-16L complex is necessary but insufficient to mediate mammalian autophagy and that an additional function of the middle region (especially around amino acid residues 229–242) of Atg16L1 (e.g., interaction with an unidentified binding partner on phagophores) is required for autophagosome formation.

Rab GTPases including Rab27a, Rab38 and Rab32 function in melanosome maturation or trafficking in melanocytes. A screen to identify additional Rabs involved in these processes revealed the localization of GFP-Rab17 on recycling endosomes (REs) and melanosomes in melanocytic cells. Rab17 mRNA expression is regulated by microphthalmia transcription factor (MITF), a characteristic of known pigmentation genes. Rab17 siRNA knockdown in melanoma cells quantitatively increased melanosome concentration at the cell periphery. Rab17 knockdown did not inhibit melanosome maturation nor movement, but it caused accumulation of melanin inside cells. Double knockdown of Rab17 and Rab27a indicated that Rab17 acts on melanosomes downstream of Rab27a. Filopodia are known to play a role in melanosome transfer, and in Rab17 knockdown cells filopodia formation was inhibited. Furthermore, we show that stimulation of melanoma cells with α-melanocyte-stimulating hormone induces filopodia formation, supporting a role for filopodia in melanosome release. Cell stimulation also caused redistribution of REs to the periphery, and knockdown of additional RE-associated Rabs 11a and 11b produced a similar accumulation of melanosomes and melanin to that seen after loss of Rab17. Our findings reveal new functions for RE and Rab17 in pigmentation through a distal step in the process of melanosome release via filopodia.

Primary cilia are antenna-like sensory organelles protruding from the plasma membrane. Defects in ciliogenesis cause diverse genetic disorders. NDR2 was identified as the causal gene for a canine ciliopathy, early retinal degeneration, but its role in ciliogenesis remains unknown. Ciliary membranes are generated by transport and fusion of Golgi-derived vesicles to the pericentrosome, a process requiring Rab11-mediated recruitment of Rabin8, a GDP-GTP exchange factor (GEF) for Rab8, and subsequent Rab8 activation and Rabin8 binding to Sec15, a component of the exocyst that mediates vesicle tethering. This study shows that NDR2 phosphorylates Rabin8 at Ser-272 and defects in this phosphorylation impair preciliary membrane assembly and ciliogenesis, resulting in accumulation of Rabin8-/Rab11-containing vesicles at the pericentrosome. Rabin8 binds to and colocalizes with GTP-bound Rab11 and phosphatidylserine (PS) on pericentrosomal vesicles. The phospho-mimetic S272E mutation of Rabin8 decreases affinity for PS but increases affinity for Sec15. These results suggest that NDR2-mediated Rabin8 phosphorylation is crucial for ciliogenesis by triggering the switch in binding specificity of Rabin8 from PS to Sec15, thereby promoting local activation of Rab8 and ciliary membrane formation.

Small GTPase Rab35 is an important molecular switch for endocytic recycling that regulates various cellular processes, including cytokinesis, cell migration, and neurite outgrowth. We previously showed that active Rab35 promotes nerve growth factor (NGF)-induced neurite outgrowth of PC12 cells by recruiting MICAL-L1, a multiple Rab-binding protein, to Arf6-positive recycling endosomes. However, the physiological significance of the multiple Rab-binding ability of MICAL-L1 during neurite outgrowth remained completely unknown. Here we report that Rab35 and MICAL-L1 promote the recruitment of Rab8, Rab13, and Rab36 to Arf6-positive recycling endosomes during neurite outgrowth. We found that Rab35 functions as a master Rab that determines the intracellular localization of MICAL-L1, which in turn functions as a scaffold for Rab8, Rab13, and Rab36. We further showed by functional ablation experiments that each of these downstream Rabs regulates neurite outgrowth in a non-redundant manner downstream of Rab35 and MICAL-L1, e.g. by showing that knockdown of Rab36 inhibited recruitment of Rab36-specific effector JIP4 to Arf6-positive recycling endosomes, and caused inhibition of neurite outgrowth without affecting accumulation of Rab8 and Rab13 in the same Arf6-positive area. Our findings suggest the existence of a novel mechanism that recruits multiple Rab proteins at the Arf6-positive compartment by MICAL-L1.

Phagosome formation and subsequent maturation are complex sequences of events that involve actin cytoskeleton remodeling and membrane trafficking. Here, we demonstrate that the Ras-related protein Rab35 is involved in the early stage of FcγR-mediated phagocytosis in macrophages. Live-cell image analysis revealed that Rab35 was markedly concentrated at the membrane where IgG-opsonized erythrocytes (IgG-Es) are bound. Rab35 silencing by RNA interference (RNAi) or the expression of GDP- or GTP-locked Rab35 mutant drastically reduced the rate of phagocytosis of IgG-Es. Actin-mediated pseudopod extension to form phagocytic cups was disturbed by the Rab35 silencing or the expression of GDP-Rab35, although initial actin assembly at the IgG-E binding sites was not inhibited. Furthermore, GTP-Rab35-dependent recruitment of ACAP2, an ARF6 GTPase-activating protein, was shown in the phagocytic cup formation. Concomitantly, overexpression of ACAP2 along with GTP-locked Rab35 showed a synergistic inhibitory effect on phagocytosis. It is likely that Rab35 regulates actin-dependent phagosome formation by recruiting ACAP2, which might control actin remodeling and membrane traffic through ARF6.

Autophagy is an evolutionarily conserved catabolic mechanism that targets intracellular molecules and damaged organelles to lysosomes. Autophagy is achieved by a series of membrane trafficking events, but their regulatory mechanisms are poorly understood. Here, we report small GTPase Rab12 as a new type of autophagic regulator that controls the degradation of an amino‐acid transporter. Knockdown of Rab12 results in inhibition of autophagy and in increased activity of mTORC1 (mammalian/mechanistic target of rapamycin complex 1), an upstream regulator of autophagy. We also found that Rab12 promotes constitutive degradation of PAT4 (proton‐coupled amino‐acid transporter 4), whose accumulation in Rab12‐knockdown cells modulates mTORC1 activity and autophagy. Our findings reveal a new mechanism of regulation of mTORC1 signalling and autophagy, that is, quality control of PAT4 by Rab12.

Small GTPase Rab functions as a molecular switch that drives membrane trafficking through specific interaction with its effector molecule. Thus, identification of its specific effector domain is crucial to revealing the molecular mechanism that underlies Rab-mediated membrane trafficking. Because of the large numbers of Rab isoforms in higher eukaryotes, however, the effector domains of most of the vertebrate- or mammalian-specific Rabs have yet to be determined. In this study we screened for effector molecules of Rab36, a previously uncharacterized Rab isoform that is largely conserved in vertebrates, and we succeeded in identifying nine Rab36-binding proteins, including RILP (Rab interacting lysosomal protein) family members. Sequence comparison revealed that five of nine Rab36-binding proteins, i.e. RILP, RILP-L1, RILP-L2, and JIP3/4, contain a conserved coiled-coil domain. We identified the coiled-coil domain as a RILP homology domain (RHD) and characterized it as a common Rab36-binding site. Site-directed mutagenesis of the RHD of RILP revealed the different contributions by amino acids in the RHD to binding activity toward Rab7 and Rab36. Expression of RILP in melanocytes, but not expression of its Rab36 binding-deficient mutants, induced perinuclear aggregation of melanosomes, and this effect was clearly attenuated by knockdown of endogenous Rab36 protein. Moreover, knockdown of Rab36 in Rab27A-deficient melanocytes, which normally exhibit perinuclear melanosome aggregation because of increased retrograde melanosome transport activity, caused dispersion of melanosomes from the perinucleus to the cell periphery, but knockdown of Rab7 did not. Our findings indicated that Rab36 mediates retrograde melanosome transport in melanocytes through interaction with RILP.

Weibel-Palade bodies (WPBs) are endothelial-cell-specific organelles that, upon fusion with the plasma membrane, release cargo molecules that are essential in blood vessel abnormalities, such as thrombosis and inflammation, as well as in angiogenesis. Despite the importance of WPBs, the basic mechanisms that mediate their secretion are only poorly understood. Rab GTPases play fundamental role in the trafficking of intracellular organelles. Yet, the only known WPB-associated Rabs are Rab27a and Rab3d. To determine the full spectrum of WPB-associated Rabs we performed a complete Rab screening by analysing the localisation of all Rabs in WPBs and their involvement in the secretory process in endothelial cells. Apart from Rab3 and Rab27, we identified three additional Rabs, Rab15 (a previously reported endocytic Rab), Rab33 and Rab37, on the WPB limiting membrane. A knockdown approach using siRNAs showed that among these five WPB Rabs only Rab3, Rab27 and Rab15 are required for exocytosis. Intriguingly, we found that Rab15 cooperates with Rab27a in WPB secretion. Furthermore, a specific effector of Rab27, Munc13-4, appears to be also an effector of Rab15 and is required for WPB exocytosis. These data indicate that WPB secretion requires the coordinated function of a specific group of Rabs and that, among them, Rab27a and Rab15, as well as their effector Munc13-4, cooperate to drive exocytosis.

Many aspects of membrane-trafficking events are regulated by Rab-family small GTPases. Neurite outgrowth requires massive addition of proteins and lipids to the tips of growing neurites by membrane trafficking, and although several Rabs, including Rab8, Rab10, and Rab11, have been implicated in this process, their regulatory mechanisms during neurite outgrowth are poorly understood. Here, we show that Rabin8, a Rab8-guanine nucleotide exchange factor (GEF), regulates nerve growth factor (NGF)-induced neurite outgrowth of PC12 cells. Knockdown of Rabin8 results in inhibition of neurite outgrowth, whereas overexpression promotes it. We also find that Rab10 is a novel substrate of Rabin8 and that both Rab8 and Rab10 function during neurite outgrowth downstream of Rabin8. Surprisingly, however, a GEF activity-deficient isoform of Rabin8 also promotes neurite outgrowth, indicating the existence of a GEF activity-independent role of Rabin8. The Arf6/Rab8-positive recycling endosomes (Arf6/Rab8-REs) and Rab10/Rab11-positive REs (Rab10/Rab11-REs) in NGF-stimulated PC12 cells are differently distributed. Rabin8 localizes on both RE populations and appears to activate Rab8 and Rab10 there. These localizations and functions of Rabin8 are Rab11 dependent. Thus Rabin8 regulates neurite outgrowth both by coordinating with Rab8, Rab10, and Rab11 and by a GEF activity-independent mechanism.

Chemokines promote the delivery and proper organization of an integrin at the leading edge of migrating lymphocytes.

Rab small GTPases are highly conserved master regulators of membrane traffic in all eukaryotes. The same as the activation and inactivation of other small GTPases, the activation and inactivation of Rabs are tightly controlled by specific GEFs (guanine nucleotide exchange factors) and GAPs (GTPase-activating proteins), respectively. Although almost all Rab-GAPs reported thus far have a TBC (Tre-2/Bub2/Cdc16)/Rab-GAP domain in common, recent accumulating evidence has indicated the existence of a number of structurally unrelated types of Rab-GEFs, including DENN proteins, VPS9 proteins, Sec2 proteins, TRAPP complexes, heterodimer GEFs (Mon1–Ccz1, HPS1–HPS4 (BLOC-3 complex), Ric1–Rgp1 and Rab3GAP1/2), and other GEFs (e.g., REI-1 and RPGR). In this review article we provide an up-to-date overview of the structures and functions of all putative Rab-GEFs in mammals, with a special focus on their substrate Rabs, interacting proteins, associations with genetic diseases, and intracellular localizations.

The Rab family of small GTPases comprises the largest number of proteins (∼60 in mammals) among the regulators of intracellular membrane trafficking, but the precise function of many Rabs and the functional redundancy and diversity of Rabs remain largely unknown. Here, we generated a comprehensive collection of knockout (KO) MDCK cells for the entire Rab family. We knocked out closely related paralogs simultaneously (Rab subfamily knockout) to circumvent functional compensation and found that Rab1A/B and Rab5A/B/C are critical for cell survival and/or growth. In addition, we demonstrated that Rab6-KO cells lack the basement membrane, likely because of the inability to secrete extracellular matrix components. Further analysis revealed the general requirement of Rab6 for secretion of soluble cargos. Transport of transmembrane cargos to the plasma membrane was also significantly delayed in Rab6-KO cells, but the phenotype was relatively mild. Our Rab-KO collection, which shares the same background, would be a valuable resource for analyzing a variety of membrane trafficking events.

α-Synuclein (aS) is a major constituent of Lewy bodies, which are not only a pathological marker for Parkinson disease but also a trigger for neurodegeneration. Cumulative evidence suggests that aS spreads from cell to cell and thereby propagates neurodegeneration to neighboring cells. Recently, Nedd4-1 (neural precursor cell expressed developmentally down-regulated protein 4-1), an E3 ubiquitin ligase, was shown to catalyze the Lys-63-linked polyubiquitination of intracellular aS and thereby facilitate aS degradation by the endolysosomal pathway. Because Nedd4-1 exerts its activity in close proximity to the inner leaflet of the plasma membrane, we speculate that after the internalization of aS the membrane resident aS is preferentially ubiquitinated by Nedd4-1. To clarify the role of Nedd4-1 in aS internalization and endolysosomal sequestration, we generated aS mutants, including ΔPR1(1-119 and 129-140), ΔC(1-119), and ΔPR2(1-119 and 134-140), that lack the proline-rich sequence, a putative Nedd4-1 recognition site. We show that wild type aS, but not ΔPR1, ΔPR2, or ΔC aS, is modified by Nedd4-1 in vitro, acquiring a Lys-63-linked ubiquitin chain. Compared with the mutants lacking the proline-rich sequence, wild type-aS is preferentially internalized and translocated to endosomes. The overexpression of Nedd4-1 increased aS in endosomes, whereas RNAi-mediated silencing of Nedd4-1 decreased endosomal aS. Although aS freely passes through plasma membranes within minutes, a pulse-chase experiment revealed that the overexpression of Nedd4-1 markedly decreased the re-secretion of internalized aS. Together, these findings demonstrate that Nedd4-1-linked Lys-63 ubiquitination specifies the fate of extrinsic and de novo synthesized aS by facilitating their targeting to endosomes.

Rab27A regulates transport of lysosome-related organelles (LROs) and release of secretory granules in various types of cells. Here, we identified up-regulation of Rab27A during differentiation of osteoclasts (OCLs) from bone-marrow macrophages (BMMs), by DNA microarray analysis. Rab27A deficiency in OCLs, using small interfering RNA (siRNA) knockdown in RAW-D cell line or BMMs derived from ashen mice, which display genetic defects in Rab27A expression, induced multinucleated and giant cells. Upon stimulation with macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL), essential cytokines for OCL differentiation, phosphorylation levels of extracellular signal-regulated kinase (Erk), proto-oncogene tyrosine-protein kinase (Src), and p-38 were slightly enhanced in ashen BMMs than in wild-type BMMs. The cell surface level of c-fms, an M-CSF receptor, was slightly higher in ashen BMMs than in wild-type BMMs, and down-regulation of RANK, a RANKL receptor, was delayed. In addition to receptors, OCLs derived from ashen mice exhibited aberrant actin ring formation, abnormal subcellular localization of lysosome-associated membrane protein (LAMP2) and cathepsin K (CTSK), and marked reduction in resorbing activity. Thus, these findings suggest that Rab27A regulates normal transport of cell surface receptors modulating multinucleation and LROs in OCLs.

Drug resistance, metastasis, and a mesenchymal transcriptional program are central features of aggressive breast tumors. The GTPase Arf6, often overexpressed in tumors, is critical to promote epithelial-mesenchymal transition and invasiveness. The metabolic mevalonate pathway (MVP) is associated with tumor invasiveness and known to prenylate proteins, but which prenylated proteins are critical for MVP-driven cancers is unknown. We show here that MVP requires the Arf6-dependent mesenchymal program. The MVP enzyme geranylgeranyl transferase II (GGT-II) and its substrate Rab11b are critical for Arf6 trafficking to the plasma membrane, where it is activated by receptor tyrosine kinases. Consistently, mutant p53, which is known to support tumorigenesis via MVP, promotes Arf6 activation via GGT-II and Rab11b. Inhibition of MVP and GGT-II blocked invasion and metastasis and reduced cancer cell resistance against chemotherapy agents, but only in cells overexpressing Arf6 and components of the mesenchymal program. Overexpression of Arf6 and mesenchymal proteins as well as enhanced MVP activity correlated with poor patient survival. These results provide insights into the molecular basis of MVP-driven malignancy.

Melanosomes are specialized intracellular organelles that produce and store melanin pigments in melanocytes, which are present in several mammalian tissues and organs, including the skin, hair, and eyes. Melanosomes form and mature stepwise (stages I-IV) in melanocytes and then are transported toward the plasma membrane along the cytoskeleton. They are subsequently transferred to neighboring keratinocytes by a largely unknown mechanism, and incorporated melanosomes are transported to the perinuclear region of the keratinocytes where they form melanin caps. Melanocytes also extend several dendrites that facilitate the efficient transfer of the melanosomes to the keratinocytes. Since the melanosome biogenesis, transport, and transfer steps require multiple membrane trafficking processes, Rab GTPases that are conserved key regulators of membrane traffic in all eukaryotes are crucial for skin and hair pigmentation. Dysfunctions of two Rab isoforms, Rab27A and Rab38, are known to cause a hypopigmentation phenotype in human type 2 Griscelli syndrome patients and in chocolate mice (related to Hermansky-Pudlak syndrome), respectively. In this review article, I review the literature on the functions of each Rab isoform and its upstream and downstream regulators in mammalian melanocytes and keratinocytes.

Transmembrane proteins are internalized by clathrin- and caveolin-dependent endocytosis. Both pathways converge on early endosomes and are thought to share the small GTPase Rab5 as common regulator. In contrast to this notion, we show here that the clathrin- and caveolin-mediated endocytic pathways are differentially regulated. Rab5 and Rab21 localize to distinct populations of early endosomes in cortical neurons and preferentially regulate clathrin- and caveolin-mediated pathways, respectively, suggesting heterogeneity in the early endosomes, rather than a converging point. Suppression of Rab21, but not Rab5, results in decreased plasma membrane localization and total protein levels of caveolin-1, which perturbs immature neurite pruning of cortical neurons, an in vivo-specific step of neuronal maturation. Taken together, our data indicate that clathrin- and caveolin-mediated endocytic pathways run in parallel in early endosomes, which show different molecular regulation and physiological function.

The ubiquitous C2 domain is a conserved Ca2+-triggered membrane-docking module that targets numerous signaling proteins to membrane surfaces where they regulate diverse processes critical for cell signaling. In this study, we quantitatively compared the equilibrium and kinetic parameters of C2 domains isolated from three functionally distinct signaling proteins: cytosolic phospholipase A2-α (cPLA2-α), protein kinase C-β (PKC-β), and synaptotagmin-IA (Syt-IA). The results show that equilibrium C2 domain docking to mixed phosphatidylcholine and phosphatidylserine membranes occurs at micromolar Ca2+ concentrations for the cPLA2-α C2 domain, but requires 3- and 10-fold higher Ca2+ concentrations for the PKC-β and Syt-IA C2 domains ([Ca2+]1/2 = 4.7, 16, 48 μM, respectively). The Ca2+-triggered membrane docking reaction proceeds in at least two steps: rapid Ca2+ binding followed by slow membrane association. The greater Ca2+ sensitivity of the cPLA2-α domain results from its higher intrinsic Ca2+ affinity in the first step compared to the other domains. Assembly and disassembly of the ternary complex in response to rapid Ca2+ addition and removal, respectively, require greater than 400 ms for the cPLA2-α domain, compared to 13 ms for the PKC-β domain and only 6 ms for the Syt-IA domain. Docking of the cPLA2-α domain to zwitterionic lipids is triggered by the binding of two Ca2+ ions and is stabilized via hydrophobic interactions, whereas docking of either the PKC-β or the Syt-IA domain to anionic lipids is triggered by at least three Ca2+ ions and is maintained by electrostatic interactions. Thus, despite their sequence and architectural similarity, C2 domains are functionally specialized modules exhibiting equilibrium and kinetic parameters optimized for distinct Ca2+ signaling applications. This specialization is provided by the carefully tuned structural and electrostatic parameters of their Ca2+- and membrane-binding loops, which yield distinct patterns of Ca2+ coordination and contrasting mechanisms of membrane docking.

Article1 October 1997free access The first C2 domain of synaptotagmin is required for exocytosis of insulin from pancreatic β-cells: action of synaptotagmin at low micromolar calcium Jochen Lang Corresponding Author Jochen Lang Division de Biochimie Clinique, Departement de Médecine Interne, Centre Médical Universitaire, CH-1211 Genève 4, Switzerland Search for more papers by this author Mitsunori Fukuda Mitsunori Fukuda Molecular Neurobiology Laboratory, Tsukuba Life Science Center, Institute of Physical, University of Tokyo, Japan Chemical Research (RIKEN), 3-1-1 Koyadai, Tsukuba, Ibaraki 305 and Department of Molecular Neurobiology, Institute of Medical Science, University of Tokyo, Japan Search for more papers by this author Hui Zhang Hui Zhang Division de Biochimie Clinique, Departement de Médecine Interne, Centre Médical Universitaire, CH-1211 Genève 4, Switzerland Search for more papers by this author Katsuhiko Mikoshiba Katsuhiko Mikoshiba Molecular Neurobiology Laboratory, Tsukuba Life Science Center, Institute of Physical, University of Tokyo, Japan Chemical Research (RIKEN), 3-1-1 Koyadai, Tsukuba, Ibaraki 305 and Department of Molecular Neurobiology, Institute of Medical Science, University of Tokyo, Japan Search for more papers by this author Claes B. Wollheim Claes B. Wollheim Division de Biochimie Clinique, Departement de Médecine Interne, Centre Médical Universitaire, CH-1211 Genève 4, Switzerland Search for more papers by this author Jochen Lang Corresponding Author Jochen Lang Division de Biochimie Clinique, Departement de Médecine Interne, Centre Médical Universitaire, CH-1211 Genève 4, Switzerland Search for more papers by this author Mitsunori Fukuda Mitsunori Fukuda Molecular Neurobiology Laboratory, Tsukuba Life Science Center, Institute of Physical, University of Tokyo, Japan Chemical Research (RIKEN), 3-1-1 Koyadai, Tsukuba, Ibaraki 305 and Department of Molecular Neurobiology, Institute of Medical Science, University of Tokyo, Japan Search for more papers by this author Hui Zhang Hui Zhang Division de Biochimie Clinique, Departement de Médecine Interne, Centre Médical Universitaire, CH-1211 Genève 4, Switzerland Search for more papers by this author Katsuhiko Mikoshiba Katsuhiko Mikoshiba Molecular Neurobiology Laboratory, Tsukuba Life Science Center, Institute of Physical, University of Tokyo, Japan Chemical Research (RIKEN), 3-1-1 Koyadai, Tsukuba, Ibaraki 305 and Department of Molecular Neurobiology, Institute of Medical Science, University of Tokyo, Japan Search for more papers by this author Claes B. Wollheim Claes B. Wollheim Division de Biochimie Clinique, Departement de Médecine Interne, Centre Médical Universitaire, CH-1211 Genève 4, Switzerland Search for more papers by this author Author Information Jochen Lang 1, Mitsunori Fukuda2,3, Hui Zhang1, Katsuhiko Mikoshiba2,3 and Claes B. Wollheim1 1Division de Biochimie Clinique, Departement de Médecine Interne, Centre Médical Universitaire, CH-1211 Genève 4, Switzerland 2Molecular Neurobiology Laboratory, Tsukuba Life Science Center, Institute of Physical, University of Tokyo, Japan 3Chemical Research (RIKEN), 3-1-1 Koyadai, Tsukuba, Ibaraki 305 and Department of Molecular Neurobiology, Institute of Medical Science, University of Tokyo, Japan *Corresponding author. E-mail: [email protected] The EMBO Journal (1997)16:5837-5846https://doi.org/10.1093/emboj/16.19.5837 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info The Ca2+- and phospholipid-binding protein synaptotagmin is involved in neuroexocytosis. Its precise role and Ca2+-affinity in vivo are unclear. We investigated its putative function in insulin secretion which is maximally stimulated by 10 μM cytosolic free Ca2+. The well-characterized synaptotagmin isoforms I and II are present in pancreatic β-cell lines RINm5F, INS-1 and HIT-T15 as shown by Northern and Western blots. Subcellular fractionation and confocal microscopy revealed their presence mainly on insulin-containing secretory granules whereas only minor amounts were found on synaptic vesicle-like microvesicles. Antibodies or Fab-fragments directed against the Ca2+-dependent phospholipid binding site of the first C2 domain of synaptotagmin I or II inhibited Ca2+-stimulated, but not GTPγS-induced exocytosis from streptolysin-O-permeabilized INS-1 and HIT-T15 cells. Transient expression of wild-type synaptotagmin II did not alter exocytosis in HIT-T15 cells. However, mutations in the Ca2+-dependent phospholipid binding site of the first C2 domain (Δ180–183, D231S) again inhibited only Ca2+-, but not GTPγS-evoked exocytosis. In contrast, mutations in the IP4-binding sites of the second C2 domain (Δ325–341; K327,328,332Q) did not alter exocytosis. Synaptotagmin II mutated in both C2 domains (Δ180–183/K327,328,332Q) induced greater inhibition than mutant Δ180–183, suggesting a discrete requirement for the second C2 domain. Thus, synaptotagmin isoforms regulate exocytotic events occurring at low micromolar Ca2+. Introduction The secretion of insulin proceeds by exocytosis, i.e. fusion between secretory granules and plasma membranes with subsequent release of the granule content into the extracellular space (Wollheim et al., 1996). Exposure of β-cells to secretagogues increases the cytosolic Ca2+ to micromolar levels, which in turn triggers insulin secretion (Theler et al., 1992; Ammala et al., 1993). How Ca2+ triggers exocytosis in pancreatic β-cells is still not identified. In neuroexocytosis, a pivotal role for the Ca2+- and phospholipid-binding protein synaptotagmin has been demonstrated (Sudhof, 1995). Currently, 11 isoforms of synaptotagmin are known in rodents (Geppert et al., 1991; Hilbush and Morgan, 1994; Mizuta et al., 1994; Craxton and Goedert, 1995; Li et al., 1995; Kwon et al., 1996; Babity et al., 1997). Antibody and peptide injections in PC12 cells provided early evidence for a role of synaptotagmin in exocytosis (Elferink et al., 1993). The deletion mutation of one of these isoforms, i.e. synaptotagmin I, in transgenic mice or of Drosophila synaptotagmin, led to a major impairment of fast Ca2+-induced neuroexocytosis. Nonetheless, the precise role and Ca2+affinity in vivo of synaptotagmin I are unknown (DiAntonio et al., 1993; Broadie et al., 1994; DiAntonio and Schwarz, 1994; Geppert et al., 1994). Synaptotagmin is an integral membrane protein of synaptic vesicles (Brose et al., 1992) and of neurohormone-containing secretory vesicles (Walch Solimena et al., 1993). It is composed of a short intravesicular N-terminal sequence, a single transmembrane region and a large cytosolic portion (Sudhof, 1995). The main features of the cytosolic part are two Ca2+-binding repeats, the C2 domains, whose basic motif was first identified in protein kinase C and has subsequently been found in several other proteins. They mediate Ca2+-induced attachment to membrane lipids (Brose et al., 1992). Several functions have been assigned to the C2 domains in synaptotagmin. The first C2 domain (C2A) binds to phospholipids at ∼5 μM free Ca2+ (Brose et al., 1992; Li et al., 1995; Fukuda et al., 1996) and to the membrane protein syntaxin 1 in the presence of several hundred micromolar Ca2+ (Li et al., 1995). The latter Ca2+ requirement coincides with that for exocytosis in bipolar neurones (Heidelberger et al., 1994). In addition to Ca2+-sensitive properties, the second C2 domain (C2B) binds inositol-1,3,4,5-tetrakisphosphate (IP4) independent of Ca2+ (Fukuda et al., 1994). The injection of IP4 into a preterminal of the squid giant synapse inhibited neuroexocytosis. This effect can be prevented by concomitant injection of an antibody directed against the C2B domain (Llinas et al., 1994; Fukuda et al., 1995b). Thus, an IP4-binding site in the C2B domain is seemingly required in neuroexocytosis and may mediate attachment to membrane lipids (Fukuda et al., 1995b). Endocrine exocytosis, such as that of insulin in the pancreatic β-cells, is maximally stimulated by 7–10 μM free Ca2+ with an EC50 of 1.6 μM (Vallar et al., 1987, Ullrich et al., 1990, Bokvist et al., 1995; Proks et al., 1996). β-Cells therefore provide a good model to test the action of synaptotagmin domains in situ at low micromolar Ca2+. Although the occurrence of several synaptotagmin isoforms has been described in pancreatic β-cells at the RNA level, their subcellular localization is unresolved (Mizuta et al., 1994; Wheeler et al., 1996). This is of crucial importance as insulin-secreting cells contain not only insulin-containing secretory granules, but also small synaptic vesicle-like microvesicles (SLMVs) containing GABA (Reetz et al., 1991). We have therefore investigated the subcellular distribution of two well-characterized isoforms of synaptotagmin, i.e. synaptotagmin I and II, and characterized their putative function. Our findings demonstrate that synaptotagmin I and II are mainly localized on secretory granules. Domains essential for Ca2+-mediated phospholipid binding in the C2A domain of synaptotagmin I and II are required for endocrine exocytosis of insulins operating at low micromolar Ca2+. Results We first investigated the expression and subcellular distribution of synaptotagmin I and II. RNA blot analysis for synaptotagmin I and II revealed the presence of a 4.8 kb transcript for the synaptotagmin I in rat brain RNA and RNA from the insulin-secreting cells RINm5F, HIT-T15 and INS-1 as well as primary islet cells (Figure 1, upper panel). Similarly, a 7 kb band was detected in blots probed for synaptotagmin II (Figure 1, lower panel) and, in addition, a minor transcript migrating at ∼9 kb was apparent. The abundance of mRNAs for synaptotagmin I and II was less in insulin-secreting cells as compared with rat. Figure 1.RNA blot analysis of synaptotagmin I and II mRNAs in insulin-secreting β-cell lines. 30 μg of total RNA from whole rat brain or indicated cell lines and primary islet cells were denatured and electrophoresed as described in the text. Upper panel, expression of synaptotagmin I RNA; Lower panel, expression of synaptotagmin II RNA. For autoradiography, the nylon membrane was exposed to X-ray film with an intensifying screen at −80°C for 3 days. Arrowheads indicate the position of 18S and 28S RNA. Download figure Download PowerPoint Western blot demonstrated the presence of the corresponding proteins in crude membrane preparations (Figure 2). Monoclonal antibodies directed against the C2A domain (mab 41.1) or against the intravesicular N-terminus of synaptotagmin I (mab 604) both detected a band of ∼60 kDa in the three cell lines. Furthermore, two other antibodies recognizing synaptotagmin I and II (mab 48 and anti-sytI/II) gave positive signals. In contrast, a monoclonal antibody directed against the N-terminus of synaptotagmin II (mab 8G2b) recognized a 60 kDa protein only in RINm5F and INS-1 cells, derived from a rat insulinoma, but not in the hamster HIT-T15 cells. Interspecies variations in the synaptotagmin II sequence can be excluded as mab 8G2b strongly reacted with a 60 kDa band in immunoblots from hamster brain (data not shown). We therefore prepared secretory granule membranes and detergent extracts of crude membranes from HIT-T15 cells. In those preparations synaptotagmin II is clearly detectable. This isoform is therefore also expressed in this cell line, albeit at lower levels than in the other insulin-secreting cells. Figure 2.Immunoblot analysis of synaptotagmin I and II in insulin-secreting β-cell lines. (A) Crude membranes (30 μg) from rat brain (1) or the indicated insulin-secreting cell lines RINm5F (2), HIT-T15 (3) or INS-1 (4) were separated on 12% SDS–PAGE, electrotransferred to PVDF and probed with antibodies against the first C2-domain (C2A) of synaptotagmin I (mab 41.1) or of synaptotagmin I and II (anti-sytI/II), against synaptotagmin I and II (mab 48) or against the intravesicular N-terminus of synaptotagmin I (mab 604) or synaptotagmin II (mab 8G2b). (B) Crude membranes (5 μg) from rat brain (5), 200 μg of Triton X-100 extract from crude HIT-T15 membranes (6) and 20 or 50 μg of enriched HIT-T15 insulin granules (7 and 8) were separated by 12% SDS–PAGE, electrotransferred to PVDF and probed with antibody against synaptotagmin II (mab 8G2b). Download figure Download PowerPoint Insulin-secreting cells contain two types of exocytotic vesicles: small synaptic-like mirovesicles, SLMVs (Reetz et al., 1991) and insulin-containing granules. Exocytotic vesicle proteins may be localized on both of them (Regazzi et al., 1995). Therefore, subcellular fractionation was performed on INS-1 cells using a continuous sucrose gradient to distinguish between the two vesicle types. Using this method, insulin-containing granules migrated at ∼1.4 M sucrose (Figure 3, lower panel) as indicated by the distribution of immunoreactive insulin in fractions 12–15. The distribution of the SLMVs is indicated by the vesicle protein synaptophysin, which is recovered at ∼0.8 M sucrose in fractions 5 and 6 (Figure 3, middle panel). The distribution of synaptotagmin I and II was tested with mab 41.1, directed against synaptotagmin I, and with anti-sytI/II, which recognizes synaptotagmin I and II. As indicated in Figure 3 (upper panel), immunoreactivity for synaptotagmin I and II was mainly concentrated in the fractions 12–15 containing secretory granules. In contrast, only a small amount of immunoreactivity was found in the synaptophysin-reactive fractions 5 and 6 containing SLMVs. In addition, some synaptotagmin immunoreactivity sedimented in a region previously identified as containing plasma (Lang et al., 1995). Figure 3.Subcellular distribution of synaptotagmin I and II in INS-1 cells. INS-1 cells were homogenized by nitrogen cavitation and centrifuged through a continuous sucrose gradient. Lower panel: distribution of protein (▪) and sucrose density (dashed line). Middle panel: distribution of synaptophysin (SVP38, •) and insulin (○) as measured by immunoblots with subsequent densitometry or by RIA. Upper panel: distribution of synaptotagmin I (SYT I; mab 41.1) and synaptotagmin I and II (anti-sytI/II). Download figure Download PowerPoint The subcellular distribution of synaptotagmin in INS-1 cells was further examined by histocytochemistry and confocal microscopy (Figure 4). The upper panel demonstrates that insulin and synaptophysin—markers for secretory granules and synaptic-like microvesicles, respectively—do not co-localize. As already observed in subcellular fractionation, synaptotagmin does stain synaptophysin-positive structures (Figure 4). A different situation was encountered in the co-staining for insulin (green) and synaptotagmin (red) (Figure 4B). Synaptotagmin was found in a granular pattern and outlining the cell surface. Most of the immunoreactivity for insulin co-localizes with synaptotagmin, as indicated by the yellow stain. A similar co-localization between synaptotagmin and insulin, but not with synaptophysin, was observed in HIT-T15 cells (data not shown). Immunocytochemistry on pancreatic islets extended previous observations for synaptotagmin I (Jacobsson et al., 1994) to synaptotagmin II. Immunoreactivities for both isoforms were absent from primary β-cells but were found on non-insulin endocrine islet cells (data not shown). Figure 4.Immunofluorescence of synaptotagmin in insulin-secreting INS-1 cells. INS-1 cells were fixed, permeabilized and subsequently incubated with anti-synaptophysin (1:400), anti-sytI/II (1:10 000) or anti-insulin (1:400). After processing with fluorescent second antibodies, pictures were taken by confocal microscopy at the mid-cellular level. (A) Anti-synaptophysin (green) and anti-insulin (red); (B) anti-insulin (green) and anti-synaptotagmin I/II (red); (C) anti-synaptophysin (green) and anti-synaptotagmin I/II (red). Left panels: immunofluorescence; right panels: phase contrast. Bars equal 5 μm. Download figure Download PowerPoint The above results suggested that synaptotagmin I and II are concentrated on insulin-containing secretory granules. We have therefore studied whether they could play a functional role in exocytosis, the final step of insulin secretion. To this end we initially used two antibodies which bind to defined regions of synaptotagmin. Affinity-purified anti-sytI/II binds to the first C2 domain (C2A) of synaptotagmin and inhibits Ca2+-dependent interaction of synaptotagmin II with phospholipid vesicles (Fukuda et al., 1995a; Mikoshiba et al., 1995). The monoclonal antibody mab 41.1 has been shown to require a short stretch of nine amino acids in the C2A domain of synaptotagmin I for binding (Chapman and Jahn, 1994) and these amino acids are required for Ca2+-dependent interaction with phospholipid vesicles (Chapman and Jahn, 1994; Fukuda et al., 1996). In the upper panel of Figure 5, the isoform specificity of these two antibodies is illustrated from experiments with recombinant fusion proteins. Both antibodies interacted with the first C2 domain (sytI–C2A), but not with the second C2 domain of synaptotagmin I (sytI–C2B). Anti-sytI/II reacted equally well with synaptotagmin II, but none of the two antibodies recognized synaptotagmin III, synaptotagmin IV or GST alone, the tag of the fusion protein. To test the functional activity of the antibodies, INS-1 or HIT-T15 cells were permeabilized with streptolysin-O (SL-O) and subsequently preincubated at 0.1 μM Ca2+ with the indicated concentration of affinity-purified IgG (anti-sytI/II) or Fab-fragments of mab 41.1 (Fab 41.1) as shown in Figure 5 (lower panel). After aspiration of antibody solutions, cells were exposed to basal levels of free Ca2+ (0.1 μM), or maximally stimulatory levels of Ca2+ (10 μM) as previously demonstrated for β-cells and derived cell lines (Vallar et al., 1987; Ullrich et al., 1990; Ammala et al., 1993; Bokvist et al., 1995). Alternatively, cells were stimulated with GTPγS. This stable GTP analogue induces Ca2+-independent exocytosis (Vallar et al., 1987; Jonas et al., 1994; Kiraly-Borri et al., 1996). The difference between insulin release at basal Ca2+ conditions and at stimulatory conditions (10 μM Ca2+, 100 μM GTPγS) in the absence of antibodies was normalized to 100%. In both cell lines, HIT-T15 and INS-1, the Fab-fragment and the affinity-purified IgG inhibited Ca2+-evoked insulin release by ∼60%, whereas the GTPγS-induced release remained unchanged (Figure 5, lower panel). In contrast to the inhibitory action of native Fab 41.1 or anti-sytI/II IgG, preheated preparations were inactive (Table I). Furthermore, control IgG or Fab did not alter insulin release, and a monoclonal antibody against the intravesicular N-terminus, which is located inside the secretory granule, did not affect hormone levels. A possible effect of Fab 41.1 or anti-sytI/II IgG on the cell surface could be excluded as Fab fragments or IgG were inactive in cells permeabilized by Staphylococcus aureus α-toxin (Table I). This toxin produces only small pores in the plasma membrane which do not allow the entry of molecules larger than 0.5–2 kDa. The inefficacy of the specific antibodies in α-toxin-permeabilized cells also excludes any contribution by antibody buffers to the inhibitory effect observed in SL-O-permeabilized cells. Figure 5.Effect of functional anti-synaptotagmin antibodies on insulin release from permeabilized INS-1 and HIT-T15 cells. Upper panel: reactivity of antibodies against recombinant proteins. Recombinant GST (GST), first (sytI-C2A) or second C2 domain (sytI-C2B) or both C2 domains of synaptotagmin II, III and IV (100 ng/lane) were separated by SDS-PAGE, immunoblotted and incubated with mab 41.1 (left part) or anti-sytI/II (right part). Lower panel: insulin-secreting cells (INS-1, ▪, □; HIT-T15, •, ○) were permeabilized with streptolysin-O, preincubated at 0.1 μM Ca2+ with the indicated concentrations of Fab-fragments of mab 41.1 (Fab 41.1) or affinity-purified polyclonal IgG against the C2A domain of synaptotagmin II (anti-sytI/II). Subsequently, supernatants were aspirated and replaced by antibody-free solution containing 0.1 μM, 10 μM Ca2+ or 100 μM GTPγS. After 7 min, supernatants were sampled and prepared for the determination of released insulin by radioimmunoassay. The Ca2+- or GTPγS-induced stimulation of insulin release in the absence of IgG or Fab was normalized to 100%. For fold-increase upon stimulation see Table I. n = 6–21 from at least three separate experiments for each point; *, 2P <0.05. Download figure Download PowerPoint Table 1. Effect of IgG or Fab-fragments on exocytosis in SL-O- or α-toxin-permeabilized cells Preincubation SL-O permeabilization α-toxin permeabilization 0.1 μM Ca2+ GTPγS 10 μM Ca2+ 0.1 μM Ca2+ GTPγS 10 μM Ca2+ Control 100 ± 23 256 ± 45 375 ± 28 100 ± 5 375 ± 12 621 ± 43 Anti-sytI/II 112 ± 7 248 ± 41 231 ± 17* 98 ± 12 405 ± 56 604 ± 56 Anti-sytI/II heated 93 ± 30 n.d. 398 ± 21 – – – IgG 105 ± 12 289 ± 58 345 ± 9 114 ± 17 371 ± 4 658 ± 13 Fab 41.1 97 ± 28 234 ± 66 217 ± 28* 103 ± 11 352 ± 31 599 ± 25 Fab 41.1 heated 114 ± 6 n.d. 405 ± 13 – – – Fab 89 ± 17 265 ± 39 402 ± 5 105 ± 9 368 ± 8 683 ± 38 Mab604.4 132 ± 31 276 ± 7 398 ± 31 121 ± 21 389 ± 24 595 ± 37 2+ Cells were permeabilized with streptolysin-O or with α-toxin and pretreated with IgG (anti-sytIIC2A, mab 604.4, non-specific IgG, 40 μg/ml) or Fab-fragments (Fab 41.1, non-specific Fab, 40 μg/ml) as in Figure 6. Heat inactivation of antibodies was performed for 20 min at 60°C. Cells were subsequently exposed to 0.1 μM Ca, 0.1 μM Ca with 100 μM GTPγS (GTPγS) or 10 μM Ca for 7 min and insulin release determined in the supernatants. Values are expressed as percent of controls (0.1 μM Ca in the absence of IgG or Fab) and means ± SEM are given for n = 6–9 from three separate experiments * , 2P <0.05; n.d., not determined. These results suggest a specific role for the C2A domain of synaptotagmin I in Ca2+-stimulated exocytosis from insulin-secreting cells. As the functional antibody anti-sytI/II reacted with both isoforms—that is, synaptotagmin I and II—we used a separate approach to evaluate further the role of synaptotagmin II in the secretion of insulin. In HIT-T15 cells we transiently expressed wild-type synaptotagmin II or synaptotagmin II mutated in the C2 domains. To determine the subcellular localization of transiently expressed synaptotagmin II, we analysed its distribution by confocal microscopy. Thus, we took advantage of the finding that only low endogenous levels of synaptotagmin II are present in HIT-T15 cells (see also Figure 2). Therefore, a monoclonal antibody could be used that is directed against the N-terminus (Nishiki et al., 1996) which is highly isoform-specific (Li et al., 1995). At the dilution used, the antibody did not react with endogenous synaptotagmin II (data not shown). Transiently expressed synaptotagmin II again co-localized largely with secretory granules as indicated by the co-distribution of insulin (Figure 6). In addition, synaptotagmin immunoreactivity was observed on the cell border (Figure 6) similar to endogenous synaptotagmin in INS-1 cells (see Figure 4). To determine whether this localization corresponds indeed to the plasma membrane, we used double staining with anti-synaptotagmin II and an antibody against syntaxin, a known plasma membrane protein. As syntaxin and synaptotagmin are differentially distributed, the rim-like stain observed with anti-synaptotagmin II corresponds probably not to localization at the plasma membrane, but to a subplasmalemmal region (Figure 6). All mutants used in this study exhibited a similar subcellular distribution (data not shown). To determine the levels of overexpressed protein, cells were co-transfected with a plasmid encoding the green fluorescent protein (GFP). This allows subsequent purification of the small population of the transiently transfected cells (∼8–13% of all cells) by fluorescence-activated cell sorting. As shown in the upper panel of Figure 7, again using a monoclonal antibody specific for synaptotagmin II (Nishiki et al., 1996), wild-type protein and its different mutants were expressed at comparable levels. Figure 6.Localization of transiently expressed synaptotagmin II in HIT-T15 cells. Upper panel: immunostaining of transiently transfected HIT-T15 cells for synaptotagmin II (red) and insulin (green). Lower panel: immunostaining for transiently expressed synaptotagmin II (red) and for syntaxin 1 (green). The monoclonal antibody against synaptotagmin II was used at a dilution (1:800) which did not stain endogenous synaptotagmin II. A similar distribution was found for the different synaptotagmin mutants used in this study (data not shown). Bars equal 5 μm. Download figure Download PowerPoint Figure 7.Effect of transiently expressed mutants of synaptotagmin II on insulin release from intact insulin-secreting HIT-T15 cells. Upper panel: HIT-T15 cells transiently expressing synaptotagmin II wild-type or mutants were purified by fluorescence-activated cell sorting using co-transfected green fluorescent protein. Control cells and fluorescent cells were solubilized in SDS-sample buffer and subjected to Western blot analysis (6×104 cells/lane) using an antibody specific for synaptotagmin II (mab 8G2b). Lower panel: HIT-T15 cells were co-transfected with plasmid encoding for human preproinsulin and control plasmid (pcDNA3, CON) or pCDNA3 containing the inserts for synaptotagmin II wild-type (WT), mutants in the first C2 domain (Δ180–183; D231S), in the second C2 domain (Δ325–341; KQ, where K327,328 and 332 were exchanged for Q) or in both domains (double mutant DM carrying the mutations Δ180–183 and the exchange of K327,328,332 to Q). Secretion experiments were performed 48–72 h later using Krebs–Ringer buffer with 3.4 mM KCl (BASAL) or with 50 mM KCl (50 mM KCl). Subsequently, the amount of human insulin C-peptide in the supernatants was measured reflecting the secretion from co-transfected cells only. n = 12–24 from at least three separate experiments for each point; ☆, 2P <0.05. Download figure Download PowerPoint To study the role of synaptotagmin II in secretion and exocytosis, the GFP-encoding plasmid was replaced by a construct expressing human preproinsulin, and human insulin C-peptide release was measured from hamster HIT-T15 cells (Figure 7, lower panel). This approach reliably reflects release from co-transfected cells as published previously (Lang et al., 1995). Wild-type synaptotagmin II (wt) did not alter basal insulin release from intact cells or hormone release induced by stimulation due to depolarization of membrane potential by KCl leading to Ca2+ entry through voltage-dependent Ca2+ channels (Wollheim and Sharp, 1981; Wollheim et al., 1996). Deletion mutation Δ180–183 and the point mutation D231S in the C2A domain of synaptotagmin II have been shown to inhibit Ca2+-dependent phospholipid binding (Fukuda et al., 1996). Here they significantly reduced stimulated insulin secretion by some 50% and 35%, respectively (Figure 7, lower panel). Two mutations in the C2B domain, i.e. Δ325–341 or the exchange of K326,327,332, to Q (KQ), abolishes the binding of IP4 and other inositol high-polyphosphates to synaptotagmin II (Fukuda et al., 1995a), a process which is thought to mimic binding of the C2B domain of synaptotagmin to membranes (Llinas et al., 1994; Mochida et al., 1997). In our hands these mutants did not alter the secretion of insulin. However, a double mutant (DM) containing Δ180–183 and KQ mutations of the C2A and C2B domains inhibited the release to a much larger extent than any mutation in the C2A domain alone (Figure 7). To study whether the observed effects of synaptotagmin II mutants on secretion occur at the level of exocytosis, or occur indirectly, we also measured their effect in SL-O-permeabilized cells. As shown in Figure 8, under this condition the effects of all tested constructs were comparable with those observed in intact cells. Similar to the effect of the functional antibodies, again only the Ca2+-, but not the GTPγS-stimulated exocytosis, was affected. Figure 8.Effect of transiently expressed mutants of synaptotagmin II on exocytosis of insulin from permeabilized HIT-T15 cells. Cells were transfected with the same constructs as in Figure 6. Cells were permeabilized 48–72 h later with recombinant streptolysin-O. Cells were subsequently exposed for 7 min to 0.1 μM Ca2+, 100 μM GTPγS or 10 μM Ca2+ and human insulin C-peptide measured from the supernatants. n = 10–27 from at least three separate experiments for each point; *, 2P <0.05. Download figure Download PowerPoint Discussion Exocytosis from pancreatic β-cells shares a number of features with exocytosis in neurones and neuroendocrine cells. We and others have previously shown that the release of insulin involves essential components of the general docking and fusion machinery. The process is ATP-dependent (Vallar et al., 1987; Lang et al., 1995) and requires the SNARE proteins synaptobrevin/VAMP (Regazzi et al., 1995, 1996), SNAP-25 (Sadoul et al., 1995), syntaxin (Martin et al., 1995, 1996) and α-SNAP (Kiraly-Borri et al., 1996). Insulin release is maximal at low micromolar free Ca2+ (Vallar et al., 1987; Ullrich et al., 1990; Bokvist et al., 1995; Proks et al., 1996). Therefore, insulin-secreting cells provide a suitable system to test the Ca2+-dependency of putative Ca2+-sensing proteins in vivo. Here, we demonstrate that the neuronal Ca2

Synaptotagmins are a family of membrane proteins that are characterized by a single transmembrane region and tandem C2 domains and that are likely to regulate constitutive and/or regulated vesicle traffic. We have shown that a subclass of synaptotagmins (III, V, VI, and X) forms homo- and heterodimers through an evolutionarily conserved cysteine motif at their N termini (Fukuda, M., Kanno, E., and Mikoshiba, K. (1999) J. Biol. Chem. 274, 31421–31427). In this study, we identified a novel alternatively spliced variant of synaptotagmin (Syt) VI that lacks the N-terminal 85 amino acids including the transmembrane region (thus designated as Syt VIΔTM). Because it lacks the cysteine motif responsible for self-dimerization, Syt VIΔTM could not associate with Syt VI even in the presence of Ca2+. Despite lacking the transmembrane region, Syt VIΔTM can associate with the plasma membrane through the C-terminal 29 amino acids. In adult mouse brain, two closely comigrating bands at M r ∼50,000, which closely corresponded to the molecular weight of recombinant Syt VIΔTM, were detected by anti-Syt VI antibody. These immunoreactive bands were found in both soluble and membrane fractions of mouse brain, indicating that they are membrane-associated proteins (Syt VIΔTM), but not transmembrane proteins (Syt VI). Expression of Syt VI and Syt VIΔTM in PC12 or COS-7 cells indicated that the two molecules have a distinct subcellular distribution: Syt VIΔTM is present in the cytosol or is associated with the plasma membrane or internal membrane structures, whereas Syt VI is localized to the endoplasmic reticulum and/or Golgi-like perinuclear compartment. These results suggest that Syt VI and Syt VIΔTM may play distinct roles in vesicular trafficking. Synaptotagmins are a family of membrane proteins that are characterized by a single transmembrane region and tandem C2 domains and that are likely to regulate constitutive and/or regulated vesicle traffic. We have shown that a subclass of synaptotagmins (III, V, VI, and X) forms homo- and heterodimers through an evolutionarily conserved cysteine motif at their N termini (Fukuda, M., Kanno, E., and Mikoshiba, K. (1999) J. Biol. Chem. 274, 31421–31427). In this study, we identified a novel alternatively spliced variant of synaptotagmin (Syt) VI that lacks the N-terminal 85 amino acids including the transmembrane region (thus designated as Syt VIΔTM). Because it lacks the cysteine motif responsible for self-dimerization, Syt VIΔTM could not associate with Syt VI even in the presence of Ca2+. Despite lacking the transmembrane region, Syt VIΔTM can associate with the plasma membrane through the C-terminal 29 amino acids. In adult mouse brain, two closely comigrating bands at M r ∼50,000, which closely corresponded to the molecular weight of recombinant Syt VIΔTM, were detected by anti-Syt VI antibody. These immunoreactive bands were found in both soluble and membrane fractions of mouse brain, indicating that they are membrane-associated proteins (Syt VIΔTM), but not transmembrane proteins (Syt VI). Expression of Syt VI and Syt VIΔTM in PC12 or COS-7 cells indicated that the two molecules have a distinct subcellular distribution: Syt VIΔTM is present in the cytosol or is associated with the plasma membrane or internal membrane structures, whereas Syt VI is localized to the endoplasmic reticulum and/or Golgi-like perinuclear compartment. These results suggest that Syt VI and Syt VIΔTM may play distinct roles in vesicular trafficking. synaptotagmin(s) endoplasmic reticulum polymerase chain reaction polyacrylamide gel electrophoresis phosphate-buffered saline Synaptotagmins are a family of membrane proteins that are suggested to be involved in regulated and/or constitutive vesicle traffic. All members share a short amino terminus, a single transmembrane region, and tandem C2 domains (named C2A and C2B) (reviewed in Refs. 1Südhof T.C. Rizo J. Neuron. 1996; 17: 379-388Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar, 2Fukuda M. Mikoshiba K. Bioessays. 1997; 19: 593-603Crossref PubMed Scopus (87) Google Scholar, 3Linial M. J. Neurochem. 1997; 69: 1781-1792Crossref PubMed Scopus (81) Google Scholar, 4Schiavo G. Osborne S.L Sgouros J.G. Biochem. Biophys. Res. Commun. 1998; 248: 1-8Crossref PubMed Scopus (97) Google Scholar). 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Commun. 1998; 248: 1-8Crossref PubMed Scopus (97) Google Scholar), isoforms that are involved in vesicle traffic other than secretory vesicle exocytosis have yet to be determined. It is also unknown whether synaptotagmin mRNAs are alternatively spliced because only a single isoform of synaptotagmins has been reported in rat or mouse to date. In the accompanying article (48Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar), we cloned mouse Syt I–XI cDNAs and found that Syts III, V, VI, and X form stable homo- and/or heterodimers via a conserved cysteine motif at the N terminus through disulfide bonds. In this study, we have identified a novel alternatively spliced variant of Syt VI (designated as Syt VIΔTM) that lacks the N-terminal domain including the transmembrane region (amino acids 1–85), which falls outside the classical synaptotagmin category described above (1Südhof T.C. Rizo J. Neuron. 1996; 17: 379-388Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar, 2Fukuda M. Mikoshiba K. Bioessays. 1997; 19: 593-603Crossref PubMed Scopus (87) Google Scholar, 3Linial M. J. Neurochem. 1997; 69: 1781-1792Crossref PubMed Scopus (81) Google Scholar, 4Schiavo G. Osborne S.L Sgouros J.G. Biochem. Biophys. Res. Commun. 1998; 248: 1-8Crossref PubMed Scopus (97) Google Scholar). Because it lacks the conserved cysteine motif at the N terminus, Syt VIΔTM did not interact with Syts III, V, VI, and X even in the presence of Ca2+. Expression of Syt VI and Syt VIΔTM in PC12 or COS-7 cells indicated that the two molecules show distinct subcellular distribution: Syt VI is mainly localized to the endoplasmic reticulum (ER) and/or Golgi-like perinuclear compartment, whereas Syt VIΔTM is localized to the plasma membrane, internal membrane structures, and cytosolic fraction. On the basis of these results, we discuss the functional differences between these two Syt VI proteins and a possible role for Syt VI in constitutive vesicle traffic, especially ER-to-Golgi or Golgi-to-ER vesicle transport. ExTaq and AmpliTaq DNA polymerases were obtained from Takara Biomedicals and Perkin-Elmer, respectively. Polyclonal and monoclonal antibodies (M2) against the FLAG peptide were purchased fromZymed Laboratories Inc. and Sigma, respectively. Horseradish peroxidase-conjugated anti-T7 tag antibody and anti-His6 antibody were from Novagen and Roche Molecular Biochemicals, respectively. All other chemicals were commercial products of reagent grade. Solutions were made in deionized water. Alternatively spliced variants of Syt VI were screened by reverse transcriptase-polymerase chain reaction (PCR) from mouse cerebellum cDNAs (8Fukuda M. Aruga J. Niinobe M. Aimoto S. Mikoshiba K. J. Biol. Chem. 1994; 269: 29206-29211Abstract Full Text PDF PubMed Google Scholar) using two sets of primers designed on the basis of rat sequences (14Li C. Ullrich B. Zhang J.Z. Anderson R.G.W. Brose N. Südhof T.C. Nature. 1995; 375: 594-599Crossref PubMed Scopus (541) Google Scholar): N-terminal half, 5′-GCATGAGCGGAGTTTGG-3′ (sense; amino acids 1–5) and 5′-CGAATTCAGTAGCGTACTGGATGTCCT-3′ (antisense; amino acids 351–357); and C-terminal half, 5′-CGGATCCGCCGCCAAGAGCTGTGGGAA-3′ (sense; amino acids 227–233) and 5′-GAATGAAATCACAACCG-3′ (antisense; amino acids 510–511 and 3′-noncoding regions). Reactions were carried out in the presence of Perfect Match PCR Enhancer (Stratagene) for 30 cycles, each consisting of denaturation at 94 °C for 1 min, annealing at 50 °C for 2 min, and extension at 72 °C for 2 min. For the N-terminal half of Syt VI, a second reaction was run to highlight the difference in size between three splice variants using internal sense (5′-CGGGATCCATGAGCGGAGTTTGGGGGGCCG-3′, amino acids 1–8) and antisense (5′-TGATCTTCACAGCTGCCT-3′, amino acids 129–135) primers. The cycling conditions were denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min, and extension at 72 °C for 1 min for 20 cycles. The PCR products, purified from an agarose gel by a MicroSpin column (Amersham Pharmacia Biotech), were directly inserted into the pGEM-T Easy vector (Promega). Both strands were completely sequenced by the ThermoSequenase premixed cycle sequencing kit (Amersham Pharmacia Biotech) using a Hitachi SQ-5500 DNA sequencer. pEF-T7-Syt VI and pEF-FLAG-Syt VI were prepared as described previously (27Mizushima S. Nagata S. Nucleic Acids Res. 1990; 18: 5332Crossref Scopus (1499) Google Scholar, 48Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). pEF-T7 (or FLAG)-Syt VIΔTM2, pEF-Syt VIΔN1–N4-His, pEF-Syt VIΔC1-His, pEF-Syt VIΔTM1-His, pEF-Syt VIΔTM2-His, and pEF-Syt VI-His were constructed similarly by PCR using the following sets of primers: ΔTM primer, 5′-CGGATCCATGCCCTGGAGGAAGAAAGA-3′ (sense, amino acids 86–92); ΔN1 primer, 5′-CCACCATGGCGGATAAGCTGAAG-3′ (sense; amino acids 115–120); ΔN2 primer, 5′-CCACCATGTCGGTCAAAGAGCAC-3′ (sense; amino acids 147–152); ΔN3 primer, 5′-CCACCATGCATGTCTCCAGCGTG-3′ (sense; amino acids 183–188); ΔN4 primer, 5′-CCACCATGGCCAAGTCGGAGGCCGCC-3′ (sense, amino acids 223–228); ΔC1 primer, 5′-CGAATTCCTCATTCCAGTGGTCCCTGC-3′ (antisense, amino acids 476–482), and His primer, 5′-TCAATGATGATGATGATGATG CAATTGCAACCGAGGGGTCCCCTC-3′ (antisense, amino acids 506–511), respectively. Italics, underlining, and boldface in the above sequences indicate restriction enzyme sites (BamHI or MunI site), Kozak sequence (28Kozak M. Nucleic Acids Res. 1984; 12: 857-872Crossref PubMed Scopus (2383) Google Scholar), and hexahistidine residues, respectively. All constructs were verified by DNA sequencing as described above. Glass-bottomed dishes (35-mm dish; Mattek Corp., Ashland, MA) were coated with collagen type IV (Becton Dickinson Labware). PC12 cells were cultured on these dishes in Dulbecco's modified Eagle's medium containing 10% horse serum and 10% fetal bovine serum at 37 °C and 5% CO2. Transfection was done using LipofectAMINE Plus reagent (Life Technologies, Inc.) according to the manufacturer's instructions. Transfection of various pEF-T7 (or FLAG)-Syt constructs into COS-7 cells was carried out by the DEAE-dextran method, and the expressed proteins were analyzed by immunoprecipitation following immunoblotting as described (48Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). COS-7 cells transfected with pEF-Syt constructs (5 × 105cells/10-cm dish) were harvested 3 days after transfection and homogenized in 1 ml of 0.32 m sucrose, 1 mmEDTA, 0.1 mm phenylmethylsulfonyl fluoride, 10 μm leupeptin, 10 μm pepstatin A, 1 mm β-mercaptoethanol, and 5 mm Tris-HCl, pH 7.5, in a glass-Teflon Potter homogenizer with 10 strokes at 900–1000 rpm. The homogenate was centrifuged at 1000 × g for 10 min at 4 °C. The supernatant was further centrifuged at 100,000 × g for 1 h at 4 °C to precipitate membrane fractions. Equal proportions of supernatant and membrane fractions were subjected to 10% SDS-polyacrylamide gel electrophoresis (PAGE) and analyzed by immunoblotting as described (48Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Subcellular fractionation of adult mouse olfactory bulb was similarly performed. New Zealand White rabbits were immunized with the purified Syt VI C2A domain fused to glutathione S-transferase (amino acids 151–281) (29Fukuda M. Kojima T. Mikoshiba K. J. Biol. Chem. 1996; 271: 8430-8434Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar) by subcutaneous injection with RIBI adjuvant at intervals of 28 days. Antisera were collected after the third booster injection. Crude IgG fractions were obtained by adding an equal amount of saturated ammonium sulfate following centrifugation at 9000 rpm for 15 min at 4 °C (GRX-220 high speed refrigerated centrifuge, TOMY, Saitama, Japan). The precipitates were dissolved in a minimum volume of phosphate-buffered saline (PBS) and then extensively dialyzed against PBS for 1 night. Since this crude IgG weakly recognized Syts I–III, V, IX, and X expressed in COS-7 cells (48Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar), the cross-reactive component was removed by incubation with glutathione-Sepharose (wet volume 1 ml; Amersham Pharmacia Biotech) coupled to the glutathioneS-transferase-Syt I–III, V, IX, and X C2A domain fusion proteins (>1 mg each) (30Ibata K. Fukuda M. Mikoshiba K. J. Biol. Chem. 1998; 273: 12267-12273Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Then, the anti-Syt VI antibody was affinity-purified by exposure to antigen bound to Affi-Gel 10 beads (Bio-Rad) according to the manufacturer's instructions. The protein concentration was determined by the Bio-Rad protein assay kit using bovine serum albumin as a reference. Immunoblotting was performed as described previously (48Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Three days after transfection, PC12 cells were washed twice with PBS and then fixed in 4% paraformaldehyde in 0.1 m sodium phosphate buffer for 20 min at room temperature, followed by washing with 0.1 m glycine. The fixed cells were permeabilized with 0.3% Triton X-100 in PBS for 2 min and immediately washed with blocking solution (1% bovine serum albumin and 0.1% Triton X-100 in PBS) three times for 5 min. The cells were incubated in blocking solution for 1 h at room temperature and then incubated with anti-BiP (Grp78, 1:200 dilution; Stressgen Biotech Corp.) and anti-TGN38 (1:500 dilution; Transduction Laboratories) mouse monoclonal antibodies and/or anti-Syt VI polyclonal antibody (0.38 μg/ml) for 1 h at room temperature. Primary antibodies were washed out with blocking solution three times for 5 min; and then the cells were incubated with appropriate secondary antibodies, anti-Alexa 488 rabbit or anti-Alexa 568 mouse (Molecular Probes, Inc.), for 1 h at room temperature. After washing out the secondary antibodies with blocking solution five times for 5 min, immunoreactivity was analyzed using a fluorescence microscope (TE300, Nikon) attached to a laser confocal scanner unit (CSU 10, Yokogawa Electric Corp.) and a HiSCA CCD camera (C6790, Hamamatsu Photonics). Images were pseudo-colored and superimposed using Adobe Photoshop software (Version 4.0). To date, 12 isoforms of synaptotagmin (Syts I-XI and Srg1) have been reported in rat and mouse (6Perin M.S. Fried V.A. Mignery G.A. Jahn R. Südhof T.C. Nature. 1990; 345: 260-263Crossref PubMed Scopus (650) Google Scholar, 7Geppert M. Archer III, B.T. Südhof T.C. J. Biol. Chem. 1991; 266: 13548-13552Abstract Full Text PDF PubMed Google Scholar, 8Fukuda M. Aruga J. Niinobe M. Aimoto S. Mikoshiba K. J. Biol. Chem. 1994; 269: 29206-29211Abstract Full Text PDF PubMed Google Scholar, 9Mizuta M. Inagaki N. Nemoto Y. Matsukura S. Takahashi M. Seino S. J. Biol. Chem. 1994; 269: 11675-11678Abstract Full Text PDF PubMed Google Scholar, 10Hilbush B.S. Morgan J.I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8195-8199Crossref PubMed Scopus (77) Google Scholar, 11Fukuda M. Kojima T. Aruga J. Niinobe M. Mikoshiba K. J. Biol. Chem. 1995; 270: 26523-26527Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 12Craxton M. Goedert M. FEBS Lett. 1995; 361: 196-200Crossref PubMed Scopus (68) Google Scholar, 13Hudson A.W. Birnbaum M.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5895-5899Crossref PubMed Scopus (78) Google Scholar, 14Li C. Ullrich B. Zhang J.Z. Anderson R.G.W. Brose N. Südhof T.C. Nature. 1995; 375: 594-599Crossref PubMed Scopus (541) Google Scholar, 15Babity J.M. Armstrong J.N. Plumier J.C. Currie R.W. Robertson H.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2638-2641Crossref PubMed Scopus (73) Google Scholar, 16von Poser C. Ichtchenko K. Shao X. Rizo J. Südhof T.C. J. Biol. Chem. 1997; 272: 14314-14319Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 17Thompson C.C. J. Neurosci. 1996; 16: 7832-7840Crossref PubMed Google Scholar, 48Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar), but no splice variants have yet been described. To examine whether synaptotagmin genes produce splice variants, we divided the coding region of synaptotagmins into two parts (from the N terminus to the end of the C2A domain and from the C2A domain to the C terminus) and amplified them separately by reverse transcriptase-PCR using custom-designed oligonucleotides. The PCR products were purified from an agarose gel, subcloned into the pGEM-T Easy vector, and completely sequenced. We obtained several cDNAs containing insertions or deletions as compared with the original sequences previously reported (Fig. 1) (data not shown). Among them, Syt VI has at least four splice variants, one major (∼85%) and three minor, in adult mouse cerebellum (Fig. 1). Sequence analysis of the four splice variants indicated that the longest PCR products (Fig. 1,asterisk) contained 47-base pair insertions just upstream of the transmembrane region of Syt VI (Fig.2 A, arrowhead). However, since the content of this isoform was quite low and the insertion might be an unspliced intron, we did not examine this form of Syt VI further. The second longest Syt VI cDNA corresponds to the rat Syt VI previously described (14Li C. Ullrich B. Zhang J.Z. Anderson R.G.W. Brose N. Südhof T.C. Nature. 1995; 375: 594-599Crossref PubMed Scopus (541) Google Scholar), and the two shorter forms were produced by alternative splicing around the transmembrane region, which resulted in a frameshift of the protein translation (Fig.2 A). We therefore designated the middle and shortest Syt VI splice variants as Syt VIΔTM1 and Syt VIΔTM2, respectively. Syt VIΔTM2 was generated by the splicing out of 83 base pairs from Syt VI, and Syt VIΔTM1 included another exon containing 66 base pairs at the same position. This frameshift generated new 5′-in-frame stop codons in the Syt VIΔTM1 and Syt VIΔTM2 cDNAs (Fig.2 A, asterisk). However, the sequence around the putative first methionine residue of Syt VIΔTM1 and Syt VIΔTM2 (methionine at position 86 of Syt VI) did not correspond well to the Kozak sequence (28Kozak M. Nucleic Acids Res. 1984; 12: 857-872Crossref PubMed Scopus (2383) Google Scholar).Figure 2Schematic representation of the synaptotagmin VI variants generated by alternative splicing. A, sequence analysis of the Syt VI, Syt VIΔTM1, and Syt VIΔTM2 PCR products obtained from Fig. 1. The nucleotide sequences of the region of difference between Syt VI, Syt VIΔTM1, and Syt VIΔTM2 areboxed. Because of the frameshift, Syt VIΔTM1 and Syt VIΔTM2 proteins were translated from just after the transmembrane region of Syt VI (methionine at position 86; compare top and bottom amino acid sequences). The transmembrane region (TM), predicted first methionine residues (circled), and in-frame stop codon preceding the first methionine (asterisk) are indicated. Nucleotide sequences shown by lowercase lettersindicate the 5′-untranslated region. The arrowhead indicates the position of 47-base pair insertions in the longest Syt VI splice variant (see Fig. 1, asterisk). B, schematic representations of Syt VI-His and Syt VIΔTM-His: transmembrane region (TM; open box), two C2 domains (hatched boxes), and His6 tag (black boxes).View Large Image Figure ViewerDownload Hi-res image Download (PPT) To elucidate whether Syt VIΔTM1 and Syt VIΔTM2 cDNAs are translated into proteins, we expressed C-terminal His6-tagged Syt VI, Syt VIΔTM1, or Syt VIΔTM2 (Fig.2 B) in COS-7 cells. Total cell homogenates were subjected to SDS-PAGE, followed by immunoblot analysis using anti-His6antibody. Two immunoreactive bands (apparent M r= 60,000 and 50,000) were detected in the cells transfected with pEF-Syt VI-His; one band (apparent M r = 50,000) was detected in the cells transfected with pEF-Syt VIΔTM1-His or pEF-Syt VIΔTM2-His; but no bands were seen in control cells (Fig.3). The apparentM r values of the two bands were almost identical to the calculated M r values of 58,300 (Syt VI-His) and 49,400 (Syt VIΔTM-His), respectively. This result indicates that the Syt VIΔTM1 and Syt VIΔTM2 cDNAs are indeed translated into the same protein (hereafter simply designated as Syt VIΔTM protein), and Syt VI cDNA is most likely to have alternative initiation of translation, which produces the two proteins (Syt VI and Syt VIΔTM), although we could not completely rule out the possibility that the M r 50,000 band seen in Fig.3 (lane 2) was a degradation product of Syt VI-His. We initially thought that Syt VIΔTM protein would be present in the cytosol because it lacks the transmembrane region as well as putative palmitoylation sites (cysteines at positions 68 and 84 of Syt VI) (31Veit M. Söllner T.H. Rothman J.E. FEBS Lett. 1996; 385: 119-123Crossref PubMed Scopus (205) Google Scholar,32Chapman E.R. Blasi J. An S. Brose N. Johnston P.A. Südhof T.C. Jahn R. Biochem. Biophys. Res. Commun. 1996; 225: 326-332Crossref PubMed Scopus (61) Google Scholar). However, the subcellular fractionation of COS-7 cells transiently expressing pEF-Syt VI-His or pEF-Syt VIΔTM2-His indicated that both proteins were membrane-associated, although small amounts of Syt VIΔTM protein (<5%) were present in the soluble fraction (Fig.4 A). The membrane-associated Syt VIΔTM protein was easily released from the membrane by incubation with buffer containing 1 m NaCl, whereas Syt VI protein was tightly associated with the membrane fractions even in the presence of 1 m NaCl (Fig. 4 B). Interestingly, Syt VIΔTM protein derived from pEF-Syt VI-His was also sensitive to NaCl concentration (Fig. 4 B, lower panel,arrowhead). Thus, we concluded that Syt VI is an integral membrane protein, whereas Syt VIΔTM is a peripheral membrane protein. To delineate which region of Syt VIΔTM is responsible for the membrane association, we produced five deletion mutants of Syt VIΔTM (Fig. 5 A). Deletion of the N-terminal region up to 137 amino acids (Syt VIΔN1–N4) had almost no effect on the membrane association, but deletion of only 29 amino acids at the C terminus (Syt VIΔC1) resulted in reduced membrane association (>60% of the protein present in the soluble fraction) (Fig. 5 B). About 40% of Syt VIΔC1 protein probably associates with phospholipids through the two C2 domains that are thought to bind negatively charged phospholipids (8Fukuda M. Aruga J. Niinobe M. Aimoto S. Mikoshiba K. J. Biol. Chem. 1994; 269: 29206-29211Abstract Full Text PDF PubMed Google Scholar, 14Li C. Ullrich B. Zhang J.Z. Anderson R.G.W. Brose N. Südhof T.C. Nature. 1995; 375: 594-599Crossref PubMed Scopus (541) Google Scholar, 29Fukuda M. Kojima T. Mikoshiba K. J. Biol. Chem. 1996; 271: 8430-8434Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 33Fukuda M. Kojima T. Mikoshiba K. Biochem. J. 1997; 323: 421-425Crossref PubMed Scopus (38) Google Scholar). In the accompanying article (48Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar), we showed that Syt VI can form stable homodimers through the N-terminal cysteine residue (at position 12) in cells and that substitution of this residue for alanine abolishes homodimer formation. Because Syt VIΔTM lacks this cysteine residue, it could not form an SDS-resistant homodimer (Figs. 3 and4 A) or a β-mercaptoethanol-sensitive homodimer (Fig.6). In the next set of experiments, we sought to determine whether Syt VIΔTM functions in concert with or independently of Syt VI because it has been reported that Syt I or II self-dimerizes via C2B domains in the presence of Ca2+(34Sugita S. Hata Y. Südhof T.C. J. Biol. Chem. 1996; 271: 1262-1265Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 35Chapman E.R. An S. Edwardson J.M. Jahn R. J. Biol. Chem. 1996; 271: 5844-5849Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 36Damer C.K. Creutz C.E. J. Neurochem. 1996; 67: 1661-1668Crossref PubMed Scopus (51) Google Scholar, 37Chapman E.R. Desai R.C. Davis A.F. Tornehl C.K. J. Biol. Chem. 1998; 273: 32966-32972Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 38Osborne S.L. Herreros J.

Small GTPase Rab is a large family of putative membrane trafficking proteins, and each member is thought to regulate a specific type(s) of membrane trafficking. However, little is known about the involvement of Rab protein(s) in secretory granule exocytosis in exocrine cells or the molecular mechanism underlying this process. We show that Rab27B, a closely related isoform of Rab27A that regulates lysosome-related granule exocytosis in cytotoxic T lymphocytes, is abundantly expressed on amylase-containing secretory granules in rat parotid gland acinar cells. We also identify the putative Rab27B effector protein, Slac2-c (Slp homologue lacking C2 domains-c)/MyRIP, which was originally described as a myosin Va/VIIa and actin binding protein, in rat parotid glands. The results of subcellular fractionation, immunoprecipitation and immunohistochemical studies indicate that the Rab27B-Slac2-c complex is formed on secretory granules in vivo. The introduction of either a specific Rab27 binding domain (i.e. a recombinant Slp homology domain of Slac2-b that specifically binds Rab27A/B but not other Rabs) or functionally blocking antibodies that specifically disrupt Rab27B-Slac2-c complex in vitro strongly inhibited isoproterenol-stimulated amylase release from streptolysin O-permeabilized parotid acinar cells. Our results indicate that the Rab27B-Slac2-c complex is an important constituent of secretory granule exocytosis in parotid acinar cells.

Synaptotagmin (Syt) is involved in Ca2+ -regulated secretion and has been suggested to serve as a general Ca2+ sensor on the membrane of secretory vesicles in neuronal cells. Insulin exocytosis from the pancreatic beta-cell is an example of a Ca2+ -dependent secretory process. Previous studies have yielded conflicting results as to which Syt isoform is present on the secretory granules in the native beta-cell. Here we show by western blotting and RT-PCR analysis, the presence of both Syt V and Syt IX in rat pancreatic islets and in the clonal beta-cell line INS-1E. The subcellular distribution of the two Syt isoforms was assessed by confocal microscopy and by sedimentation in a continuous sucrose density gradient in INS-1E cells. These experiments show that both proteins colocalize with insulin-containing secretory granules but are absent from synaptic-like microvesicles. Further immunofluorescence studies performed in primary pancreatic endocrine cells revealed that Syt V is present in glucagon-secreting alpha-cells, whereas Syt IX is associated with insulin granules in beta-cells. Transient overexpression of Syt V and Syt IX did not alter exocytosis in INS-1E cells. Finally, reduction of the expression of both Syt isoforms by RNA interference did not change basal secretion. Remarkably, hormone release in response to glucose was selectively and strongly reduced, indicating that Syt V and Syt IX are directly involved in the Ca2+ -dependent stimulation of exocytosis.

Rabphilin is a membrane trafficking protein on secretory vesicles that consists of an N-terminal Rab-binding domain and C-terminal tandem C2 domains. The N-terminal part of rabphilin has recently been shown to function as an effector domain for both Rab27A and Rab3A in PC12 cells (Fukuda, M., Kanno, E., and Yamamoto, A. (2004) <i>J. Biol. Chem.</i> 279, 13065–13075), but the function of the C2 domains of rabphilin during secretory vesicle exocytosis is largely unknown. In this study we investigated the interaction between rabphilin and SNAREs (soluble <i>N</i>-ethylmaleimide-sensitive factor attachment protein receptors, VAMP-2/synaptobrevin-2, syntaxin IA, and SNAP-25) and SNARE-associated proteins (Munc18-1 and Munc13-1) and found that the C2B domain of rabphilin, but not of other Rab27A-binding proteins with tandem C2 domains (<i>i.e.</i> Slp1-5), directly interacts with a plasma membrane protein, SNAP-25. The interaction between rabphilin and SNAP-25 occurs even in the absence of Ca²⁺ (EC₅₀ = 0.817 μm SNAP-25), but 0.5 mm Ca²⁺ increases the affinity for SNAP-25 2-fold (EC₅₀ = 0.405 μm SNAP-25) without changing the <i>B</i>_max value (1.06 mol of SNAP-25/mol of rabphilin). Furthermore, vesicle dynamics were imaged by total internal reflection fluorescence microscopy in a single PC12 cell expressing a lumen-targeted pH-insensitive yellow fluorescent protein (Venus), neuropeptide Y-Venus. Expression of the wild-type rabphilin in PC12 cells significantly increased the number of docked vesicles to the plasma membrane without altering the kinetics of individual secretory events, whereas expression of the mutant rabphilin lacking the C2B domain, rabphilin-ΔC2B, decreased the number of docked vesicle or fusing at the plasma membrane. These findings suggest that rabphilin is involved in the docking step of regulated exocytosis in PC12 cells, possibly through interaction between the C2B domain and SNAP-25.

The inositol phosphate metabolism network has been found to be much more complex than previously thought, as more and more inositol phosphates and their metabolizing enzymes have been discovered. Some of the inositol phosphates have been shown to have biological activities, but little is known about their signal transduction mechanisms except for that of inositol 1,4,5-trisphosphate. The recent discovery, however, of a number of binding proteins for inositol high polyphosphate [inositol 1,3,4,5-tetrakisphosphate (IP4), inositol 1,3,4,5,6-pentakisphosphate, or inositol hexakisphosphate] enables us to speculate on the physiological function of these compounds. In this article we focus on two major issues: (1) the roles of inositol high polyphosphates in vesicular trafficking, especially exocytosis, and (2) pleckstrin homology domain-containing IP4 binding proteins involved in the Ras signaling pathway.

Synaptotagmins (Syt), rabphilin-3A, and Doc2 belong to a family of carboxyl terminal type (C-type) tandem C2 proteins and are thought to be involved in vesicular trafficking. We have cloned and characterized a novel family of C-type tandem C2 proteins, designated Slp1-3 (synaptotagmin-like protein 1-3). The Slp1-3 C2 domains show high homology to granuphilin-a C2 domains, but the amino-terminal domain of Slp1-3 does not contain any known protein motifs or a transmembrane domain. A subcellular fractionation study indicated that Slp1-3 proteins are peripheral membrane proteins. Phospholipid binding experiments indicated that Slp3 is a Ca(2+)-dependent isoform, but Slp1 and Slp2 are Ca(2+)-independent isoforms, because only the Slp3 C2A domain showed Ca(2+)-dependent phospholipid binding activity. The C-terminus of Slp1-3 also bound neurexin Ialpha in vitro, in the same manner as Syt family proteins, which may be important for the membrane association of Slp1-3. In addition, Slp family proteins are differentially distributed in different mouse tissues and at different developmental stages.

Synaptotagmin (Syt) I-deficient phaeochromocytoma (PC12) cell lines show normal Ca²⁺-dependent norepinephrine (NE) release (Shoji-Kasai, Y., Yoshida, A., Sato, K., Hoshino, T., Ogura, A., Kondo, S., Fujimoto, Y., Kuwahara, R., Kato, R., and Takahashi, M. (1992) <i>Science</i> 256, 1821–1823). To identify an alternative Ca²⁺ sensor, we searched for other Syt isoforms in Syt I-deficient PC12 cells and identified Syt IX, an isoform closely related to Syt I, as an abundantly expressed dense-core vesicle protein. Here we show that Syt IX is required for the Ca²⁺-dependent release of NE from PC12 cells. Antibodies directed against the C2A domain of either Syt IX or Syt I inhibited Ca²⁺-dependent NE release in permeable PC12 cells indicating that both Syt proteins function in dense-core vesicle exocytosis. Our results support the idea that Syt family proteins that co-reside on secretory vesicles may function cooperatively and redundantly as potential Ca²⁺ sensors for exocytosis.

Recent emerging biochemical data indicate that several important neuroregulatory genes and proteins may be involved in the etiology of schizophrenia and bipolar disorder. Additionally, the same genes appear to be targets of several psychotropic medications that are used to treat these disorders. Recent DNA microarray studies show that genes involved in synaptic neurotransmission, signal transduction, and glutamate/GABA regulation may be differentially regulated in brains of subjects with schizophrenia. We hypothesized that chronic administration of olanzapine to rats would alter expression of various genes that may be involved in the etiology of schizophrenia and mood disorders. Rats were administered olanzapine (N=20, 2 mg/kg/day) or sterile saline intraperitoneally (N=20) daily for 21 days. Control and olanzapine-treated frontal cortices were analyzed using cDNA microarray technology. The results showed significant downregulation of 31 genes and upregulation of 38 genes by greater than two-fold in the drug-treated brains vs controls. Our results provide evidence for altered regulation of genes involved with signal transduction and cell communication, metabolism and energy pathways, transport, immune response, nucleic acid metabolism, and neuronal growth factors. Real-time quantitative RT-PCR analysis verified the direction and magnitude of change in six genes of interest: calbindin 3, homer 1, regulator of G-protein signaling (RGS) 2, pyruvate kinase, Reelin and insulin 2. Western blotting showed significant upregulation in protein products for Reelin 410 and Reelin 180 kDa and downregulation for NMDA3B and RGS2. Our results show for the first time that olanzapine causes changes in levels of several important genes that may be involved in the etiology and treatment of schizophrenia and other psychiatric disorders.

Synaptotagmins I and II are Ca2+- and phospholipid-binding proteins of synaptic vesicles that may function as Ca2+ receptors for neurotransmitter release via their first C2 domains. Herein, we describe the phospholipid binding properties of C2A domains of multiple synaptotagmins (II-VI). We demonstrate that all synaptotagmins can bind negatively charged phospholipids (phosphatidylserine (PS) and phosphatidylinositol (PI)) in a Ca2+-dependent manner, although it was previously reported that synaptotagmins IV and VI do not bind phospholipids. The Ca2+-dependent interaction of the C2A domain of synaptotagmin IV with PS was found to have two components with EC50 values of approximately 5 and 120 μM free Ca2+ and exhibited positive cooperativity (Hill coefficient of approximately 2 for both components). This value is lower than that of the C2A domain of synaptotagmin II (Hill coefficient of approximately 3). All other isoforms bound PS with high affinity (EC50 of 0.3-1 μM free Ca2+; Hill coefficient of 3-3.5). In addition, the C2A domain of synaptotagmin IV cannot bind liposomes consisting of PS (or PI) and phosphatidylcholine, PC (or phosphatidylethanolamine, PE) (1:1, w/w), indicating that the binding to negatively charged phospholipids is inhibited by the presence of PC or PE. In contrast, other isoforms bound all of the liposomes, which include either PS or PI, in a Ca2+-dependent manner. Mutational analysis indicated that this phospholipid composition-dependent Ca2+ binding of synaptotagmin IV results in the substitution of Asp for Ser at position 244. The cytoplasmic domain of synaptotagmin IV also shows this unique phospholipid binding. However, it binds PS with a positive cooperativity and an affinity similar to those of the C2A domains of other isoforms. Our results suggest that synaptotagmin IV is also a potential Ca2+ sensor for neurotransmitter release. Synaptotagmins I and II are Ca2+- and phospholipid-binding proteins of synaptic vesicles that may function as Ca2+ receptors for neurotransmitter release via their first C2 domains. Herein, we describe the phospholipid binding properties of C2A domains of multiple synaptotagmins (II-VI). We demonstrate that all synaptotagmins can bind negatively charged phospholipids (phosphatidylserine (PS) and phosphatidylinositol (PI)) in a Ca2+-dependent manner, although it was previously reported that synaptotagmins IV and VI do not bind phospholipids. The Ca2+-dependent interaction of the C2A domain of synaptotagmin IV with PS was found to have two components with EC50 values of approximately 5 and 120 μM free Ca2+ and exhibited positive cooperativity (Hill coefficient of approximately 2 for both components). This value is lower than that of the C2A domain of synaptotagmin II (Hill coefficient of approximately 3). All other isoforms bound PS with high affinity (EC50 of 0.3-1 μM free Ca2+; Hill coefficient of 3-3.5). In addition, the C2A domain of synaptotagmin IV cannot bind liposomes consisting of PS (or PI) and phosphatidylcholine, PC (or phosphatidylethanolamine, PE) (1:1, w/w), indicating that the binding to negatively charged phospholipids is inhibited by the presence of PC or PE. In contrast, other isoforms bound all of the liposomes, which include either PS or PI, in a Ca2+-dependent manner. Mutational analysis indicated that this phospholipid composition-dependent Ca2+ binding of synaptotagmin IV results in the substitution of Asp for Ser at position 244. The cytoplasmic domain of synaptotagmin IV also shows this unique phospholipid binding. However, it binds PS with a positive cooperativity and an affinity similar to those of the C2A domains of other isoforms. Our results suggest that synaptotagmin IV is also a potential Ca2+ sensor for neurotransmitter release.

Presynaptic injection of inositol 1,3,4,5-tetraphosphate, inositol 1,3,4,5,6-pentakisphosphate, or inositol 1,2,3,4,5,6-hexakisphosphate--which we denote here the inositol high-polyphosphate series (IHPS)--is shown to block synaptic transmission when injected into the preterminal of the squid giant synapse. This effect is not produced by injection of inositol 1,4,5-trisphosphate. The synaptic block is characterized by a time course in the order of 15-45 min, depending on the injection site in the preterminal fiber; the fastest block occurs when the injection is made at the terminal release site. Presynaptic voltage clamp during transmitter release demonstrates that IHPS block did not modify the presynaptic inward, calcium current. Analysis of synaptic noise at the postsynaptic axon shows that both the evoked and spontaneous transmitter release are blocked by the IHPS. Tetanic stimulation of the presynaptic fiber at frequencies of 100 Hz indicates that block is accompanied by gradual reduction of the postsynaptic response, demonstrating that the block interferes with vesicular fusion rather than with vesicular docking. These results, in combination with the recently demonstrated observation that the IHPS bind the C2B domain in synaptotagmin [Fukada, M., Aruga, J., Niinobe, M., Aimoto, S. &amp; Mikoshiba, K. (1994) J. Biol. Chem. 269, 29206-29211], suggest that IHPS elements are involved in vesicle fusion and exocytosis. In addition, a scheme is proposed in which synaptotagmin triggers transmitter release directly by promoting the fusion of synaptic vesicles with the presynaptic plasmalemma, in agreement with the very rapid nature of transmitter release in chemical synapses.

We previously reported that the pleckstrin homology (PH) domain of Bruton's tyrosine kinase (Btk) binds Ins(1,3,4,5)P4 and that missense mutations in this domain which cause either human X-linked agammaglobulinemia (XLA) or murine X-linked immunodeficiency (Xid) also dramatically reduce the Ins(1,3,4,5)P4 binding activity. In this paper, we describe the inositol phosphate binding specificity of the Btk PH domain and different inositol polyphosphate binding properties among the PH domains of Tec family kinases. Our results suggest that certain inositol phosphates and/or phosphoinositides are physiological ligands of some Tec family kinases and that Tec family members are differently regulated by inositol molecules.

Rabphilin and Noc2 were originally described as Rab3A effector proteins involved in the regulation of secretory vesicle exocytosis, however, recently both proteins have been shown to bind Rab27A in vitro in preference to Rab3A (Fukuda, M. (2003) J. Biol. Chem. 278, 15373-15380), suggesting that Rab3A is not their major ligand in vivo. In the present study we showed by means of deletion and mutation analyses that rabphilin and Noc2 are recruited to dense-core vesicles through specific interaction with Rab27A, not with Rab3A, in PC12 cells. Rab3A binding-defective mutants of rabphilin(E50A) and Noc2(E51A) were still localized in the distal portion of the neurites (where dense-core vesicles had accumulated) in nerve growth factor-differentiated PC12 cells, the same as the wild-type proteins, whereas Rab27A binding-defective mutants of rabphilin(E50A/I54A) and Noc2(E51A/I55A) were present throughout the cytosol. We further showed that expression of the wild-type or the E50A mutant of rabphilin-RBD, but not the E50A/I54A mutant of rabphilin-RBD, significantly inhibited high KCl-dependent neuropeptide Y secretion by PC12 cells. We also found that rabphilin and its binding partner, Rab27 have been highly conserved during evolution (from nematoda to humans) and that Caenorhabditis elegans and Drosophila rabphilin (ce/dm-rabphilin) specifically interact with ce/dm-Rab27, but not with ce/dm-Rab3 or ce/dm-Rab8, suggesting that rabphilin functions as a Rab27 effector across phylogeny. Based on these findings, we propose that the N-terminal Rab binding domain of rabphilin and Noc2 be referred to as "RBD27 (Rab binding domain for Rab27)", the same as the synaptotagmin-like protein homology domain (SHD) of Slac2-a/melanophilin.

Rab27a is required for polarized secretion of lysosomes from cytotoxic T lymphocytes (CTLs) at the immunological synapse. A series of Rab27a-interacting proteins have been identified; however, only Munc13-4 has been found to be expressed in CTL. In this study, we screened for expression of the synaptotagmin-like proteins (Slps): Slp1/JFC1, Slp2-a/exophilin4, Slp3-a, Slp4/granuphilin, Slp5 and rabphilin in CTL. We found that both Slp1 and Slp2-a are expressed in CTL. Isoforms of Slp2-a in CTL showed variation of the linker region but conserved the C2A and C2B and Slp homology (SHD) domains. Both Slp1 and Slp2-a interact with Rab27a in CTL, and Slp2-a, but not Slp1, is rapidly degraded when Rab27a is absent. Slp2-a contains PEST-like sequences within its linker region, which render it susceptible to degradation. Both Slp1 and Slp2-a localize predominantly to the plasma membrane of both human and mouse CTLs, and we show that Slp2-a can focus tightly at the immunological synapse formed with a target cell. Individual knockouts of either Slp2-a or Slp1 fail to impair CTL-mediated killing of targets; however, overexpression of a dominant-negative construct consisting of the SHD of Slp2-a, which is 56% identical to that of Slp1, reduces target cell death, suggesting that both Slp1 and Slp2-a contribute to secretory lysosome exocytosis from CTL. These results suggest that both Slp1 and Slp2-a may form part of a docking complex, capturing secretory lysosomes at the immunological synapse.

Rab27A is required for actin-based melanosome transport in mammalian skin melanocytes through its interaction with a specific effector, Slac2-a/melanophilin. Mutations that disrupt the Rab27A/Slac2-a interaction cause human Griscelli syndrome. The other Rab27 isoform, Rab27B, also binds all of the known effectors of Rab27A. In this study, we determined the crystal structure of the constitutively active form of Rab27B complexed with GTP and the effector domain of Slac2-a. The Rab27B/Slac2-a complex exhibits several intermolecular hydrogen bonds that were not observed in the previously reported Rab3A/rabphilin complex. A Rab27A mutation that disrupts one of the specific hydrogen bonds with Slac2-a resulted in the dramatic reduction of Slac2-a binding activity. Furthermore, we generated a Rab3A mutant that acquires Slac2-a binding ability by transplanting four Rab27-specific residues into Rab3A. These findings provide the structural basis for the exclusive association of Slac2-a with the Rab27 subfamily, whereas rabphilin binds several subfamilies, including Rab3 and Rab27.

The RUN domain is a less conserved protein motif that consists of approximately 70 amino acids, and because RUN domains are often found in proteins involved in the regulation of Rab small GTPases, the RUN domain has been suggested to be involved in Rab-mediated membrane trafficking, possibly as a Rab-binding site. However, since the Rab binding activity of most RUN domains has never been investigated, in this study we performed a genome-wide analysis of the Rab binding activity of the RUN domains of 19 different RUN domain-containing proteins by yeast two-hybrid assays with 60 different Rabs as bait. The results showed that only six of them interact with specific Rab isoforms with different Rab binding specificity, i.e., DENND5A/B with Rab6A/B, PLEKHM2 with Rab1A, RUFY2/3 with Rab33, and RUSC2 with Rab1/Rab35/Rab41. We also identified the minimal functional Rab35-binding site of RUSC2 (amino acid residues 982–1199) and succeeded in developing a novel GTP-Rab35-specific trapper, which we named RBD35 (Rab-binding domain specific for Rab35). Recombinant RBD35 was found to trap GTP-Rab35 specifically both in vitro and in PC12 cells, and overexpression of fluorescently tagged RBD35 in PC12 cells strongly inhibited nerve growth factor-dependent neurite outgrowth.

Because Varp (VPS9-ankyrin-repeat protein)/Ankrd27 specifically binds two small GTPases, Rab32 and Rab38, which redundantly regulate the trafficking of melanogenic enzymes in mammalian epidermal melanocytes, it has recently been implicated in the regulation of trafficking of a melanogenic enzyme tyrosinase-related protein 1 (Tyrp1) to melanosomes. However, the functional interaction between Rab32/38 and Varp and the involvement of the VPS9 domain (i.e. Rab21-GEF domain) in Tyrp1 trafficking have never been elucidated. In this study, we succeeded in identifying critical residues of Rab32/38 and Varp that are critical for the formation of the Rab32/38·Varp complex by performing Ala-based site-directed mutagenesis, and we discovered that a conserved Val residue in the switch II region of Rab32(Val-92) and Rab38(Val-78) is required for Varp binding activity and that its point mutant, Rab38(V78A), does not support Tyrp1 trafficking in Rab32/38-deficient melanocytes. We also identified two critical residues for Rab32/38 binding in the Varp ANKR1 domain and demonstrated that their point mutants, Varp(Q509A) and Varp(Y550A), do not support peripheral melanosomal distribution of Tyrp1 in Varp-deficient cells. Interestingly, the VPS9 domain point mutants, Varp(D310A) and Varp(Y350A), did support Tyrp1 trafficking in Varp-deficient cells, and knockdown of Rab21 had no effect on Tyrp1 distribution. We also found evidence for the functional interaction between a vesicle SNARE VAMP7/TI-VAMP and Varp in Tyrp1 trafficking. These results collectively indicated that both the Rab32/38 binding activity and VAMP7 binding activity of Varp are essential for trafficking of Tyrp1 in melanocytes but that activation of Rab21 by the VPS9 domain is not necessary for Tyrp1 trafficking.

Autophagy is a bulk degradation system in all eukaryotic cells and regulates a variety of biological activities in higher eukaryotes. Recently involvement of autophagy in the regulation of the secretory pathway has also been reported, but the molecular mechanism linking autophagy with the secretory pathway remains largely unknown. Here we show that Atg16L1, an essential protein for canonical autophagy, is localized on hormone-containing dense-core vesicles in neuroendocrine PC12 cells and that knockdown of Atg16L1 causes a dramatic reduction in the level of hormone secretion independently of autophagic activity. We also find that Atg16L1 interacts with the small GTPase Rab33A and that this interaction is required for the dense-core vesicle localization of Atg16L1 in PC12 cells. Our findings indicate that Atg16L1 regulates not only autophagy in all cell types, but also secretion from dense-core vesicles, presumably by acting as a Rab33A effector, in particular cell types.

Axonal outgrowth is a coordinated process of cytoskeletal dynamics and membrane trafficking; however, little is known about proteins responsible for regulating the membrane supply. LMTK1 (lemur kinase 1)/AATYK1 (apoptosis-associated tyrosine kinase 1) is a serine/threonine kinase that is highly expressed in neurons. We recently reported that LMTK1 plays a role in recycling endosomal trafficking in CHO-K1 cells. Here we explore the role of LMTK1 in axonal outgrowth and its regulation by Cdk5 using mouse brain cortical neurons. LMTK1 was expressed and was phosphorylated at Ser34, the Cdk5 phosphorylation site, at the time of axonal outgrowth in culture and colocalized with Rab11A, the small GTPase that regulates recycling endosome traffic, at the perinuclear region and in the axon. Overexpression of the unphosphorylated mutant LMTK1-S34A dramatically promoted axonal outgrowth in cultured neurons. Enhanced axonal outgrowth was diminished by the inactivation of Rab11A, placing LMTK1 upstream of Rab11A. Unexpectedly, the downregulation of LMTK1 by knockdown or gene targeting also significantly enhanced axonal elongation. Rab11A-positive vesicles were transported anterogradely more quickly in the axons of LMTK1 -deficient neurons than in those of wild-type neurons. The enhanced axonal outgrowth was reversed by LMTK1-WT or the LMTK1-S34D mutant, which mimics the phosphorylated state, but not by LMTK1-S34A. Thus, LMTK1 can negatively control axonal outgrowth by regulating Rab11A activity in a Cdk5-dependent manner, and Cdk5–LMTK1–Rab11 is a novel signaling pathway involved in axonal outgrowth.

Endocytic recycling is a process in which molecules that have been internalized are recycled back to the plasma membrane, and although it is crucial for regulating various cellular events, the molecular nexus underlying this process remains poorly understood. Here we report a molecular link between two gatekeepers for endocytic recycling, the molecular switch Rab35 and the molecular scissors EHD1, that is mediated by two distinct Rab35 effectors during neurite outgrowth of PC12 cells. Rab35 forms a tripartite complex with MICAL-L1 and centaurin-β2/ACAP2 and recruits them to perinuclear Arf6-positive endosomes in response to nerve growth factor stimulation. MICAL-L1 and centaurin-β2 then cooperatively recruit EHD1 to the same compartment by functioning as a scaffold for EHD1 and as an inactivator of Arf6, respectively. We propose that Rab35 regulates the formation of an EHD1-association site on Arf6-positive endosomes by integrating the functions of two distinct Rab35 effectors for successful neurite outgrowth.

Background: Rab small GTPases are membrane trafficking proteins in eukaryotes.Results: Comprehensive knockdown screening identified six Rab isoforms that are involved in regulating Golgi morphology in HeLa-S3 cells.Conclusion: Five of the six Rabs, including Rab2A and Rab2B, non-redundantly regulate Golgi morphology.A Rab2B-specific effector, GARI-L4, also regulates it.Significance: This is the first study to systematically analyze all human Rabs in the Golgi.Rab small GTPases are crucial regulators of the membrane traffic that maintains organelle identity and morphology.Several Rab isoforms are present in the Golgi, and it has been suggested that they regulate the compacted morphology of the Golgi in mammalian cells.However, the functional relationships among the Golgi-resident Rabs, e.g.whether they are functionally redundant or different, are poorly understood.In this study, we used specific siRNAs to perform genome-wide screening for human Rabs that are involved in Golgi morphology in HeLa-S3 cells.The results showed that knockdown of any one of the six Rab isoforms (Rab1A/1B/2A/2B/6B/8A) induced fragmentation of the Golgi in HeLa-S3 cells and that its phenotype was rescued by re-expression of their respective siRNA-resistant construct.We then performed systematic knockdown-rescue experiments in relation to each of the six Rabs.Interestingly, with the exception of the Rab8A knockdown, the Golgi fragmentation phenotype induced by knockdown of a single Rab isoform, e.g.Rab2B, was efficiently rescued by re-expression of its siRNA-resistant Rab alone, not by any of the other five Rabs, e.g.Rab2A, which is highly homologous to Rab2B, indicating that these Rab isoforms non-redundantly regulate Golgi morphology possibly through interaction with isoform-specific effector molecules.In addition, we identified Golgi-associated Rab2B interactor-like 4 (GARI-L4) as a novel Golgi-resident Rab2Bspecific binding protein whose knockdown also induced fragmentation of the Golgi.Our findings suggest that the compacted Golgi morphology of mammalian cells is finely tuned by multiple sets of Rab (or Rab-effector complexes) that for the most part function independently.

Abstract Synaptotagmins (Syts) are a group of type I membrane proteins that regulate vesicle docking and fusion in processes such as exocytosis and phagocytosis. All Syts possess a single transmembrane domain, and two conserved tandem Ca2+-binding C2 domains. However, Syts IV and XI possess a conserved serine in their C2A domain that precludes these Syts from binding Ca2+ and phospholipids, and from mediating vesicle fusion. Given the importance of vesicular trafficking in macrophages, we investigated the role of Syt XI in cytokine secretion and phagocytosis. We demonstrated that Syt XI is expressed in murine macrophages, localized in recycling endosomes, lysosomes, and recruited to phagosomes. Syt XI had a direct effect on phagocytosis and on the secretion of TNF and IL-6. Whereas small interfering RNA–mediated knockdown of Syt XI potentiated secretion of these cytokines and particle uptake, overexpression of an Syt XI construct suppressed these processes. In addition, Syt XI knockdown led to decreased recruitment of gp91phox and lysosomal-associated membrane protein–1 to phagosomes, suggesting attenuated microbicidal activity. Remarkably, knockdown of Syt XI ensued in enhanced bacterial survival. Our data reveal a novel role for Syt XI as a regulator of cytokine secretion, particle uptake, and macrophage microbicidal activity.

Synaptotagmins (Syts) are type-I membrane proteins that regulate vesicle docking and fusion in processes such as exocytosis and phagocytosis. We recently discovered that Syt XI is a recycling endosome- and lysosome-associated protein that negatively regulates the secretion of TNF and IL-6. In this study, we show that Syt XI is directly degraded by the zinc metalloprotease GP63 and excluded from Leishmania parasitophorous vacuoles by the promastigotes surface glycolipid lipophosphoglycan. Infected macrophages were found to release TNF and IL-6 in a GP63-dependent manner. To demonstrate that cytokine release was dependent on GP63-mediated degradation of Syt XI, small interfering RNA-mediated knockdown of Syt XI before infection revealed that the effects of small interfering RNA knockdown and GP63 degradation were not cumulative. In mice, i.p. injection of GP63-expressing parasites led to an increase in TNF and IL-6 secretion and to an augmented influx of neutrophils and inflammatory monocytes to the inoculation site. Both of these cell types have been shown to be infection targets and aid in the establishment of infection. In sum, our data revealed that GP63 induces proinflammatory cytokine release and increases infiltration of inflammatory phagocytes. This study provides new insight on how Leishmania exploits the immune response to establish infection.