Takeshi Noda

During the process of autophagy, autophagosomes undergo a maturation process consisting of multiple fusions with endosomes and lysosomes, which provide an acidic environment and digestive function to the interior of the autophagosome. Here we found that a fusion protein of monomeric red-fluorescence protein and LC3, the most widely used marker for autophagosomes, exhibits a quite different localization pattern from that of GFP-LC3. GFP-LC3 loses fluorescence due to lysosomal acidic and degradative conditions but mRFP-LC3 does not, indicating that the latter can label the autophagic compartments both before and after fusion with lysosomes. Taking advantage of this property, we devised a novel method for dissecting the maturation process of autophagosomes. mRFP-GFP tandem fluorescent-tagged LC3 (tfLC3) showed a GFP and mRFP signal before the fusion with lysosomes, and exhibited only the mRFP signal subsequently. Using this method, we provided evidence that overexpression of a dominant negative form of Rab7 prevented the fusion of autophagosomes with lysosomes, suggesting that Rab7 is involved in this step. This method will be of general utility for analysis of the autophagosome maturation process.

Autophagy is a bulk protein degradation process that is induced by starvation. The control mechanism for induction of autophagy is not well understood. We found that Tor, a phosphatidylinositol kinase homologue, is involved in the control of autophagy in the yeast, <i>Saccharomyces cerevisiae.</i> When rapamycin, an inhibitor of Tor function, is added, autophagy is induced even in cells growing in nutrient-rich medium. A temperature-sensitive<i>tor</i> mutant also leads to induction of autophagy at a nonpermissive temperature. These results indicate that Tor negatively regulates the induction of autophagy. Tor is the first molecule that is identified as a pivotal player in the starvation-signaling pathway of autophagy. Furthermore, we found that a high concentration of cAMP is inhibitory for induction of autophagy. <i>APG</i> gene products are involved in autophagy induced by starvation. Autophagy was not induced in <i>apg</i> mutants in the presence of rapamycin, indicating that the site of action of Tor is upstream of those of Apg proteins. In nutrient-rich medium, Apg proteins are involved also in the transport of aminopeptidase I from the cytosol to the vacuole. Tor may act to switch Apg function between autophagy and transport of aminopeptidase I.

For determination of the physiological role and mechanism of vacuolar proteolysis in the yeast Saccharomyces cerevisiae, mutant cells lacking proteinase A, B, and carboxypeptidase Y were transferred from a nutrient medium to a synthetic medium devoid of various nutrients and morphological changes of their vacuoles were investigated. After incubation for 1 h in nutrient-deficient media, a few spherical bodies appeared in the vacuoles and moved actively by Brownian movement. These bodies gradually increased in number and after 3 h they filled the vacuoles almost completely. During their accumulation, the volume of the vacuolar compartment also increased. Electron microscopic examination showed that these bodies were surrounded by a unit membrane which appeared thinner than any other intracellular membrane. The contents of the bodies were morphologically indistinguishable from the cytosol; these bodies contained cytoplasmic ribosomes, RER, mitochondria, lipid granules and glycogen granules, and the density of the cytoplasmic ribosomes in the bodies was almost the same as that of ribosomes in the cytosol. The diameter of the bodies ranged from 400 to 900 nm. Vacuoles that had accumulated these bodies were prepared by a modification of the method of Ohsumi and Anraku (Ohsumi, Y., and Y. Anraku. 1981. J. Biol. Chem. 256:2079-2082). The isolated vacuoles contained ribosomes and showed latent activity of the cytosolic enzyme glucose-6-phosphate dehydrogenase. These results suggest that these bodies sequestered the cytosol in the vacuoles. We named these spherical bodies "autophagic bodies." Accumulation of autophagic bodies in the vacuoles was induced not only by nitrogen starvation, but also by depletion of nutrients such as carbon and single amino acids that caused cessation of the cell cycle. Genetic analysis revealed that the accumulation of autophagic bodies in the vacuoles was the result of lack of the PRB1 product proteinase B, and disruption of the PRB1 gene confirmed this result. In the presence of PMSF, wild-type cells accumulated autophagic bodies in the vacuoles under nutrient-deficient conditions in the same manner as did multiple protease-deficient mutants or cells with a disrupted PRB1 gene. As the autophagic bodies disappeared rapidly after removal of PMSF from cultures of normal cells, they must be an intermediate in the normal autophagic process. This is the first report that nutrient-deficient conditions induce extensive autophagic degradation of cytosolic components in the vacuoles of yeast cells.

Vps30p/Apg6p is required for both autophagy and sorting of carboxypeptidase Y (CPY). Although Vps30p is known to interact with Apg14p, its precise role remains unclear. We found that two proteins copurify with Vps30p. They were identified by mass spectrometry to be Vps38p and Vps34p, a phosphatidylinositol (PtdIns) 3-kinase. Vps34p, Vps38p, Apg14p, and Vps15p, an activator of Vps34p, were coimmunoprecipitated with Vps30p. These results indicate that Vps30p functions as a subunit of a Vps34 PtdIns 3-kinase complex(es). Phenotypic analyses indicated that Apg14p and Vps38p are each required for autophagy and CPY sorting, respectively, whereas Vps30p, Vps34p, and Vps15p are required for both processes. Coimmunoprecipitation using anti-Apg14p and anti-Vps38p antibodies and pull-down experiments showed that two distinct Vps34 PtdIns 3-kinase complexes exist: one, containing Vps15p, Vps30p, and Apg14p, functions in autophagy and the other containing Vps15p, Vps30p, and Vps38p functions in CPY sorting. The vps34 and vps15 mutants displayed additional phenotypes such as defects in transport of proteinase A and proteinase B, implying the existence of another PtdIns 3-kinase complex(es). We propose that multiple Vps34p-Vps15p complexes associated with specific regulatory proteins might fulfill their membrane trafficking events at different sites.

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.

Autophagy and the Cvt pathway are examples of nonclassical vesicular transport from the cytoplasm to the vacuole via double-membrane vesicles. Apg8/Aut7, which plays an important role in the formation of such vesicles, tends to bind to membranes in spite of its hydrophilic nature. We show here that the nature of the association of Apg8 with membranes changes depending on a series of modifications of the protein itself. First, the carboxy-terminal Arg residue of newly synthesized Apg8 is removed by Apg4/Aut2, a novel cysteine protease, and a Gly residue becomes the carboxy-terminal residue of the protein that is now designated Apg8FG. Subsequently, Apg8FG forms a conjugate with an unidentified molecule “X” and thereby binds tightly to membranes. This modification requires the carboxy-terminal Gly residue of Apg8FG and Apg7, a ubiquitin E1-like enzyme. Finally, the adduct Apg8FG-X is reversed to soluble or loosely membrane-bound Apg8FG by cleavage by Apg4. The mode of action of Apg4, which cleaves both newly synthesized Apg8 and modified Apg8FG, resembles that of deubiquitinating enzymes. A reaction similar to ubiquitination is probably involved in the second modification. The reversible modification of Apg8 appears to be coupled to the membrane dynamics of autophagy and the Cvt pathway.

We characterized Apg8/Aut7p essential for autophagy in yeast. Apg8p was transcriptionally upregulated in response to starvation and mostly existed as a protein bound to membrane under both growing and starvation conditions. Immunofluorescence microscopy revealed that the intracellular localization of Apg8p changed drastically after shift to starvation. Apg8p resided on unidentified tiny dot structures dispersed in the cytoplasm at growing phase. During starvation, it was localized on large punctate structures, some of which were confirmed to be autophagosomes and autophagic bodies by immuno-EM. Besides these structures, we found that Apg8p was enriched on isolation membranes and in electron less-dense regions, which should contain Apg8p-localized membrane- or lipid-containing structures. These structures would represent intermediate structures during autophagosome formation. Here, we also showed that microtubule does not play an essential role in the autophagy in yeast. The result does not match with the previously proposed role of Apg8/Aut7p, delivery of autophagosome to the vacuole along microtubule. Moreover, it is revealed that autophagosome formation is severely impaired in the apg8 null mutant. Apg8p would play an important role in the autophagosome formation.

Microbial nucleic acids are critical for the induction of innate immune responses, a host defense mechanism against infection by microbes. Recent studies have indicated that double-stranded DNA (dsDNA) induces potent innate immune responses via the induction of type I IFN (IFN) and IFN-inducible genes. However, the regulatory mechanisms underlying dsDNA-triggered signaling are not fully understood. Here we show that the translocation and assembly of the essential signal transducers, stimulator of IFN genes (STING) and TANK-binding kinase 1 (TBK1), are required for dsDNA-triggered innate immune responses. After sensing dsDNA, STING moves from the endoplasmic reticulum (ER) to the Golgi apparatus and finally reaches the cytoplasmic punctate structures to assemble with TBK1. The addition of an ER-retention signal to the C terminus of STING dampens its ability to induce antiviral responses. We also show that STING co-localizes with the autophagy proteins, microtubule-associated protein 1 light chain 3 (LC3) and autophagy-related gene 9a (Atg9a), after dsDNA stimulation. The loss of Atg9a, but not that of another autophagy-related gene (Atg7), greatly enhances the assembly of STING and TBK1 by dsDNA, leading to aberrant activation of the innate immune response. Hence Atg9a functions as a regulator of innate immunity following dsDNA stimulation as well as an essential autophagy protein. These results demonstrate that dynamic membrane traffic mediates the sequential translocation and assembly of STING, both of which are essential processes required for maximal activation of the innate immune response triggered by dsDNA.

Autophagy is an intracellular process for vacuolar bulk degradation of cytoplasmic components. The molecular machinery responsible for yeast and mammalian autophagy has recently begun to be elucidated at the cellular level, but the role that autophagy plays at the organismal level has yet to be determined. In this study, a genome-wide search revealed significant conservation between yeast and plant autophagy genes. Twenty-five plant genes that are homologous to 12 yeast genes essential for autophagy were discovered. We identified an Arabidopsis mutant carrying a T-DNA insertion within AtAPG9, which is the only ortholog of yeast Apg9 in Arabidopsis (atapg9-1). AtAPG9 is transcribed in every wild-type organ tested but not in the atapg9-1 mutant. Under nitrogen or carbon-starvation conditions, chlorosis was observed earlier in atapg9-1 cotyledons and rosette leaves compared with wild-type plants. Furthermore, atapg9-1 exhibited a reduction in seed set when nitrogen starved. Even under nutrient growth conditions, bolting and natural leaf senescence were accelerated in atapg9-1 plants. Senescence-associated genes SEN1 and YSL4 were up-regulated in atapg9-1 before induction of senescence, unlike in wild type. All of these phenotypes were complemented by the expression of wild-type AtAPG9 in atapg9-1 plants. These results imply that autophagy is required for maintenance of the cellular viability under nutrient-limited conditions and for efficient nutrient use as a whole plant.

Autophagy is an intracellular process for vacuolar degradation of cytoplasmic components. Thus far, plant autophagy has been studied primarily using morphological analyses. A recent genome-wide search revealed significant conservation among autophagy genes (ATGs) in yeast and plants. It has not been proved, however, that Arabidopsis thaliana ATG genes are required for plant autophagy. To evaluate this requirement, we examined the ubiquitination-like Atg8 lipidation system, whose component genes are all found in the Arabidopsis genome. In Arabidopsis, all nine ATG8 genes and two ATG4 genes were expressed ubiquitously and were induced further by nitrogen starvation. To establish a system monitoring autophagy in whole plants, we generated transgenic Arabidopsis expressing each green fluorescent protein–ATG8 fusion (GFP-ATG8). In wild-type plants, GFP-ATG8s were observed as ring shapes in the cytoplasm and were delivered to vacuolar lumens under nitrogen-starved conditions. By contrast, in a T-DNA insertion double mutant of the ATG4s (atg4a4b-1), autophagosomes were not observed, and the GFP-ATG8s were not delivered to the vacuole under nitrogen-starved conditions. In addition, we detected autophagic bodies in the vacuoles of wild-type roots but not in those of atg4a4b-1 in the presence of concanamycin A, a V-ATPase inhibitor. Biochemical analyses also provided evidence that autophagy in higher plants requires ATG proteins. The phenotypic analysis of atg4a4b-1 indicated that plant autophagy contributes to the development of a root system under conditions of nutrient limitation.

Article6 August 2013free access Source Data Autophagy sequesters damaged lysosomes to control lysosomal biogenesis and kidney injury Ikuko Maejima Ikuko Maejima Department of Genetics, Graduate School of Medicine, Osaka University, Osaka, Japan Japan Science and Technology Agency CREST, Tokyo, Japan Search for more papers by this author Atsushi Takahashi Atsushi Takahashi Department of Geriatric Medicine and Nephrology, Graduate School of Medicine, Osaka University, Osaka, Japan Search for more papers by this author Hiroko Omori Hiroko Omori Research Institute for Microbial Diseases, Osaka University, Osaka, Japan Search for more papers by this author Tomonori Kimura Tomonori Kimura Department of Geriatric Medicine and Nephrology, Graduate School of Medicine, Osaka University, Osaka, Japan Search for more papers by this author Yoshitsugu Takabatake Yoshitsugu Takabatake Department of Geriatric Medicine and Nephrology, Graduate School of Medicine, Osaka University, Osaka, Japan Search for more papers by this author Tatsuya Saitoh Tatsuya Saitoh Laboratory of Host Defense, WPI Immunology Frontier Research Center (IFReC), Osaka University, Osaka, Japan Search for more papers by this author Akitsugu Yamamoto Akitsugu Yamamoto Faculty of Bioscience, Nagahama Institute of BioScience and Technology, Shiga, Japan Search for more papers by this author Maho Hamasaki Maho Hamasaki Department of Genetics, Graduate School of Medicine, Osaka University, Osaka, Japan Search for more papers by this author Takeshi Noda Takeshi Noda Center for Frontier Oral Science, Graduate School of Dentistry, Osaka University, Osaka, Japan Search for more papers by this author Yoshitaka Isaka Yoshitaka Isaka Department of Geriatric Medicine and Nephrology, Graduate School of Medicine, Osaka University, Osaka, Japan Search for more papers by this author Tamotsu Yoshimori Corresponding Author Tamotsu Yoshimori Department of Genetics, Graduate School of Medicine, Osaka University, Osaka, Japan Laboratory of Intracellular Membrane Dynamics, Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan Japan Science and Technology Agency CREST, Tokyo, Japan Search for more papers by this author Ikuko Maejima Ikuko Maejima Department of Genetics, Graduate School of Medicine, Osaka University, Osaka, Japan Japan Science and Technology Agency CREST, Tokyo, Japan Search for more papers by this author Atsushi Takahashi Atsushi Takahashi Department of Geriatric Medicine and Nephrology, Graduate School of Medicine, Osaka University, Osaka, Japan Search for more papers by this author Hiroko Omori Hiroko Omori Research Institute for Microbial Diseases, Osaka University, Osaka, Japan Search for more papers by this author Tomonori Kimura Tomonori Kimura Department of Geriatric Medicine and Nephrology, Graduate School of Medicine, Osaka University, Osaka, Japan Search for more papers by this author Yoshitsugu Takabatake Yoshitsugu Takabatake Department of Geriatric Medicine and Nephrology, Graduate School of Medicine, Osaka University, Osaka, Japan Search for more papers by this author Tatsuya Saitoh Tatsuya Saitoh Laboratory of Host Defense, WPI Immunology Frontier Research Center (IFReC), Osaka University, Osaka, Japan Search for more papers by this author Akitsugu Yamamoto Akitsugu Yamamoto Faculty of Bioscience, Nagahama Institute of BioScience and Technology, Shiga, Japan Search for more papers by this author Maho Hamasaki Maho Hamasaki Department of Genetics, Graduate School of Medicine, Osaka University, Osaka, Japan Search for more papers by this author Takeshi Noda Takeshi Noda Center for Frontier Oral Science, Graduate School of Dentistry, Osaka University, Osaka, Japan Search for more papers by this author Yoshitaka Isaka Yoshitaka Isaka Department of Geriatric Medicine and Nephrology, Graduate School of Medicine, Osaka University, Osaka, Japan Search for more papers by this author Tamotsu Yoshimori Corresponding Author Tamotsu Yoshimori Department of Genetics, Graduate School of Medicine, Osaka University, Osaka, Japan Laboratory of Intracellular Membrane Dynamics, Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan Japan Science and Technology Agency CREST, Tokyo, Japan Search for more papers by this author Author Information Ikuko Maejima1,8, Atsushi Takahashi2, Hiroko Omori3, Tomonori Kimura2, Yoshitsugu Takabatake2, Tatsuya Saitoh4, Akitsugu Yamamoto5, Maho Hamasaki1, Takeshi Noda6, Yoshitaka Isaka2 and Tamotsu Yoshimori 1,7,8 1Department of Genetics, Graduate School of Medicine, Osaka University, Osaka, Japan 2Department of Geriatric Medicine and Nephrology, Graduate School of Medicine, Osaka University, Osaka, Japan 3Research Institute for Microbial Diseases, Osaka University, Osaka, Japan 4Laboratory of Host Defense, WPI Immunology Frontier Research Center (IFReC), Osaka University, Osaka, Japan 5Faculty of Bioscience, Nagahama Institute of BioScience and Technology, Shiga, Japan 6Center for Frontier Oral Science, Graduate School of Dentistry, Osaka University, Osaka, Japan 7Laboratory of Intracellular Membrane Dynamics, Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan 8Japan Science and Technology Agency CREST, Tokyo, Japan *Corresponding author. Department of Genetics, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, 565-0871 Osaka, Japan. Tel:+81 6 6879 3580; Fax:+81 6 6879 3589; E-mail: [email protected] The EMBO Journal (2013)32:2336-2347https://doi.org/10.1038/emboj.2013.171 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Diverse causes, including pathogenic invasion or the uptake of mineral crystals such as silica and monosodium urate (MSU), threaten cells with lysosomal rupture, which can lead to oxidative stress, inflammation, and apoptosis or necrosis. Here, we demonstrate that lysosomes are selectively sequestered by autophagy, when damaged by MSU, silica, or the lysosomotropic reagent L-Leucyl-L-leucine methyl ester (LLOMe). Autophagic machinery is recruited only on damaged lysosomes, which are then engulfed by autophagosomes. In an autophagy-dependent manner, low pH and degradation capacity of damaged lysosomes are recovered. Under conditions of lysosomal damage, loss of autophagy causes inhibition of lysosomal biogenesis in vitro and deterioration of acute kidney injury in vivo. Thus, we propose that sequestration of damaged lysosomes by autophagy is indispensable for cellular and tissue homeostasis. Introduction The lysosome, a single-membrane acidic organelle, is present in all eukaryotic cells. Lysosomes act as a cellular ‘digestive apparatus’ that degrades materials delivered either from outside via the endocytic pathway or from inside via the autophagic pathway. These compartments provide cells with nutrients, including amino acids and lipids, by degrading proteins and other macromolecules, and they also function in plasma membrane repair, defense against pathogens, antigen presentation, and cell death (Saftig and Klumperman, 2009). Lysosomal rupture results in leakage of contents, including Cathepsins, from the lysosomal lumen into the cytosol; in the worst cases, this can cause apoptotic or necrotic cell death (Boya and Kroemer, 2008). Even when the damage is not lethal, lysosomal rupture provokes oxidative stress due to the release of H+ from the lysosomal lumen into the cytosol, DNA damage, and reduction in the catabolic capacity of the lysosome, which might in turn affect the cellular functions in which lysosomes are involved (Boya and Kroemer, 2008; Hornung et al, 2008; Johansson et al, 2010). Emerging evidence indicates that lysosomal rupture activates the NLRP3 inflammasome, which induces the secretion of proinflammatory cytokines including IL-1β, promoting inflammation and enhancing pathogenesis (Hornung et al, 2008; Salminen et al, 2012). Therefore, lysosomal rupture is a potentially harmful and stressful event for cells. Diverse substances, including mineral crystals such as silica and monosodium urate (MSU), bacterial or viral toxins, lipids, β-Amyloid, lysosomotropic compounds, and cell death effectors can impair lysosomal membranes in vivo. These substances can cause pathologies, including neurodegenerative disorders such as Parkinson's disease, inflammation, and the development of hyperuricemic nephropathy (Emmerson et al, 1990; Kroemer and Jäättelä, 2005; Boya and Kroemer, 2008; Dehay et al, 2010; Salminen et al, 2012). Autophagy is an intracellular bulk degradation system that is drastically induced under several cellular stress conditions such as nutrient starvation, and plays diverse physiological and pathological roles as a prosurvival mechanism of cells through maintaining cellular and tissue homeostasis (Mizushima and Levine, 2010). Autophagy is initiated by de novo generation of the isolation membrane in the cytoplasm. This membrane elongates to engulf cytoplasmic macromolecules and organelles, and encloses these cargos to form the autophagosome. The autophagosome itself does not have the ability to degrade its contents. Fusion with the lysosome provides an acidic environment and hydrolases, enabling degradation of autophagosomal contents. LC3, a mammalian Atg8 homologue, is a ubiquitin-like protein that is essential for autophagy. The cysteine proteinase Atg4 processes the C-terminal 22 residues of newly synthesized precursor LC3, producing a soluble form of LC3 (LC3-I) that exposes the C-terminal glycine residue (Kabeya et al, 2000). The exposed C-terminus of LC3 is conjugated to the head group amine of phosphatidylethanolamine (PE) by a ubiquitin-like system composed of Atg7 (E1 enzyme), Atg3 (E2 enzyme), and the Atg12–Atg5–Atg16L complex (E3 enzyme) (Ichimura et al, 2000; Hanada et al, 2007; Fujita et al, 2008b). Lipidated LC3 (LC3-II), a widely used autophagic marker, is anchored to the forming autophagosome membrane. Upon membrane closure, LC3-II is cleaved by Atg4, and LC3 dissocites from PE (Kirisako et al, 2000; Kim et al, 2001). Thus, Atg4 is involved in both lipidation and delipidation of LC3. Furthermore, overexpression of Atg4BC74A, an inactive mutant of Atg4B (a mammalian Atg4 homologue), strongly inhibits lipidation of LC3 by sequestering LC3 orthologues including LC3A, LC3B, GABARAP, GATE16, and Atg8L prior to lipidation (Fujita et al, 2008a). Although autophagy is considered to be a non-selective degradation system, growing evidence has revealed autophagic pathways that selectively degrade aggregation-prone proteins, invading pathogens, and damaged or superfluous organelles such as mitochondria, peroxisomes, and endoplasmic reticulum (ER) in order to maintain cellular homeostasis (Komatsu and Ichimura, 2010; Tanida, 2011). In mammalian cells, ubiquitination plays a crucial role in determining the target of selective autophagy, working in conjunction with adaptor proteins such as p62/SQSTM1, which enable interaction between ubiquitin and LC3 paralogues. Following interaction with adaptors, ubiquitinated substrates are engulfed by autophagosomes via binding between adaptors and LC3. Here, we examined the possibility that cells induce autophagy in order to avoid the associated risks that is caused by lysosomal rupture. We found that lysosomes damaged by lysosomotropic reagents are selectively isolated by autophagy. Furthermore, we suggest a possibility that this selective isolation plays critical roles in lysosomal biogenesis in cells and in suppression of acute kidney injury in vivo. Results Lysosomal rupture induces autopahgy To disrupt the lysosomal membrane, we used either the lysosomotropic compound L-Leucyl-L-leucine methyl ester (LLOMe) or crystalline silica. LLOMe accumulates in lysosomes and is converted into its membranolytic form (Leu-Leu)n-OMe (n>3) by a lysosomal thiol protease, dipeptidyl peptidase I (DPPI) (Thiele and Lipsky, 1990; Uchimoto et al, 1999). Crystalline silica, also known as silicon dioxide (SiO2), exists in nature as sand or quartz. Although silica itself is usually harmless, crystalline silica has been shown to disrupt the lysosomal membrane in alveolar macrophages and activate lung inflammation in vivo (Mossman and Churg, 1998; Hornung et al, 2008). To test whether autophagy is induced by lysosomal rupture, we measured lipidation of endogenous LC3 by immunoblotting in the murine macrophage cell line J774 cells and mouse embryonic fibroblasts (MEFs). LLOMe treatment stimulated cytosolic release of Cathepsin D in a dose-dependent manner (Supplementary Figure S1A), and the level of lipidated LC3 dose dependently increased upon either LLOMe or silica treatment (Supplementary Figure S1B and C). These results suggest that lysosomal rupture induces autophagy. Then, we found the colocalization of LC3 and Lamp1 in LLOMe- or silica-treated J774 cells (Supplementary Figure S1D) using confocal microscopy. In particular, silica-treated cells exhibited obvious recruitment of LC3 to silica particles surrounded by Lamp1. These results raise the possibility that autophagy selectively targets damaged lysosomes. Autophagic machinery is selectively recruited to damaged lysosomes To determine whether LC3 is specifically recruited only to damaged lysosomes, we used Galectin-3 (Gal3), a recently established marker of damaged endomembranes (Paz et al, 2010). Galectin-3/Mac-2 is a member of the Galectins, a lectin protein family defined by conserved sequence and affinity for β-galactosides. Gal3 is distributed throughout the cytoplasm and nucleus, whereas β-galactose-containing glycoconjugates are present only on the cell surface and in the lumens of endocytic compartments, the Golgi apparatus, and post-Golgi secretory compartments (Houzelstein et al, 2004). Therefore, these proteins normally do not interact with each other; however, endosomal membrane rupture allows Gal3 to access the lumenal glycoproteins of these compartments (Paz et al, 2010). We generated Atg7-deficient MEFs, in which Atg5–Atg12 conjugation is impaired, stably expressing GFP-Gal3 to investigate the recruitment of LC3 to membrane-damaged lysosomes. Under untreated conditions, GFP-Gal3 and LC3 were diffusely distributed throughout the cytosol and did not colocalize with Lamp1 (Figure 1A). However, upon LLOMe or silica treatment, several GFP-Gal3 puncta appeared in the cytosol. The number of these punctate structures increased in an LLOMe dose-dependent manner (Supplementary Figure S2A) and Gal3 puncta extensively colocalized with Lamp1 in LLOMe- or silica-treated Atg7+/+ and Atg7−/− MEFs; conversely, we could not observe Lamp1-negative Gal3 puncta. GFP-Gal3-positive Lamp1 puncta were not stained with Lysotracker (Supplementary Figure S2B). Video microscopy observations showed that GFP-Gal3 puncta were never stained with Lysotracker throughout its recruitment in the presence of LLOMe (Supplementary Figure S3A; Supplementary Movie1), indicating that Gal3 is recruited to lysosomes that have lost their acidic interior environment following membrane damage. Gal3 was also recruited to Lamp1 and colocalized with LC3 under oxidative stress conditions, which is also known to cause lysosomal membrane damage (Supplementary Figure S2C) (Boya and Kroemer, 2008), suggesting that Gal3 can be used as a general marker of damaged lysosomes. LC3 was specifically recruited to GFP-Gal3-positive Lamp1 puncta upon LLOMe or silica treatment in Atg7+/+ MEFs, but not in Atg7−/− MEFs (Figure 1A). Since LC3 was recruited to Gal3 puncta, even when only a few lysosomes were damaged at the low concentration of LLOMe (Supplementary Figure S2D), autophagy is highly sensitive to lysosomal damage. Figure 1.The recruitment of ubiquitin and LC3 to GFP-Gal3-positive damaged lysosomes. (A–C) Atg7+/+ and Atg7–/– MEFs stably expressing GFP-Gal3 (A, C) or GFP-Ub (B) were treated with 1000 μM LLOMe or 250 μg/ml silica for 3 h. Cells were subjected to immunocytochemistry using the following antibodies: anti-LC3 and anti-Lamp1 (A), anti-p62 and anti-Lamp1 (B), or anti-FK2 and anti-p62 (C). Bars: 10 μm. Download figure Download PowerPoint Gal3-positive damaged membranes were ubiquitinated and colocalized with p62 in both MEFs (Figure 1B and C). These results suggest that ubiquitination and the recruitment of p62 occurs in a manner similar to that observed for selective autophagy against invading bacteria. We also confirmed the colocalization of other GFP-tagged upstream Atg proteins (ULK1, Atg9L1, Atg14L, WIPI1, and Atg5) and Gal3-positive lysosomes upon either LLOMe or silica treatment (Supplementary Figure S3C). Futhermore, to clarify the cause–effect relationship between lysosomal rupture and autophagy induction, we observed puncta formation of GFP-Atg5, a marker of the isolation membrane, and mStrawberry-Gal3 using video microscopy (Supplementary Figure S3B; Supplementary Movie 2). Upon LLOMe treatment, the recruitment of GFP-Atg5 always followed the formation of mStrawberry-Gal3 puncta, suggesting that autophagy is induced after lysosomal rupture. Taken together, these results indicate that the core autophagy machinery is selectively recruited to damaged lysosomes. GFP-Gal3-positive lysosomes decrease in an autophagy-dependent manner What happens in damaged lysosomes after specific recruitment of Atg proteins? We exposed cells to 1000 μM LLOMe for 1 h, and after washing out the reagent cultured the cells for an additional 24 h in the absence of LLOMe. Then, we evaluated the percentage of GFP-Gal3-positive Lamp1 puncta at the indicated time points (Figure 2A and B; Supplementary Figure S4A and B). Three hours after LLOMe washout, almost 30% of Lamp1 puncta colocalized with GFP-Gal3 (Figure 2B). These Gal3-positive Lamp1 puncta dramatically decreased within 10 h, and completely disappeared until 24 h after LLOMe washout. In contrast to control cells, in cells stably expressing an inactive Atg4B mutant, Atg4BC74A, that sequesters LC3 paralogues prior to lipidation and strongly blocks autophagy (Fujita et al, 2008a), GFP-Gal3-positive Lamp1 puncta slightly decreased but were then maintained at a high level even 24 h after LLOMe washout (Figure 2B). These results suggest that disappearance of GFP-Gal3 puncta is due to autophagy. The total number of Lamp1 puncta remained stable in both control and Atg4B-mutant cells (Supplementary Figure S4A). Similar results were obtained in Atg7-deficient MEFs (Supplementary Figure S5A–C). Lysotracker staining showed that there remained Lysotracker-positive Lamp1 puncta representing acidic intact lysosomes at 3 h after LLOMe washout (Supplementary Figure S5D). Figure 2.Decrease in the number of GFP-Gal3 puncta is dependent on time and autophagy. (A, B) NIH3T3 cells stably expressing GFP-Gal3 and either empty vector (control) or mStrawberry-Atg4BC74A (Atg4B mutant) were treated with 1000 μM LLOMe for 1 h. After LLOMe washout, cells were fixed at the indicated time points and subjected to immunocytochemistry for Lamp1 and DAPI (blue) (A). The number of GFP-Gal3 or Lamp1 puncta per cell was quantified using G-Count (see also Supplementary Figure S4A and B). Then, the percent of GFP-Gal3-positive Lamp1 puncta was determined (B). The data represent means±s.d. At least 70 cells were counted (n=3). Bars: 20 μm.Source data for this figure is available on the online supplementary information page. Source Data for Figure 2b&s4ab [embj2013171-sup-0001-SourceData-S1.xls] Download figure Download PowerPoint We also tested continuous treatment of cells with LLOMe for longer periods of time. NIH3T3 cells were treated with 1000 μM LLOMe for the indicated time, and analysed the percentage of GFP-Gal3-positive Lamp1 puncta (Supplementary Figure S6A–C). Interestingly, even in the continuous presence of LLOMe, GFP-Gal3-positive Lamp1 puncta significantly decreased, although there remained some at 24 h. Presumably, autophagic sequestration of damaged lysosomes overcomes continuous damage of them. Acidity of damaged lysosomes recovers in an autophagy-dependent manner At least two phenomena could explain the disappearance of GFP-Gal3 puncta after LLOMe washout: release of GFP-Gal3 from the damaged membrane, and quenching of the GFP signal in an acidic environment such as that generated during autophagy. To distinguish between these two possibilities, we constructed mRFP- and GFP-tandem-tagged Gal3 (tandem fluorescent-tagged Galectin-3, tfGal3) (Figure 3A). GFP and mRFP are differentially sensitive to acidic environments (Kneen et al, 1998; Campbell et al, 2002). GFP fluorescence is rapidly quenched, and GFP is degraded by lysosomal hydrolases, whereas mRFP fluorescence remains relatively stable. Thus, as shown in Figure 3B, tfGal3 makes it possible to monitor the pH change in damaged lysosomes. The change in the surrounding environment from neutral to acidic pH causes attenuation of GFP puncta signals, while mRFP puncta stably persist. Therefore, if GFP signal is quenched after LLOMe washout, the number of mRFP+GFP+ puncta should decrease, whereas the number of mRFP+ puncta (i.e., regardless of the presence or absence of GFP signal) should not be changed. In contrast, if Gal3 is released from damaged lysosomes, then the number of both mRFP+GFP+ and mRFP+ puncta should decrease. Figure 3.tfGal3 GFP signal in puncta attenuates in an autophagy-dependent manner. (A) Diagram of the primary structure of tandem fluorescence-tagged Galectin-3 (tfGal3). (B) Schematic diagram of the fate of tfGal3 recruited to damaged lysosomes. (C–E) HeLa cells transfected with tfGal3 and either One-STrEP-FLAG-tagged Atg4BC74A (Atg4B mutant) or empty vector (control) were observed by confocal microscopy after treatment as shown in Figure 2A and B. The number of GFP±RFP+ or GFP+RFP+ puncta per cell was quantified using G-Count (D). Then, the percent of GFP+RFP+ tfGal3 puncta was calculated (E). The data represent means±s.d. At least 30 cells were counted (n=3). Bar: 10 μm. (F–H) NIH3T3 cells stably expressing empty vector (control) or mStrawberry-Atg4BC74A (Atg4B mutant) were treated with 1000 μM LLOMe for 1 h. After LLOMe washout, cells were cultured in the presence or absence of both 10 μg/ml E64d and Pepstatin A, fixed at the indicated time points, and subjected to immunocytochemistry for Gal3 (green) and DAPI (blue) (F). The number of endogenous Gal3 puncta per control (G) or Atg4B-mutant (H) cells was quantified by G-Count. The data represent means±s.d. At least 50 cells were counted (n=3). Bars: 20 μm.Source data for this figure is available on the online supplementary information page. Source Data for Figure 3de [embj2013171-sup-0002-SourceData-S2.xls] Source Data for Figure 3gh&s5g [embj2013171-sup-0003-SourceData-S3.xls] Download figure Download PowerPoint We transfected HeLa cells with a plasmid encoding tfGal3, with or without a plasmid encoding Atg4BC74A, and then subjected the cells to the experimental procedure as in Figure 2. As we expected, upon LLOMe treatment, tfGal3 formed several mRFP+GFP+ puncta in both control and Atg4B-mutant cells (Figure 3C). In control cells, the GFP+ puncta were almost completely abolished 24 h after LLOMe washout, whereas the mRFP+ puncta could be observed at the same level as at time 0 (Figure 3D and E). The attenuation of GFP puncta signals was cancelled by Bafilomycin A1, a specific inhibitor of the vacuolar-type ATPase. By contrast, tfGal3 puncta did not lose GFP signal in Atg4B-mutant cells. The total number of mRFP+ puncta did not decrease even 24 h after LLOMe depletion in either cell types (Figure 3D). From these data, we conclude that GFP-Gal3 is not released from damaged membranes, and that the acidity of damaged lysosomes recovers in an autophagy-dependent manner. We also tried to corroborate our conclusion by other approaches than those using tagged Gal3. In Supplementary Figure S5D, Lysotracker-positive intact lysosomes seemed to increase while Lysotracker-negative lysosomes were colocalized with decreased GFP-Gal3. Therefore, we next measured % of Lysotracker-negative lysosomes in total lysosomes representing damaged lysosomes leaking protons (Supplementary Figure S5E). Lysotracker-negative lysosomes increased after 1.5 h from LLOMe washout but decreased after 6 h from LLOMe washout in control cells, suggesting recovery of low pH in damaged lysosomes. On the other hand, in autophagy-deficient Atg7−/− MEFs, the increase in Lysotracker-negative lysosomes was higher after 1.5 h from LLOMe washout but their decrease after 6 h from LLOMe washout was small compared to control. The result also supports that acidity is recovered by autophagy. Endogenous Gal3 on damaged lysosomes is degraded by autophagy Endogenous Gal3 puncta that appeared upon LLOMe treatment exhibited similar decrease as exogenous-tagged Gal3 puncta in control and Atg4B-mutant cells (Figure 3F–H), and in Atg7+/+ and Atg7−/− MEFs (Supplementary Figure S5F and G). The number of Gal3 puncta was not affected in Atg4B-mutant cells by protease inhibitors, whereas Gal3 puncta accumulated in control cells in the presence of protease inhibitors (Figure 3G and H). Most likely, these results indicate that endogenous Gal3 on damaged lysosomes is degraded in an autophagy-dependent manner. We also measured the amount of endogenous Lamp1 in the presence of cycloheximide to inhibit synthesis of new proteins up to 10 h after LLOMe washout (Supplementary Figure S5H). There was no significant change observed. Presumably, Lamp1 on damaged lysosomes turns over very slowly due to its resistance to lysosomal proteases. Indeed, the half-life of Lamp1 was reported to be 1.6 days (Meikle et al, 1999). In addition, the increase in LC3 lipidation upon LLOMe treatment decreased after LLOMe washout, returning to the basal level within 10 h after LLOMe washout (Supplementary Figure S4C and D), consistent with the kinetics of change in the number of Gal3-positive lysosomes. This is not due to impaired autophagy flux, since we could observe autophagy flux by using mRFP-GFP-LC3, which is an established probe for autophagy flux (Kimura et al, 2007). In this assay, mRFP+GFP+ LC3 puncta represent forming autophagosomes or autophagosomes, and mRFP+-only puncta indicate autolysosomes. After LLOMe washout, the percentage of mRFP+GFP+ LC3 puncta decreased with increased mRFP+-only puncta in a time-dependent manner (Supplementary Figure S4E and F), indicating that autophagy flux (formation of autolysosome) is not significantly hindered in the experimental condition. Autophagosomes engulf damaged lysosomes To reveal how autophagy restores the acidic and proteolytic environment in damaged lysosomes, we observed the ultrastructure of GFP-LC3- and mStrawberry-Gal3-positive damaged lysosomes by correlation of light and electron microscopy (CLEM). HeLa cells expressing GFP-LC3 and mStrawberry-Gal3 were treated with LLOMe for 1 h, and immediately fixed (Figure 4A–F). CLEM revealed that LC3- and Gal3-positive puncta are double membranes, a typical autophagosome structure, tightly sequester swollen lysosomes (Figure 4C–F). The single-membrane vesicles were partially or completely sequestered in double-membrane structures (Figure 4G and H). We could not find such structures in cells that were not treated with LLOMe (Supplementary Figure S7A–D). In contrast to control cells, all of the obtained images in Atg4B-mutant-expressing cells treated with LLOMe showed that lysosomes were partially attached with flattened membranous sacs but were never enclosed in double-membrane structures (Figure 4I). These results suggest that damaged lysosomes are selectively engulfed by autophagosomes. Presumably, autophagosomes containing damaged lysosomes fuse with remaining intact lysosomes, resulting in the recovery of acidity and proteolysis activity. Figure 4.CLEM analysis of mSt-Gal3- and GFP-LC3-associated membranes. (A–F) HeLa cells stably expressing GFP-LC3 were transfected with mStrawberry-Gal3, and treated with 1000 μM LLOMe for 1 h. Then, cells were fixed and observed by confocal microscopy (A). The same specimens were further examined by transmission electron microscopy (B–F). Green: LC3; magenta: Gal3; blue: DAPI. (G–I) NIH3T3 cells stably expressing CFP-Gal3 and YFP-LC3, and either empty vector (control) (G, H) or mStrawberry-Atg4BC74A (I) were treated with 1000 μM LLOMe for 2 h, fixed, and observed by confocal microscopy. The electron micrographs were taken in the same sample field as the transmission electron microscope. Green: LC3; blue: Gal3 and DAPI; black arrow: single membrane; white arrow: autophagosome; white arrowhead: ER membrane. Download figure Download PowerPoint Autophagy suppresses development of acute hyperuricemic nephropathy in mice Finally, we examined the pathophysiological importance of autophagic isolation of damaged lysosomes in vivo. Acute hyperuricemic nephropathy is a type of acute kidney injury observed in patients with tumour lysis syndrome, caused by chemotherapeutic treatment of haematopoietic malignancies (Ejaz et al, 2006). In this disease, oversaturation of uric acid (UA) in urine causes precipitation of UA and MSU in the renal tubule (Nickeleit and Mihatsch, 1997; Ejaz et al, 2006). A previous study showed that urate crystals cause the cytosolic release of lysosomal enzymes in MDCK cells, and that this lysosomal damage is involved in the development of hyperuricemic nephropa

In the process of autophagy, a ubiquitin-like molecule, LC3/Atg8, is conjugated to phosphatidylethanolamine (PE) and associates with forming autophagosomes. In mammalian cells, the existence of multiple Atg8 homologues (referred to as LC3 paralogues) has hampered genetic analysis of the lipidation of LC3 paralogues. Here, we show that overexpression of an inactive mutant of Atg4B, a protease that processes pro-LC3 paralogues, inhibits autophagic degradation and lipidation of LC3 paralogues. Inhibition was caused by sequestration of free LC3 paralogues in stable complexes with the Atg4B mutant. In mutant overexpressing cells, Atg5- and ULK1-positive intermediate autophagic structures accumulated. The length of these membrane structures was comparable to that in control cells; however, a significant number were not closed. These results show that the lipidation of LC3 paralogues is involved in the completion of autophagosome formation in mammalian cells. This study also provides a powerful tool for a wide variety of studies of autophagy in the future.

Autophagy is a catabolic process that allows cells to digest their cytoplasmic constituents via autophagosome formation and lysosomal degradation. Recently, an autophagy-specific phosphatidylinositol 3-kinase (PI3-kinase) complex, consisting of hVps34, hVps15, Beclin-1, and Atg14L, has been identified in mammalian cells. Atg14L is specific to this autophagy complex and localizes to the endoplasmic reticulum (ER). Knockdown of Atg14L leads to the disappearance of the DFCP1-positive omegasome, which is a membranous structure closely associated with both the autophagosome and the ER. A point mutation in Atg14L resulting in defective ER localization was also defective in the induction of autophagy. The addition of the ER-targeting motif of DFCP1 to this mutant fully complemented the autophagic defect in Atg14L knockout embryonic stem cells. Thus, Atg14L recruits a subset of class III PI3-kinase to the ER, where otherwise phosphatidylinositol 3-phosphate (PI3P) is essentially absent. The Atg14L-dependent appearance of PI3P in the ER makes this organelle the platform for autophagosome formation.

Article15 July 1999free access Apg16p is required for the function of the Apg12p–Apg5p conjugate in the yeast autophagy pathway Noboru Mizushima Noboru Mizushima Department of Cell Biology, National Institute for Basic Biology, Okazaki, 444-8585 Japan Search for more papers by this author Takeshi Noda Takeshi Noda Department of Cell Biology, National Institute for Basic Biology, Okazaki, 444-8585 Japan Search for more papers by this author Yoshinori Ohsumi Corresponding Author Yoshinori Ohsumi Department of Cell Biology, National Institute for Basic Biology, Okazaki, 444-8585 Japan Search for more papers by this author Noboru Mizushima Noboru Mizushima Department of Cell Biology, National Institute for Basic Biology, Okazaki, 444-8585 Japan Search for more papers by this author Takeshi Noda Takeshi Noda Department of Cell Biology, National Institute for Basic Biology, Okazaki, 444-8585 Japan Search for more papers by this author Yoshinori Ohsumi Corresponding Author Yoshinori Ohsumi Department of Cell Biology, National Institute for Basic Biology, Okazaki, 444-8585 Japan Search for more papers by this author Author Information Noboru Mizushima1, Takeshi Noda1 and Yoshinori Ohsumi 1 1Department of Cell Biology, National Institute for Basic Biology, Okazaki, 444-8585 Japan *Corresponding author. E-mail: [email protected] The EMBO Journal (1999)18:3888-3896https://doi.org/10.1093/emboj/18.14.3888 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Autophagy is an intracellular bulk degradation system that is ubiquitous for eukaryotic cells. In this process, cytoplasmic components are enclosed in autophagosomes and delivered to lysosomes/vacuoles. We recently found that a protein conjugation system, in which Apg12p is covalently attached to Apg5p, is indispensable for autophagy in yeast. Here, we describe a novel coiled-coil protein, Apg16p, essential for autophagy. Apg16p interacts with Apg12p-conjugated Apg5p and less preferentially with unconjugated Apg5p. Moreover, the coiled-coil domain of Apg16p mediates self-multimerization that leads to cross-linking of Apg5p molecules and formation of a stable protein complex. Apg16p is not essential for the Apg12p–Apg5p conjugation reaction. These results suggest that the Apg12p–Apg5p conjugate requires Apg16p to accomplish its role in the autophagy pathway, and Apg16p is a key molecule as a linker to form the Apg12p–Apg5p–Apg16p multimer. Introduction Autophagy is an intracellular degradation system in which cytoplasmic components are sequestered to the lysosome/vacuole by a membrane-mediated process (Seglen and Bohley, 1992; Dunn, 1994). There are two major classes of autophagy: microautophagy and macroautophagy. Microautophagy is a process of incorporation of cytoplasmic components by invagination of the lysosomal/vacuolar membrane. In macroautophagy, cytoplasmic components are first enclosed in double membrane structures termed autophagosomes, and then delivered to the lysosome/vacuole to be degraded. Autophagy is a relatively non-selective bulk process. Cytoplasmic proteins, carbohydrates, lipids, nucleic acids and even organelles such as mitochondria and the endoplasmic reticulum are sequestered. In mammalian hepatocytes, autophagy is regulated by extracellular nutrients and hormones, and is suggested to be essential for cellular homeostasis (Mortimore and Pösö, 1987). It has also been reported to be responsible for various physiological processes such as degradation of phenobarbital-induced endoplasmic reticulum in hepatocytes (Bolender and Weibel, 1973; Masaki et al., 1987) and post-partum regression of luteal cells (Paavola, 1978) and of uterine smooth muscle cells (Henell et al., 1983). Therefore, autophagy has been thought to be crucial also for cellular remodeling, differentiation and removal of obsolete cellular components. The molecular basis of the autophagic pathway has been poorly characterized in higher eukaryotes. We have used the yeast Saccharomyces cerevisiae as a model system since we found that macroautophagy in this yeast proceeds in a manner quite similar to that in animal cells (Takeshige et al., 1992; Baba et al., 1994). Taking advantage of yeast genetics, we isolated 14 autophagy-defective (apg) mutants (Tsukada and Ohsumi, 1993). Cloning of APG genes revealed that they were novel genes (Kametaka et al., 1996; Funakoshi et al., 1997; Matsuura et al., 1997), except APG6, which is allelic to VPS30 involved in vacuolar protein sorting (Kametaka et al., 1998). The apg mutants partially overlap with aut mutants that are also based on defects in autophagy (Thumm et al., 1994; Schlumpberger et al., 1997; Straub et al., 1997; Lang et al., 1998). These apg mutants have a common phenotype: (i) loss of bulk protein degradation during starvation; (ii) loss of viability during starvation; (iii) a defect in sporulation of the homozygous diploid; and (iv) normal vacuolar function (Tsukada and Ohsumi, 1993). A vacuolar enzyme, aminopeptidase I (API), is delivered from the cytoplasm to vacuoles constitutively by a non-classical vesicular mechanism to yield a mature active enzyme (Klionsky et al., 1992; Scott et al., 1997). This ‘Cvt pathway’ is distinct from but closely related to the autophagic pathway (Baba et al., 1997), and all the apg mutants also show defects in this pathway (Scott et al., 1996). During the systematic characterization of Apg proteins, we discovered a new protein conjugation system essential for autophagy (Mizushima et al., 1998a). Apg12p, a 186 amino acid protein, is covalently attached to Apg5p, a 294 amino acid protein, through an isopeptide bond between the C-terminal glycine of Apg12p and Lys149 of Apg5p. This conjugation reaction requires ATP and two other factors, Apg7p and Apg10p. Apg7p shows significant homology to the ubiquitin-activating enzyme (E1), and indeed it was shown to be an Apg12p-activating enzyme (Kim et al., 1999; Tanida et al., 1999; Yuan et al., 1999). Apg10p is another conjugating enzyme (T.Shintani, N.Mizushima, Y.Ogawa, A.Matsuura, T.Noda and Y.Ohsumi, in preparation). These four Apg proteins (Apg5p, 7p, 10p and 12p) function together in the protein conjugation system. Although Apg12p does not have apparent homology to ubiquitin, the Apg12 conjugation system is similar to that of ubiquitination (Hochstrasser, 1996; Varshavsky, 1997; Ciechanover, 1998; Hershko and Ciechanover, 1998), Recently, these types of covalent modifications were discovered for other ubiquitin-like molecules such as SUMO-1 (Matunis et al., 1996; Kamitani et al., 1997; Mahajan et al., 1997), Smt3p (Johnson and Blobel, 1997; Johnson et al., 1997), NEDD8 (Osaka et al., 1998), Rub1p (Lammer et al., 1998; Liakopoulos et al., 1998), UCRP (Haas et al., 1987) and Fau (Olvera and Wool, 1993; Nakamura et al., 1995). Accordingly, the importance of protein conjugation systems has increasingly been recognized. Since the Apg12 conjugation system is well conserved in human (Mizushima et al., 1998b), further analysis of the system will provide important insights into the molecular basis of autophagy in eukaryotes. In this report, we describe the cloning and characterization of a novel Apg protein, Apg16p, that binds to Apg5p and is required for Apg12p–Apg5p function. Results Two-hybrid screening with Apg12p identifies YMR159c To identify proteins related to the Apg12p conjugation system, a yeast two-hybrid screening was carried out with Apg12p as bait. This screening was aimed at obtaining proteins that bind to Apg12p and/or the Apg12p–Apg5p conjugate. The two-hybrid system we used has three criteria for positive interaction: (i) growth on Ade− plates; (ii) growth on His− plates; and (iii) positive for β-galactosidase activity (James et al., 1996). Thirty four Ade+ colonies were obtained from 2×107 transformants screened. Among them, 33 colonies were also His+ and β-gal+. DNA sequencing and restriction enzyme analysis revealed that 28 clones contained an entire open reading frame (ORF), YMR159c (Figure 1). Four contained only the N-terminal third of YMR159c. To test the specificity of the interaction, the prey plasmid containing the entire YMR159c and pGBD-APG12 or pGBD vector alone were co-transformed. Only the cells carrying both the prey plasmid and pGBD-APG12 grew on Ade− Trp− Leu− plates (see Figure 5) and His− Trp− Leu− plates [with 5 mM 3-amino-triazole (AT)], and were positive for β-gal activity (data not shown). These results suggest that the product of YMR159c specifically interacts with Apg12p. Figure 1.Sequence and structure of YMR159c. (A) Amino acid sequence of YMR159c. (B) Structural analysis of YMR159c. The amino acid sequence was analyzed using the COILS program in the 28 residue window setting (obtained from http://www.isrec.isb-sib.ch/software/coils_form.html) (Lupas et al., 1991). A putative coiled-coil region (probability of 1.0) was shown. Download figure Download PowerPoint APG16 is an essential gene for autophagy YMR159c encodes a 17 kDa hydrophilic protein (150 amino acids) (Figure 1). It contains a coiled-coil motif at the C-terminal half. To determine the null phenotype of the YMR159c gene, we disrupted the ORF by insertion of the LEU2 gene at the AccI site in the KA31 diploid background (Figure 2A) The YMR159/ymr159Δ::LEU2 diploid cells were allowed to sporulate and seven tetrads were dissected. All spores were viable and the segregation pattern of Leu+ and Leu− was 2:2. We next examined their autophagic ability by detecting autophagic body accumulation (Takeshige et al., 1992). When cultured under nitrogen starvation conditions in the presence of phenylmethylsulfonyl fluoride (PMSF), autophagic bodies accumulated in the vacuoles of all the Leu− segregants as well as of wild-type cells. On the other hand, all the Leu+ segregants showed no accumulation of autophagic bodies during starvation, indicating that they are autophagy negative. When we disrupted YMR159c by replacement of an AccI–PvuII fragment with the LEU2 gene in KA31 haploid cells, similar results were obtained. We used the latter version of the disruptant (YNM124) for further analyses. The loss of autophagic activity was also confirmed by an assay system, in which a truncated form of pro-alkaline phosphatase (ALP) expressed in the cytoplasm was delivered to the vacuoles in an autophagy-dependent manner, and processed to an active enzyme (Noda et al., 1995). The ymr159Δ pho8Δ60 strain (YNM114) showed no significant elevation of phosphatase activity after 4 h nitrogen starvation (Figure 2B). Thus, YMR159c turned out to be essential for autophagy. Since it is not allelic to the other APG genes, this gene was designated as APG16. Figure 2.Phenotype of the apg16Δ strain. (A) Disruption constructs of YMR159c (APG16). Disruption of YMR159c was achieved by insertion of the LEU2 marker gene at the AccI site or by replacing an AccI–PvuII fragment with the LEU2 gene. (B) Autophagy-negative phenotype of the apg16Δ strain. The autophagic ability of wild-type (TN125), apg12Δ (YNM107) and apg16Δ cells (YNM114) was measured by the alkaline phosphatase assay before (black bars) and after (white bars) nitrogen starvation for 4 h. Error bars indicate the standard deviation of three independent experiments. (C) Loss of viability during starvation. Wild-type (KA311B, ○), apg12Δ (YNM101, ●) and apg16Δ cells (YNM124, ▴) were cultured in nitrogen starvation medium, and their viability was determined by phloxine B staining. (D) Defect in API maturation in apg16Δ cells. Transport of pro-API to the vacuole was examined by immunoblotting with anti-API antiserum. The positions of precursor and mature API are indicated. (E) The Apg12p–Apg5p conjugation in apg16Δ cells. Total lysates were prepared by the NaOH/2-mercaptoethanol extraction method from apg12Δ (YNM107) and apg12Δapg16Δ cells (YNM115), both carrying pHA-APG12 (CEN). Western blot analysis was performed using anti-HA antibody. Download figure Download PowerPoint The apg16Δ cells grew normally in a nutrient-rich medium, YEPD (data not shown), but lost their viability during starvation (Figure 2C). The homozygous diploid cells of apg16Δ were defective in sporulation (data not shown). The vacuolar enzyme API is delivered from the cytoplasm to vacuoles to yield a mature active enzyme in a manner similar to macroautophagy (Klionsky et al., 1992; Baba et al., 1997; Scott et al., 1997). As with all the other apg mutants, the apg16Δ cells showed a complete defect in this pathway (Figure 2D). These results show that the apg16Δ strain shares the common characters with other apg mutants, and that APG16 is a typical APG gene. As we have shown previously, hemagglutinin (HA)-tagged Apg12p exists as two forms. One is a HAApg12p monomer (31 kDa), and the other is covalently conjugated to Apg5p (the 70 kDa HAApg12p–Apg5p conjugate). The conjugate is completely lost in the conjugation enzyme-deficient strains apg7Δ and apg10Δ (Mizushima et al., 1998a). In the apg16Δ cells, although it was reduced, the Apg12p–Apg5p conjugate was clearly detected (Figure 2E, and see also below), indicating that Apg16p is not essential for the conjugation reaction. Nonetheless, the apg16Δ cells showed complete defects in the Apg and Cvt pathways (Figure 2B and D). These results suggest that Apg16p functions after the conjugation reaction as an Apg12p- or Apg12p–Apg5p conjugate-interacting protein, rather than in the conjugation pathway as a conjugation-promoting factor. Apg16p interacts with the Apg12p–Apg5p conjugate, but not with the Apg12p monomer For biochemical analysis of Apg16p, we tagged Apg16p with three HA epitopes at the C-terminus. The C-terminal tagged Apg16p complemented the phenotype of the apg16Δ strain (data not shown). HAApg16p was detected as a 21 kDa band (see Figure 7), a size compatible with the predicted molecular mass (17 kDa) plus the tagged HA peptide (40 amino acids). In addition, there was another faint band at 25 kDa. Both signals of 21 and 25 kDa are intensified in a strain overexpressing Apg16p (Figure 3, lane 8). It is possible that Apg16p is partially subjected to a post-translational modification, although we have not yet characterized this. The expression level and the band pattern were not significantly changed after 3 h of nitrogen starvation (data not shown). Figure 3.Apg16p interacts with the Apg12p–Apg5p conjugate but not with the Apg12p monomer. The apg12Δapg16Δ cells (YNM115) were transformed with high copy (2μ) plasmids encoding MycApg12p and/or HAApg16p as indicated. Total lysates were immunoprecipitated with anti-Myc (lanes 1–4) or anti-HA antibody (lanes 5–8) and detected by immunoblotting using anti-Myc or anti-HA antibody. The positions of the IgG heavy chain and κ light chain are indicated. Download figure Download PowerPoint To prove the interaction between Apg12p and Apg16p in vivo, co-immunoprecipitation analysis was performed. Total lysates of cells transformed with high copy (2μ) plasmids expressing MycApg12p and/or HAApg16p from their own promotors were immunoprecipitated with anti-Myc or anti-HA antibody. In the precipitates with anti-Myc, HAApg16p was co-precipitated specifically only when cells expressed both MycApg12p and HAApg16p (Figure 3, lane 4). The 25 kDa Apg16p was also co-precipitated, but to a lesser extent. These results suggest that Apg12p interacts with Apg16p in vivo. As mentioned above, the Apg12p–Apg5p conjugate was generated in the absence of Apg16p, but its amount was reduced. This reduction is observed more readily when Apg12p and Apg16p are overexpressed by 2μ plasmids (Figure 3, lane 3). In the immunoprecipitation analysis, only the Apg12p–Apg5p conjugate and not free Apg12p was co-precipitated by the anti-HA antibody (compare Figure 3, lane 4 with lane 8). These data suggest that Apg16p interacts with the Apg12p–Apg5p conjugate but not with the Apg12p monomer, and that Apg16p may stabilize the conjugate. The interaction between Apg16p and Apg12p–Apg5p was not affected by nitrogen starvation (data not shown). Apg16p directly interacts with Apg5p The immunoprecipitation analysis in Figure 3 indicates that Apg16p either interacts with Apg12p only when it is conjugated to Apg5p or interacts directly with Apg5p. Using the two-hybrid system, we examined the relationship between these Apg proteins. The two-hybrid interaction between Apg12p and Apg16p that is seen in the wild-type strain was not observed in an apg5Δ strain (Table I). The C-terminal glycine of Apg12p is essential for the Apg12p–Apg5p conjugation, since Apg12ΔGp in which the C-terminal glycine is deleted cannot be conjugated to Apg5p (Mizushima et al., 1998a). In the two-hybrid system, Apg16p did not interact with Apg12ΔGp. These results suggest that the interaction between Apg12p and Apg16p is indirect and depends on the conjugation of Apg12p to Apg5p. In agreement with these results, when we tested Apg16p and Apg5p, we detected a strong two-hybrid interaction (Table I). Furthermore, growth of the strains bearing the Apg16p and Apg5p two-hybrid plasmids was not affected by the absence of Apg12p. This indicates that Apg16p interacts directly with Apg5p. Strong interaction was detected between pGAD-APG16N and pGBD-APG12 or pGBD-APG5 (Figure 5A), suggesting that Apg5p associates with the N-terminal region of Apg16p and that their interaction does not require the coiled-coil domain. Table 1. Two-hybrid interaction in apg5Δ and apg12Δ BD AD Growth on Trp− Leu− Ade− WT apg5Δ apg12Δ APG12 APG16 + − + APG12ΔG APG16 − n.d. n.d. APG5 APG12 + + + APG5 APG16 + + + APG16 APG16 + + + n.d., not determined. Apg16p interacts preferentially with Apg12p-modified Apg5p We next examined the in vivo interaction between Apg5p and Apg16p. HAApg5p and MycApg16p were expressed by CEN plasmid (single copy) in apg16Δ cells. In these cells, about one-fifth of Apg5p was conjugated by Apg12p (Figure 4, lane 3). Immunoprecipitation with anti-Myc showed that both Apg5p monomer and Apg12-modified Apg5p were precipitated (Figure 4, lane 2), confirming that Apg16p interacts with Apg5p. However, it is noteworthy that only a very small portion of unconjugated Apg5p was brought down by anti-Myc antibody, and most of the Apg16p-interacting Apg5p was modified by Apg12p (Figure 4, compare the level of Apg5p in lanes 2 and 3). In apg12Δapg16Δ cells in which Apg5p exists only as an unmodified form, a very small amount of Apg5p interacted with Apg16p (Figure 4, lanes 5 and 6). These data indicate that Apg16p associates preferentially with Apg12pmodified Apg5p and, without Apg12p modification, Apg16p interacts with Apg5p only weakly. Figure 4.Apg16p interacts preferentially with Apg12p-modified Apg5p. Total lysates were prepared from apg16Δ cells (YNM124) or apg12Δapg16Δ cells (YNM115) harboring both pHA-APG5 and pMyc-APG16 CEN plasmids. The lysates were immunoprecipitated and analyzed as described in Figure 3. Download figure Download PowerPoint Figure 5.The N-terminal region of Apg16p is required for interaction with Apg5p, and the C-terminal region is required for self-multimerization. (A) PJ69-4A cells were co-transformed with each pGBD and pGAD plasmid as indicated. Transformants were selected on Trp− Leu− plates, and then two-hybrid interaction (+ or −) was assessed for growth on Ade− Trp− Leu− plates. (B) Self-multimerization of Apg16p in vivo. Lysates of apg16Δ cells (YNM124) transformed with pMyc-APG16 (2μ) and/or pHA-APG16 (2μ) were immunoprecipitated with anti-Myc antibody and detected with anti-HA antibody. Download figure Download PowerPoint The coiled-coil domain of Apg16p mediates self-multimerization As shown in Figure 5A, the interaction of Apg5p and Apg16p does not require the coiled-coil domain. Since coiled-coil structures are known to be involved in protein–protein interaction, we examined whether Apg16p interacts with protein(s) other than Apg5p through its coiled-coil region. Two-hybrid study revealed that Apg16p interacted with not only Apg5p, but also with Apg16p itself (Figure 5A and Table I). Full-length Apg16p interacted with the C-terminal half of Apg16p containing the coiled-coil region, but not with the N-terminal region (Figure 5A). The Apg16p–Apg16p interaction did not require Apg5p or Apg12p (Table I). These results suggest that the coiled-coil domain mediates self-multimerization of Apg16p. To detect Apg16p multimerization in vivo, lysate from agp16Δ cells carrying both HA-APG16 and Myc-APG16 plasmids was subjected to immunoprecipitation analysis. As shown in Figure 5B, HAApg16p was precipitated by anti-Myc antibody, indicating that Apg16p forms a multimer in vivo. Apg16p cross-links the Apg12p–Apg5p conjugates Because Apg16p forms a multivalent complex, we hypothesized that Apg16p may act as a linker, and Apg16p, Apg5p and Apg12p constitute a large protein complex. Cell lysate from apg12Δ cells expressing both HAApg12p and MycApg12p was subjected to immunoprecipitation. In the immunoprecipitates with anti-Myc antibody, we detected HAApg12p–Apg5p in addition to MycApg12p–Apg5p and MycApg12p (Figure 6A, lane 3). HAApg12p monomer was not detected. Similarly, in the immunoprecipitates with anti-HA, MycApg12p–Apg5p but not MycApg12p was detected (data not shown). These results suggest that at least two Apg12p–Apg5p conjugates exist in the same complex. In contrast, HAApg12p–Apg5p was not precipitated with anti-Myc antibody in the absence of Apg16p (Figure 6A, lane 4), indicating that the interaction between the HA- and Myc-tagged Apg12p–Apg5p conjugates is mediated by Apg16p (as illustrated in Figure 6A, lower panel). Figure 6.Apg16p mediates Apg5p assembly. Immunoprecipitation was carried out as described in Figure 3, using apg12Δ (YNM107) and apg12Δapg16Δ cells (YNM115) carrying pMyc-APG12 (CEN) and pHA-APG12 (CEN) (A), or pMyc-APG12 (CEN) and pHA-APG5 (CEN) (B). Following SDS–PAGE, immunoprecipitates were subjected to immunoblot analysis using anti-HA or anti-Myc antibody as indicated. A model of the Apg12p–Apg5p–Apg16p complex is illustrated in each panel. Apg16p forms a homo-oligomer through its C-terminal coiled-coil region. It is not clear whether Apg16p forms a dimer or a larger multimer. The N-terminal region of each Apg16p interacts with Apg5p; most of these complexes are conjugated to Apg12p (A) but a small population are unmodified (B). In the absence of Apg16p, separate Apg5ps do not assemble. Download figure Download PowerPoint Figure 7.Expression levels of Apg16p are dependent on the Apg12p–Apg5p conjugate. Total lysates were prepared by the NaOH/2-mercaptoethanol extraction method from apg16Δ (YNM124), apg5Δapg16Δ (YNM126) and apg12Δapg16Δ cells (YNM115) carrying pHA-APG16 (CEN). Western blot analysis was performed using anti-HA antibody. Download figure Download PowerPoint Although Apg16p interacted preferentially with the Apg12p–Apg5p conjugate, it also interacted with unconjugated Apg5p (Figure 4). We thus examined whether unconjugated Apg5p is also included in the Apg16p-mediated complex. Cell lysate was prepared from cells expressing both HAApg5p and MycApg12p, and immunoprecipitated. A small but significant amount of unconjugated Apg5p was precipitated with anti-Myc antibody (Figure 6B, lane 2). As expected, this was not observed in the absence of Apg16p (Figure 6B, lane 5). These results indicated that a portion of unconjugated Apg5p was assembled with conjugated Apg5p through Apg16p (Figure 6B, lower panel). Taken together, these results suggest that Apg16p functions as a linker to form the (Apg12p–)Apg5p–Apg16p multimeric complex. The Apg12p–Apg5p conjugate and Apg16p depend on each other for their expression level As shown above (Figure 2E, Figure 3, lane 3, Figure 6A and B), the amount of the Apg12p–Apg5p conjugate was reduced in the absence of Apg16p, suggesting that the Apg12p–Apg5p conjugate is stabilized by interacting with Apg16p. In the immunoprecipitation analysis, the amount of Apg16p is also decreased in the absence of Apg12p (Figure 3, lane 6). To exclude the possibility that Apg16p was degraded during the immunoprecipitation procedures, we prepared the total lysates directly by the NaOH/2-mercaptoethanol extraction method from cells harboring CEN plasmids, and examined the proteins by Western blot analysis. The amount of Apg16p was still reduced in Δapg5 cells (Figure 7A). Furthermore, it was also reduced in Δapg12 cells (Figure 7B), suggesting that the stability of Apg16p depends on the Apg12p–Apg5p conjugate rather than on Apg5p itself. Together with the data in Figure 2E, these results show that the apparent steady-state levels of the Apg12p–Apg5p conjugate and Apg16p are co-dependent, supporting the idea that Apg16p interacts preferentially with Apg12p-modified Apg5p, resulting in formation of a stable complex. Membrane association of Apg16 depends on Apg5p Apg5p is a hydrophilic protein that is found in pelletable complexes (Mizushima et al., 1998a). Apg16p is also a hydrophilic protein but, because of its interaction with Apg5p, it may interact with membranes. We examined the subcellular localization of Apg5p and Apg16p by fractionation analysis. Total cell lysates were prepared in 0.2 M sorbitol, 20 mM triethanolamine (pH 7.2) and 1 mM EDTA, and were centrifuged at 100 000 g for 60 min to generate pellet and supernatant fractions. Unconjugated Apg5p existed mainly in the pellet fraction, with a low level found in the supernatant fraction (Figure 8A, Δ5). The Apg12p–Agp5p conjugate was detected exclusively in the pellet fraction. Most of the Apg16p was also in the pellet and a smaller amount was in the supernatant fraction (Figure 8B). It has been demonstrated that Apg5p is a peripheral membrane protein (M.D.George and D.J.Klionsky, in preparation). Thus, these findings suggested that Apg16p also exists mostly on the same membrane structures. These fractionation patterns were not affected significantly by nitrogen starvation (data not shown). Figure 8.Apg16p exists in a pellet fraction in an Apg5p-dependent manner. Spheroplasts were generated from cells harboring pHA-APG5 (A), pHA-APG16 (B) or pHA-APG16 (2μ) with or without pHA-APG5 (2μ) (C). They were lysed by Dounce homogenization. After unbroken spheroplasts were removed by low-speed centrifugation, the supernatant (T) was fractionated by 100 000 g centrifugation for 60 min to generate a supernatant (S) and pellet (P) fraction. Each fraction was subjected to immunoblot analysis using anti-HA antibody. Download figure Download PowerPoint As shown in Figure 8A, both the Apg12p–Apg5p conjugate and Apg5p monomer were still pelletable in Δapg16 cells (Figure 8A, Δ5Δ16). In contrast, most of the Apg16p shifted to the soluble fraction in Δapg5 cells (data not shown). Since the amount of Apg16p was reduced in Δapg5 cells (as in Figure 7B), we fractionated the lysates of cells expressing these proteins from 2μ plasmids. Similarly to the results with centromeric expression, when Apg5p was overexpressed, overexpressed Apg16p still localized to the pellet fraction (Figure 8C). However, when Apg5p was absent, most of the Apg16p was recovered in the supernatant fraction, suggesting that membrane binding of Apg16p is Apg5p dependent. Discussion By our previous morphological screening for macroautophagy-defective mutants, 14 apg mutants (apg7 and apg11 are allelic) were isolated. Here we identify a novel APG gene, APG16. The studies on the apg16Δ strain showed that it demonstrates the typical Apg− phenotype: negative for autophagic body accumulation, loss of viability during starvation, and defects in sporulation and API maturation. Apg16p is the first Apg protein identified after the mutant screening. This apparently indicates that our former screening was not saturated. We now have 15 APG genes, but there might be additional genes involved in autophagy. The amino acid sequence of Apg16p revealed that it has a coiled-coil motif encompassing most of its C-terminal half, but no other known characteristic sequences. BLAST searches have failed to identify any proteins with significant homology in other species. Previously, Apg16p was proposed as a yeast Sap18 counterpart, only because it has a weak homology to human Sap18 (16.1% identity) (Zhang et al., 1997). In human cells, Sap18 is a component of a Sin3-containing complex which contains histone deacetylases, HDAC1 and HDAC2. It has been thought that histone deacetylation leads to transcription repression. Sap18 interacts with Sin3 and enhances Sin3-mediated repression of transcription (Zhang et al., 1997). However, our data argue against this proposal. First, Sap18 is suggested to be responsible for general transcription events, whereas our apg16Δ strain showed a specific defect in autophagy. Second, if Apg16p is a functional Sap18 homolog, its function would be Sin3p-mediated. However, autophagy proceeded normally in a sin3 disruptant (data not shown). Third, Apg16p is a coiled-coil protein but the human Sap18 is not. Therefore, we believe that this protein is not a functional counterpart of human Sap18 in yeast, and the nomenclature ‘Apg16p’ is appropriate. Although Apg16p was isolated originally as an Apg12p-interacting protein by a two-hybrid screening, the interaction between Apg12p and Apg16p turned out to be indirect and mediated by Apg5p (Table I). Further two-hybrid and immunoprecipitation analyses strongly suggest that Apg16p interacts directly with Apg5p. However, we cannot rule out the possibility that oth

Autophagy is a membrane trafficking pathway that carries cytosolic components to the lysosome for degradation. During this process, the autophagosome, a double-membraned organelle, is generated de novo, sequesters cytoplasmic proteins and organelles, and delivers them to lysosomes. However, the mechanism by which autophagosomes are targeted to lysosomes has not been determined. Here, we observed the real-time behavior of microtubule-associated protein light chain 3 (LC3), which localizes to autophagosomes, and showed that autophagosomes move in a microtubule- and dynein-dynactin motor complex-dependent manner. After formation, autophagosomes show a rapid vectorial movement in the direction of the centrosome, where lysosomes are usually concentrated. Microinjection of antibodies against LC3 inhibited this movement; furthermore, using FRAP, we showed that anti-LC3 antibody injection caused a defect in targeting of autophagosomes to lysosomes. Collectively, our data demonstrate the functional significance of autophagosome movement that enables effective delivery from the cytosol to lysosomes.

ADVERTISEMENT RETURN TO ISSUEPREVCommunicationNEXTA Novel Class of Emitting Amorphous Molecular Materials as Bipolar Radical Formants: 2-{4-[Bis(4-methylphenyl)amino]phenyl}- 5-(dimesitylboryl)thiophene and 2-{4-[Bis(9,9-dimethylfluorenyl)amino]phenyl}- 5-(dimesitylboryl)thiopheneYasuhiko Shirota, Motoi Kinoshita, Tetsuya Noda, Kenji Okumoto, and Takahiro OharaView Author Information Department of Applied Chemistry Faculty of Engineering, Osaka University Yamadaoka, Suita, Osaka 565-0871, Japan Cite this: J. Am. Chem. Soc. 2000, 122, 44, 11021–11022Publication Date (Web):October 21, 2000Publication History Received28 June 2000Published online21 October 2000Published inissue 1 November 2000https://doi.org/10.1021/ja0023332Copyright © 2000 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views3977Altmetric-Citations342LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit Read OnlinePDF (40 KB) Get e-AlertscloseSUBJECTS:Amorphous materials,Diodes,Layers,Luminescence,Materials Get e-Alerts

ADVERTISEMENT RETURN TO ISSUEPREVCommunicationNEXT5,5‘-Bis(dimesitylboryl)-2,2‘-bithiophene and 5,5‘‘-Bis(dimesitylboryl)-2,2‘:5‘,2‘‘-terthiophene as a Novel Family of Electron-Transporting Amorphous Molecular MaterialsTetsuya Noda and Yasuhiko ShirotaView Author Information Department of Applied Chemistry, Faculty of Engineering Osaka University, Yamadaoka, Suita, Osaka 565-0871, Japan Cite this: J. Am. Chem. Soc. 1998, 120, 37, 9714–9715Publication Date (Web):September 5, 1998Publication History Received18 May 1998Published online5 September 1998Published inissue 1 September 1998https://doi.org/10.1021/ja9817343Copyright © 1998 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views2320Altmetric-Citations316LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit Read OnlinePDF (53 KB) Get e-AlertsSUBJECTS:Amorphous materials,Electrodes,Layers,Oligomers,Organic compounds Get e-Alerts

The yeast S. cerevisiae imports cytosolic components into the vacuole non-selectively by autophagy and degrades them by vacuolar hydrolases under nutrient starvation conditions. We developed a novel system for monitoring autophagy by constructing cells in which modified vacuolar alkaline phosphatase is expressed as an inactive precursor form in the cytosol. Under starvation conditions, the processing of the precursor to the mature form and phosphatase activity appeared gradually, and the mature form was located in the vacuole. Disruption of APG1, an essential gene for autophagy, resulted in no processing or phosphatase activity. These results indicate that the precursor form in the cytosol is transferred to the vacuole by autophagy and converted to the active form by vacuolar proteinases. Thus, autophagy could be determined easily and accurately by measuring the phosphatase activity.

Neurogenesis has been described in limited regions of the adult mammalian brain. In this study, we showed that the ependymal layer of the 3rd ventricle is a neurogenic region in the adult rat brain. DiI labeling of the 3rd ventricle revealed that neural progenitor cells were derived from cells at the ependymal layer of the adult 3rd ventricle. The mitosis of these progenitor cells at the ependymal layer was promoted by bFGF administration. Combination of BrdU administration, nestin/GFAP immunohistochemistry, and labeling by GFP-recombinant adenoviral infection (vGFP) indicated that at least some tanycytes might be neural progenitor cells in the ependymal layer of the 3rd ventricle. Tracing by vGFP indicated that neural progenitor cells may have migrated from the 3rd ventricle to the hypothalamic parenchyma, where they were integrated into neural networks by forming synapses. In addition, some BrdU(+) neurons had immunoreactivity for orexin A in the hypothalamus. These results indicate that neural progenitor cells exist in the ependymal layer of the adult rat 3rd ventricle and that they may differentiate into neurons functioning in the hypothalamus.

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.

Recent epidemiological studies have revealed a significant association between periodontitis and oral squamous cell carcinoma (OSCC). Furthermore, matrix metalloproteinase 9 (MMP9) is implicated in the invasion and metastasis of tumour cells. We examined the involvement of Porphyromonas gingivalis, a periodontal pathogen, in OSCC invasion through induced expression of proMMP and its activation. proMMP9 was continuously secreted from carcinoma SAS cells, while P. gingivalis infection increased proenzyme expression and subsequently processed it to active MMP9 in culture supernatant, which enhanced cellular invasion. In contrast, Fusobacterium nucleatum, another periodontal organism, failed to demonstrate such activities. The effects of P. gingivalis were observed with highly invasive cells, but not with the low invasivetype. P. gingivalis also stimulated proteinase-activated receptor 2 (PAR2) and enhanced proMMP9 expression, which promoted cellular invasion. P. gingivalis mutants deficient in gingipain proteases failed to activate MMP9. Infected SAS cells exhibited activation of ERK1/2, p38, and NF-kB, and their inhibitors diminished both proMMP9-overexpression and cellular invasion. Together, our results show that P. gingivalis activates the ERK1/2-Ets1, p38/HSP27, and PAR2/NF-kB pathways to induce proMMP9 expression, after which the proenzyme is activated by gingipains to promote cellular invasion of OSCC cell lines. These findings suggest a novel mechanism of progression and metastasis of OSCC associated with periodontitis.

In nutrient-rich, vegetative conditions, the yeast Saccharomyces cerevisiae transports a resident protease, aminopeptidase I (API), to the vacuole by the cytoplasm to vacuole targeting (Cvt) pathway, thus contributing to the degradative capacity of this organelle. When cells subsequently encounter starvation conditions, the machinery that recruited precursor API (prAPI) also sequesters bulk cytosol for delivery, breakdown, and recycling in the vacuole by the autophagy pathway. Each of these overlapping alternative transport pathways is specifically mobilized depending on environmental cues. The basic mechanism of cargo packaging and delivery involves the formation of a double-membrane transport vesicle around prAPI and/or bulk cytosol. Upon completion, these Cvt and autophagic vesicles are targeted to the vacuole to allow delivery of their lumenal contents. Key questions remain regarding the origin and formation of the transport vesicle. In this study, we have cloned the APG9/CVT7 gene and characterized the gene product. Apg9p/Cvt7p is the first characterized integral membrane protein required for Cvt and autophagy transport. Biochemical and morphological analyses indicate that Apg9p/Cvt7p is localized to large perivacuolar punctate structures, but does not colocalize with typical endomembrane marker proteins. Finally, we have isolated a temperature conditional allele of APG9/CVT7 and demonstrate the direct role of Apg9p/Cvt7p in the formation of the Cvt and autophagic vesicles. From these results, we propose that Apg9p/Cvt7p may serve as a marker for a specialized compartment essential for these vesicle-mediated alternative targeting pathways.

Double membrane structure, autophagosome, is formed de novo in the process of autophagy in the yeast Saccharomyces cerevisiae, and many Apg proteins participate in this process. To further understand autophagy, we analyzed the involvement of factors engaged in the secretory pathway. First, we showed that Sec18p (N-ethylmaleimide-sensitive fusion protein, NSF) and Vti1p (soluble N-ethylmaleimide-sensitive fusion protein attachment protein, SNARE), and soluble N-ethylmaleimide-sensitive fusion protein receptor are required for fusion of the autophagosome to the vacuole but are not involved in autophagosome formation. Second, Sec12p was shown to be essential for autophagy but not for the cytoplasm to vacuole-targeting (Cvt) (pathway, which shares mostly the same machinery with autophagy. Subcellular fractionation and electron microscopic analyses showed that Cvt vesicles, but not autophagosomes, can be formed in sec12 cells. Three other coatmer protein (COPII) mutants, sec16, sec23, and sec24, were also defective in autophagy. The blockage of autophagy in these mutants was not dependent on transport from endoplasmic reticulum-to-Golgi, because mutations in two other COPII genes, SEC13 and SEC31, did not affect autophagy. These results demonstrate the requirement for subgroup of COPII proteins in autophagy. This evidence demonstrating the involvement of Sec proteins in the mechanism of autophagosome formation is crucial for understanding membrane flow during the process.

The effects of bone marrow stromal cells (BMSCs) on the repair of injured spinal cord and on the behavioral improvement were studied in the rat. The spinal cord was injured by contusion using a weight-drop at the level of T8-9, and the BMSCs from the bone marrow of the same strain were infused into the cerebrospinal fluid (CSF) through the 4th ventricle. BMSCs were conveyed through the CSF to the spinal cord, where most BMSCs attached to the spinal surface although a few invaded the lesion. The BBB score was higher, and the cavity volume was smaller in the rats with transplantation than in the control rats. Transplanted cells gradually decreased in number and disappeared from the spinal cord 3 weeks after injection. The medium supplemented by CSF (250 microl in 3 ml medium) harvested from the rats in which BMSCs had been injected 2 days previously promoted the neurosphere cells to adhere to the culture dish and to spread into the periphery. These results suggest that BMSCs can exert effects by producing some trophic factors into the CSF or by contacting with host spinal tissues on the reduction of cavities and on the improvement of behavioral function in the rat. Considering that BMSCs can be used for autologous transplantation, and that the CSF infusion of transplants imposes a minimal burden on patients, the results of the present study are important and promising for the clinical use of BMSCs in spinal cord injury treatment.

A multilayer, blue-emitting electroluminescent device based on a novel, thermally stable, amorphous molecular material with electron-transport properties, BMB-2T (the title compound, see also the Figure), is described. Its fabrication and performance are reported. It is shown that the device efficiency increases when a thin layer of another new amorphous material is inserted between the BMB-2T and the hole-transporting layer to prevent exciplex formation.

The vacuolar protein aminopeptidase I (API) uses a novel cytoplasm-to-vacuole targeting (Cvt) pathway. Complementation analysis of yeast mutants defective for cytoplasm-to-vacuole protein targeting (cvt) and autophagy (apg) revealed seven overlapping complementation groups between these two sets of mutants. In addition, all 14 apg complementation groups are defective in the delivery of API to the vacuole. Similarly, the majority of nonoverlapping cvt complementation groups appear to be at least partially defective in autophagy. Kinetic analyses of protein delivery rates indicate that autophagic protein uptake is induced by nitrogen starvation, whereas Cvt is a constitutive biosynthetic pathway. However, the machinery governing Cvt is affected by nitrogen starvation as targeting defects resulting from API overexpression can be rescued by induction of autophagy.

SiC nanowires (NWs) reinforce SiC matrix composites with high efficiency. With the incorporation of ∼ 6 vol.-% randomly oriented single-crystal SiC NWs in the matrices, the fracture toughnesses and flexural strengths of the composites doubled. The composites (see Figure) were fabricated in situ by a new process based on chemical vapor infiltration. Reinforcement efficiency of the NW is affected by the amount of C deposited on the NWs as the NW/matrix interfacial layer.

Abstract Transplantation of bone marrow stromal cells (MSCs) has been regarded as a potential approach for promoting nerve regeneration. In the present study, we investigated the influence of MSCs on spinal cord neurosphere cells in vitro and on the regeneration of injured spinal cord in vivo by grafting. MSCs from adult rats were cocultured with fetal spinal cord‐derived neurosphere cells by either cell mixing or making monolayered‐feeder cultures. In the mixed cell cultures, neuroshpere cells were stimulated to develop extensive processes. In the monolayered‐feeder cultures, numerous processes from neurosphere cells appeared to be attracted to MSCs. In an in vivo experiment, grafted MSCs promoted the regeneration of injured spinal cord by enhancing tissue repair of the lesion, leaving apparently smaller cavities than in controls. Although the number of grafted MSCs gradually decreased, some treated animals showed remarkable functional recovery. These results suggest that MSCs might have profound effects on the differentiation of neurosphere cells and be able to promote regeneration of the spinal cord by means of grafting. © 2003 Wiley‐Liss, Inc.

In this chapter, we introduce several methods that rely on the analysis of LC3, a versatile marker protein of autophagic structures in mammalian cultured cells. The appearance of LC3‐positive puncta is indicative of the induction of autophagy, and it is observed either by immunofluorescence or by GFP‐based microscopy. The maturation process by which autophagosomes are converted into autolysosomes can be monitored by the GFP and RFP tandemly tagged LC3 (tfLC3) method. Lysosomal turnover of LC3 is a good index of the proceeding of autophagy and can be assessed by Western blotting. These methods will provide a relatively easy assessment of autophagy, and the details of the procedure will be described along with possible pitfalls.

Autophagy plays a crucial role in host defense, termed antimicrobial autophagy (xenophagy), as it functions to degrade intracellular foreign microbial invaders such as group A Streptococcus (GAS). Xenophagosomes undergo a stepwise maturation process consisting of a fusion event with lysosomes, after which the cargoes are degraded. However, the molecular mechanism underlying xenophagosome/lysosome fusion remains unclear. We examined the involvement of endocytic soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) in xenophagosome/lysosome fusion. Confocal microscopic analysis showed that SNAREs, including vesicle-associated membrane protein (VAMP)7, VAMP8, and vesicle transport through interaction with t-SNAREs homologue 1B (Vti1b), colocalized with green fluorescent protein-LC3 in xenophagosomes. Knockdown of Vti1b and VAMP8 with small interfering RNAs disturbed the colocalization of LC3 with lysosomal membrane protein (LAMP)1. The invasive efficiency of GAS into cells was not altered by knockdown of VAMP8 or Vti1b, whereas cellular bactericidal efficiency was significantly diminished, indicating that antimicrobial autophagy was functionally impaired. Knockdown of Vti1b and VAMP8 also disturbed colocalization of LC3 with LAMP1 in canonical autophagy, in which LC3-II proteins were negligibly degraded. In contrast, knockdown of Syntaxin 7 and Syntaxin 8 showed little effect on the autophagic fusion event. These findings strongly suggest that the combinational SNARE proteins VAMP8 and Vti1b mediate the fusion of antimicrobial and canonical autophagosomes with lysosomes, an essential event for autophagic degradation.

<h2>Abstract</h2> Autophagy – the degradation of organelles and cytoplasmic material – occurs through dynamic rearrangements of cellular membrane structures. Following the induction of autophagy, newly formed autophagosomes transfer cytosolic materials to the lysosome or vacuole for degradation. The autophagosome is an organelle destined for degradation, suggesting that the membrane structure is formed <i>de novo</i> many times. The autophagosome is formed through the nucleation, assembly and elongation of membrane structures. The concerted action of several Apg/Aut/Cvt proteins around a characteristic subcellular structure (the preautophagosomal structure) is the key to understanding this novel type of membrane-formation process.

In an analogous manner to protein ubiquitination, The C terminus of Atg8p, a yeast protein essential for autophagy, conjugates to a head group of phosphatidylethanolamine via an amide bond. Though physiological role of this reaction is assigned to membrane organization during autophagy, its molecular details are still unknown. Here, we show that Escherichia coli cells coexpressed Atg8p, Atg7p (E1), and Atg3p (E2) allowed to form conjugate of Atg8p with endogenous PE. Further, we established an in vitro Atg8p-PE reconstitution system using purified Atg8pG116, Atg7p, Atg3p, and PE-containing liposomes, demonstrating that the Atg7p and the Atg3p are minimal catalysts for Atg8p-PE conjugate reaction. Efficiency of this lipidation reaction depends on the state of the substrate, PE (phospholipid bilayer and its lipid composition). It is also suggested that the lipidation induces a conformational change in the N-terminal region of Atg8p. In vitro system developed here will provide a powerful system for further understanding the precise role of lipidation and interaction of two ubiquitin-like systems essential for autophagy. In an analogous manner to protein ubiquitination, The C terminus of Atg8p, a yeast protein essential for autophagy, conjugates to a head group of phosphatidylethanolamine via an amide bond. Though physiological role of this reaction is assigned to membrane organization during autophagy, its molecular details are still unknown. Here, we show that Escherichia coli cells coexpressed Atg8p, Atg7p (E1), and Atg3p (E2) allowed to form conjugate of Atg8p with endogenous PE. Further, we established an in vitro Atg8p-PE reconstitution system using purified Atg8pG116, Atg7p, Atg3p, and PE-containing liposomes, demonstrating that the Atg7p and the Atg3p are minimal catalysts for Atg8p-PE conjugate reaction. Efficiency of this lipidation reaction depends on the state of the substrate, PE (phospholipid bilayer and its lipid composition). It is also suggested that the lipidation induces a conformational change in the N-terminal region of Atg8p. In vitro system developed here will provide a powerful system for further understanding the precise role of lipidation and interaction of two ubiquitin-like systems essential for autophagy. Autophagy, an intracellular bulk degradation system in the lysosomes/vacuoles of eukaryotic cells is necessary for the recycling of cytoplasmic components for survival during nutrient starvation conditions (1Klionsky D.J. Ohsumi Y. Annu. Rev. Cell Dev. Biol. 1999; 15: 1-32Crossref PubMed Scopus (395) Google Scholar, 2Huang W.P. Klionsky D.J. Cell Struct. Funct. 2002; 27: 409-420Crossref PubMed Scopus (163) Google Scholar, 3Wang C.W. Klionsky D.J. Mol. Med. 2003; 9: 65-76Crossref PubMed Google Scholar). Membrane dynamics in autophagy are distinct from classical membrane trafficking. In particular, the mechanism by which this new double membrane-bounded compartment, the autophagosome, is formed has remained poorly understood. Genetic screens of the budding yeast, Saccharomyces cerevisiae, have identified 16 genes potentially involved in this process, named ATG genes (formerly APG/AUT) (4Klionsky D.J. Cregg J.M. Dunn Jr., W.A. Emr S.D. Sakai Y. Sandoval I.V. Sibirny A. Subramani S. Thumm M. Veenhuis M. Ohsumi Y. Dev. Cell. 2003; 5: 539-545Abstract Full Text Full Text PDF PubMed Scopus (1048) Google Scholar). Dissection of autophagosome biogenesis through intensive characterization of these gene products Atgps (1Klionsky D.J. Ohsumi Y. Annu. Rev. Cell Dev. Biol. 1999; 15: 1-32Crossref PubMed Scopus (395) Google Scholar) has revealed that the overall process of autophagy in yeast is similar to that in higher eukaryotes, exhibiting conservation of the molecular machinery involved (5Mizushima N. Ohsumi Y. Yoshimori T. Cell Struct. Funct. 2002; 27: 421-429Crossref PubMed Scopus (774) Google Scholar). Our recent studies have identified two ubiquitin-like conjugation systems essential for autophagosome formation; these systems utilize approximately half of the Atg protein members (6Mizushima N. Noda T. Yoshimori T. Tanaka Y. Ishii T. George M.D. Klionsky D.J. Ohsumi M. Ohsumi Y. Nature. 1998; 395: 395-398Crossref PubMed Scopus (1325) Google Scholar, 7Ichimura Y. Kirisako T. Takao T. Satomi Y. Shimonishi Y. Ishihara N. Mizushima N. Tanida I. Kominami E. Ohsumi M. Noda T. Ohsumi Y. Nature. 2000; 408: 488-492Crossref PubMed Scopus (1587) Google Scholar, 8Ohsumi Y. Nat. Rev. Mol. Cell. Biol. 2001; 2: 211-216Crossref PubMed Scopus (1071) Google Scholar). Atg12p, a ubiquitin-like protein, is covalently linked to Atg5p through an isopeptide bond between the C-terminal glycine of Atg12p and Lys149 of Atg5p by sequential reactions catalyzed by Atg7p (E1) and Atg10p (E2) (6Mizushima N. Noda T. Yoshimori T. Tanaka Y. Ishii T. George M.D. Klionsky D.J. Ohsumi M. Ohsumi Y. Nature. 1998; 395: 395-398Crossref PubMed Scopus (1325) Google Scholar, 9Tanida I. Mizushima N. Kiyooka M. Ohsumi M. Ueno T. Ohsumi Y. Kominami E. Mol. Biol. Cell. 1999; 10: 1367-1379Crossref PubMed Scopus (342) Google Scholar, 10Shintani T. Mizushima N. Ogawa Y. Matsuura A. Noda T. Ohsumi Y. EMBO J. 1999; 18: 5234-5241Crossref PubMed Scopus (245) Google Scholar). Atg12p-Atg5p conjugates then associate with Atg16p to form a multimeric complex, Atg12p-Atg5p·Atg16p, mediated by Atg16p homo-oligomerization (11Kuma A. Mizushima N. Ishihara N. Ohsumi Y. J. Biol. Chem. 2002; 277: 18619-18625Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar). A second ubiquitin-like protein, Atg8p, that was originally reported as a microtubule-associated protein (Aut7p) (12Lang T. Schaeffeler E. Bernreuther D. Bredschneider M. Wolf D.H. Thumm M. EMBO J. 1998; 17: 3597-3607Crossref PubMed Scopus (231) Google Scholar), is conjugated to a membrane phospholipid, phosphatidylethanolamine (PE) 1The abbreviations used are: PE, phosphatidylethanolamine; DTT, dithiothreitol; GST, glutathione S-transferase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PG, phosphatidylglycerol; PS, phosphatidylserine; CBB, Coomassie Brilliant Blue; PA, phosphatidic acid; DOPE, dioleoylphosphatidylethanolamine; POPC, 1-palmitoyl-2-oleoylphoshatidylcholine; DOPG, dioleoyl phosphatidylglycerol; PI, phosphatidylinositol; CL, cardiolipin. (7Ichimura Y. Kirisako T. Takao T. Satomi Y. Shimonishi Y. Ishihara N. Mizushima N. Tanida I. Kominami E. Ohsumi M. Noda T. Ohsumi Y. Nature. 2000; 408: 488-492Crossref PubMed Scopus (1587) Google Scholar). The C-terminal arginine of newly synthesized Atg8p (Atg8pR117) is initially removed by the Atg4p protease to expose a C-terminal glycine residue (Atg8pG116) (13Kirisako T. Ichimura Y. Okada H. Kabeya Y. Mizushima N. Yoshimori T. Ohsumi M. Takao T. Noda T. Ohsumi Y. J. Cell Biol. 2000; 151: 263-276Crossref PubMed Scopus (756) Google Scholar). Atg8pG116 is then activated by Atg7p (E1) and transferred to Atg3p (E2) (7Ichimura Y. Kirisako T. Takao T. Satomi Y. Shimonishi Y. Ishihara N. Mizushima N. Tanida I. Kominami E. Ohsumi M. Noda T. Ohsumi Y. Nature. 2000; 408: 488-492Crossref PubMed Scopus (1587) Google Scholar). Finally, the Atg8pG116 conjugates to PE through an amide bond between its C-terminal glycine and the amino group of PE (7Ichimura Y. Kirisako T. Takao T. Satomi Y. Shimonishi Y. Ishihara N. Mizushima N. Tanida I. Kominami E. Ohsumi M. Noda T. Ohsumi Y. Nature. 2000; 408: 488-492Crossref PubMed Scopus (1587) Google Scholar). Atg8p-PE is tightly associated with membranes, behaving as an integral membrane protein. Notably, the Atg8 conjugation system, while similar to ubiquitination, mechanically utilizes a ubiquitous phospholipid, not a protein, as a target. Liberation of the Atg8p moiety from Atg8p-PE by the action of Atg4p (deconjugation) is required for the normal progression of autophagy (13Kirisako T. Ichimura Y. Okada H. Kabeya Y. Mizushima N. Yoshimori T. Ohsumi M. Takao T. Noda T. Ohsumi Y. J. Cell Biol. 2000; 151: 263-276Crossref PubMed Scopus (756) Google Scholar). The mammalian Atg8p homologues, MAP1-LC3, GATE-16, and GABARAP, have been implicated in membrane dynamics, including autophagy, intra-Golgi transport, and GABA receptor sorting to the postsynaptic membrane (14Kabeya Y. Mizushima N. Ueno T. Yamamoto A. Kirisako T. Noda T. Kominami E. Ohsumi Y. Yoshimori T. EMBO J. 2000; 19: 5720-5728Crossref PubMed Scopus (5646) Google Scholar, 15Sagiv Y. Legesse-Miller A. Porat A. Elazar Z. EMBO J. 2000; 19: 1494-1504Crossref PubMed Scopus (213) Google Scholar, 16Wang H. Bedford F.K. Brandon N.J. Moss S.J. Olsen R.W. Nature. 1999; 397: 69-72Crossref PubMed Scopus (492) Google Scholar). These Atg8p homologues are also modified via a ubiquitin-like system analogous to Atg8p lipidation (14Kabeya Y. Mizushima N. Ueno T. Yamamoto A. Kirisako T. Noda T. Kominami E. Ohsumi Y. Yoshimori T. EMBO J. 2000; 19: 5720-5728Crossref PubMed Scopus (5646) Google Scholar, 17Tanida I. Komatsu M. Ueno T. Kominami E. Biochem. Biophys. Res. Commun. 2003; 300: 637-644Crossref PubMed Scopus (86) Google Scholar, 18He H. Dang Y. Dai F. Guo Z. Wu J. She X. Pei Y. Chen Y. Ling W. Wu C. Zhao S. Liu J.O. Yu L. J. Biol. Chem. 2003; 278: 29278-29287Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar, 19Hemelaar J. Lelyveld V.S. Kessler B.M. Ploegh H.L. J. Biol. Chem. 2003; 278: 51841-51850Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). We have observed a small number of autophagosome-like structures of abnormal morphology in the null mutant of ATG8 (20Kirisako T. Baba M. Ishihara N. Miyazawa K. Ohsumi M. Yoshimori T. Noda T. Ohsumi Y. J. Cell Biol. 1999; 147: 435-446Crossref PubMed Scopus (733) Google Scholar). Whereas Abeliovich et al. (21Abeliovich H. Dunn Jr., W.A. Kim J. Klionsky D.J. J. Cell Biol. 2000; 151: 1025-1034Crossref PubMed Scopus (239) Google Scholar) reported that significantly smaller vesicles than normal autophagosome were observed in a similar Δatg8 strain. These observations imply that the Atg8p-PE may function in normal development of autophagosomal membrane. In fact, previous immunoelectorn microscopic analyses demonstrated the specific localization of Atg8p to the isolation membrane (intermediate structure of autophagosome) under the starvation conditions (20Kirisako T. Baba M. Ishihara N. Miyazawa K. Ohsumi M. Yoshimori T. Noda T. Ohsumi Y. J. Cell Biol. 1999; 147: 435-446Crossref PubMed Scopus (733) Google Scholar). The precise roles of the two conjugates during autophagosome formation remain unclear. Atg8p-PE conjugation is severely reduced in the absence of the Atg12p-Atg5p conjugate. Thus, it appears likely that the functions of these two conjugates, Atg8p-PE and Atg12p-Atg5p, are closely related. To identify the molecular events governing autophagosome formation, we need to address not only the function of each of the two conjugates, but also the interrelationship between Ag8p-PE and Atg12p-Atg5p. In this study, to address the molecular machinery involved in membrane dynamics, we focused on Atg8p lipidation, and developed in vitro reconstitution system of Atg8p-PE. Strains, Media, and Standard Molecular Genetic Methods—The Escherichia coli strains XL1Blue and BL21 (DE3) were used for plasmid construction and protein expression, respectively. Total lipid extracts from the E. coli strain W3011 and a PE-deficient strain, GN10, were used to make liposomes. E. coli transformants were grown in Luria-Bertani (LB) medium at 37 °C, supplemented with the appropriate antibiotics (ampicillin, 50 μg/ml; chloramphenicol, 20 μg/ml). GN10 was grown in LB medium containing 50 mm MgCl2 at 37 °C (22Saha S.K. Nishijima S. Matsuzaki H. Shibuya I. Matsumoto K. Biosci. Biotechnol. Biochem. 1996; 60: 111-116Crossref PubMed Scopus (44) Google Scholar). Total cell lysates from the S. cerevisiae strain SEY6210 (MATαleu2-3 112 ura3-52 trp1-Δ901 his3-Δ200 ade2-101 lys2-801 suc2-Δ9) and its derivatives (Δatg7::HIS3 and Δatg3::TRP1) were used as Atg8p-PE mobility standards in Western blot analyses. Total lipid extracts from SEY6210 was used for making liposomes. Yeast cells were grown in YEPD (1% yeast extract, 2% polypeptone, 2% glucose) at 30 °C. Molecular biological procedures were performed in accordance with standard procedures (23Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Plasmid Construction and Protein Expression in E. coli—To construct E. coli expression plasmids encoding Atg7-Myc (C-terminal 3× Myc-tagged Atg7), Atg3, Atg8G116 (C-terminal glycine-exposed form of Atg8), and Atg8R117 (C-terminal arginine-containing form of Atg8), the appropriate genes were amplified from the following plasmids, pRS424-Atg7-Myc, pRS426Atg3, and pRS426Atg8, by PCR using the following primers: (ATG7-MYC) KpnI-S.D. (Fw), 5′-GGGGTACCCCAGGAGGAATTCACCATGTCGTCAGA-3′, SmaI (Rv), 5′-TCCCCCGGGGGAATGCAAAATATTA-3′, (ATG3) SmaI-S.D. (Fw), 5′-TCCCCCGGGGGAAGGAGGAATTCACCATGTTAGATC-3′, BamHI (Rv), 5′-CGGGATCCCGTTACCAACCTTCC-3′, (ATG8G116) BglII-S.D. (Fw), 5′-GAAGATCTTCAGGAGGAATTCACCATGAAGTCTAC-3′, XbaI (Rv), 5′-GCTCTAGAGCCTAGCCAAATGTATTTTC-3′.(ATG8R117) BglII-S.D. (Fw), 5′-GAAGATCTTCAGGAGGAATTCACCATGAAGTCTAC-3′, HindIII-S.D. (Rv), 5′-CCCAAGTTGGGCTAGCCAAATGTATTTTC-3′. The resulting PCR products all contained a Shine-Dalgarno sequence (S.D.) upstream of the start codon to facilitate efficient translation in E. coli. The amplified genes were subcloned into pUC18 and then inserted into the multiple cloning sites of the arabinose-inducible plasmids, pBAD18 and pBAD33 (24Guzman L.M. Belin D. Carson M.J. Beckwith J. J. Bacteriol. 1995; 177: 4121-4130Crossref PubMed Scopus (4031) Google Scholar). pBAD18 includes gene expression via the PBAD promoter, expresses AraC, confers Ampr (ampicillin resistance), and contains a pBR origin. pBAD33 includes gene expression via the PBAD promoter, expresses AraC, confers Cmr (chloramphenicol resistance), and contains a pACYC origin (diagram showed in Fig. 1A). Atg7C507S-Myc and Atg3C234S mutant forms were encoded by derivatives of the pBAD plasmids, containing single amino acid substitutions from cysteine to serine within the active centers of Atg7 and Atg3, respectively. These plasmids were constructed using a QuikChange site-directed mutagenesis kit (Stratagene) using the following mutagenesis oligonucleotides; (ATG7C507S) (Fw), 5′-ACTTTGGATCAAATGTCGACAGTAACTAGACC-3′, (Rv), 5′-GGGTCTAGTTACTGTCGACATTTGATCCAAAGT-3′, (ATG3C234S) (Fw), 5′-GTTTCCATTCATCCAAGCAAGCATGCTAATGTA-3′, (Rv), 5′-TACATTAGCATGCT TGCTTGGATGAATGGAAAC-3′. BL21 (DE3) cells were transformed with pBAD-Atg7-Myc and either pBADC-Atg8G116, pBADC-Atg3Atg8R117, or pBADC-Atg3 Atg8G116. Transformed cells were grown to an OD600 of 0.5 prior to the addition of 0.2% arabinose to the cultures. After induction for 1 h, cells were harvested and lysed in TBS (50 mm Tris-HCl, pH 7.5, 150 mm NaCl) containing 1 mm DTT and 1 mm phenylmethylsulfonyl fluoride by sonication (2.0 OD unit cells/100 μl). For Western blotting, cell lysates (equivalent to 0.04 OD unit cells) were subjected to SDS-PAGE containing 6 m urea (urea-SDS-PAGE). Protein Purification—To purify the recombinant Atg proteins, we constructed glutathione S-transferase (GST) fusions of the desired Atgs (GST-Atg7-Myc, GST-Atg3, GST-Atg3C234S and GST-Atg8G116) by insertion of PCR products into the cloning site (BamHI/SalI) of pGEX4-T-1 (Amersham Biosciences). Following introduction of the obtained plasmids into BL21 (DE3) E. coli, expression of each protein was induced by addition of 0.05 mm isopropyl β-d(-)-thiogalactopyranoside to the medium. After induction, cells were disrupted by sonication in TBST (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% Triton X-100) containing 2 mm DTT, and 1 mm phenylmethylsulfonyl fluoride. After removal of cell debris by centrifugation at 10,000 rpm for 10 min, obtained cell lysates were incubated with 1 ml of glutathione-Sepharose 4B (50% slurry in TBST) (Amersham Biosciences) for 30 min at 4 °C to collect recombinant proteins. The proteins bound to the Sepharose beads were washed with TBST, then suspended in 400 μl of TBS containing 2 mm DTT. The GST-fused Atg proteins were treated with thrombin (10 units) at 20 °C for 1 h; the glutathione-Sepharose 4B/GST complexes were then removed by centrifugation. Protein concentrations were determined by the Bradford method (Pierce) using bovine serum albumin as a standard. The purified Atg proteins were then diluted in TBS containing 50% glycerol for storage at –30 °C. Extraction of Total Lipids and Liposome Preparation—W3011 and GN10 E. coli strains were grown in LB medium containing 50 mm MgCl2 to stationary phase. SEY6210 yeast strain was grown in YEPD to stationary phase. Both types of cells were harvested (cells wet-weight 10 g), and total lipids were extracted by Bligh and Dyer's methods (25Bligh E.G. Dyer W.J. Can. J. Med. Sci. 1959; 37: 911-917Google Scholar). The total lipids were dissolved in 10 ml of chloroform, and the concentration of total phospholipids was quantified by a phosphorous assay using the phosphomolybdate reaction (26Bartlett G.R. Ann. N. Y. Acad. Sci. 1958; 75: 110-114Crossref PubMed Scopus (17) Google Scholar). Individual phospholipids were separated by two-dimensional thin-layer chromatography, and the composition of phospholipids was determined by the phosphorous assay (W3011; PE/PG/CL/PA = 75.4:16.6:4.3:3.57, GN10; PE/PG/CL/PA = <0.01:52.6:33.6:11.4, SEY6210; PE/PC/PS/PI = 16.6:51.7:13.7:18.0). Total lipids from E. coli and yeast were used to generate E. coli and yeast total lipid liposomes, respectively. To prepare liposomes with various phospholipid compositions, phospholipids were mixed in the appropriate ratios from stocks dissolved in chloroform as described above. PE from E. coli, dioleoylphosphatidylethanolamine (DOPE), 1-palmitoyl-2-oleoylphoshatidylcholine (POPC), dioleoyl phosphatidylglycerol (DOPG), phosphatidylinositol (PI) from bovine liver, and dioleoylphosphatidic acid (DOPA), dioleoylphosphatidylserine (DOPS) were purchased from Avanti Polar Lipids. After transfer to a glass tube, the chloroform solvent was removed by rotary evaporation. Samples were dried further in a desiccator under vacuum for 12 h. The resulting lipid film was suspended in a buffer (25 mm Tris-HCl, pH 7.5, 137 mm NaCl, 2.7 mm KCl) at a final concentration of 1 mm phospholipids by vortexing at room temperature. Samples were then subjected to sonication for 5 min to obtain small unilamellar liposomes. Reconstitution of Atg8p-PE Conjugation System in Vitro—In vitro reconstitution of Atg8 system was performed using purified Atg proteins and liposomes. Liposomes were mixed with purified Atg7p-Myc, Atg3p (or Atg3pC234S), and Atg8pG116 in reconstitution buffer (50 mm Tris-HCl, pH 8.0, 100 mm NaCl, 0.2 mm DTT) in the presence of an ATP regeneration system (1 mm ATP (Sigma), 1 mm MgCl2, 5 mm phosphocreatine (Sigma), and 2.5 μg of creatine kinase (Roche Applied Science), pH 7.0). The reaction mixture (with a final pH of 7.6) was incubated at 30 °C for 10–60 min. Antibodies and Immunoblotting—Polyclonal antibodies against full-length Atg3p and against the Atg8p N-terminal peptide have been previously described (7Ichimura Y. Kirisako T. Takao T. Satomi Y. Shimonishi Y. Ishihara N. Mizushima N. Tanida I. Kominami E. Ohsumi M. Noda T. Ohsumi Y. Nature. 2000; 408: 488-492Crossref PubMed Scopus (1587) Google Scholar). A polyclonal antibody against full-length Atg8p and a monoclonal antibody against Myc (9E10) were purchased from Rockland and BabCo, respectively. SDS-PAGE was performed according to Laemmli's method. SDS-PAGE separation in the presence of 6 m urea (urea-SDS-PAGE gel) was used to distinguish Atg8p and Atg8p-PE conjugates, as described (13Kirisako T. Ichimura Y. Okada H. Kabeya Y. Mizushima N. Yoshimori T. Ohsumi M. Takao T. Noda T. Ohsumi Y. J. Cell Biol. 2000; 151: 263-276Crossref PubMed Scopus (756) Google Scholar). To visualize the protein bands within the gel, SDS-PAGE gels were stained using GelCode Blue (Pierce). For immunoblot analyses, proteins were transferred to polyvinylidene difluoride membranes (Immobilon-PSQ, Millipore). Immunoblotting analysis was performed using rabbit or mouse antibodies against the specified proteins, then visualized using either peroxidase-conjugated goat anti-rabbit IgG or rabbit anti-mouse IgG. The immunoreactive protein bands were detected using Western Lightning Chemiluminescence Reagent Plus (PerkinElmer Life Sciences) Immunoprecipitation—In vitro Atg8p-PE conjugation was performed as described in the above. The Atg8p-PE-bound liposomes were collected by centrifugation at 15,000 rpm for 10 min. The harvested Atg8p-PE liposomes were mixed with the nearly equal amount of Atg8p, and treated with 2% CHAPS for 1 h on ice. The solubilized Atg8p-PE and Atg8p were suspended in IP buffer (50 mm Tris-HCl, pH 7.5. 150 mm NaCl, 2% CHAPS, and 0.01% bovine serum albumin (w/w)), and subjected to immunoprecipitation analysis according to previously reported methods with the following modifications (7Ichimura Y. Kirisako T. Takao T. Satomi Y. Shimonishi Y. Ishihara N. Mizushima N. Tanida I. Kominami E. Ohsumi M. Noda T. Ohsumi Y. Nature. 2000; 408: 488-492Crossref PubMed Scopus (1587) Google Scholar). Briefly, samples were incubated at 4 °C for 2 h in the presence or absence of the anti-N-terminal 14 peptide of Atg8p antibody. Protein G-Sepharose 4 Fast Flow beads (Amersham Biosciences) were subsequently added to the samples and further incubated at 4 °C for 1 h. Immune complexes were washed with TBS containing 2% CHAPS and suspended in SDS-PAGE sample buffer. The resulting immunoprecipitates were separated by urea-SDS-PAGE and visualized by SyproOrange staining (Amersham Biosciences). Trypsin Digestion—In vitro Atg8-PE conjugation was performed as described in the above. Then, Atg8-PE-conjugated liposomes were recovered by centrifugation at 15,000 rpm for 10 min, and solubilized in TBS containing 1 mm DTT and 1% CHAPS for 1 h on ice. The mixture of Atg8p-PE (2.0 μg) and Atg8p (1.5 μg), or each of them was treated with trypsin (0.01 μg) (Sigma T8802) in 20 μl of TBS containing 1 mm DTT and 1% CHAPS at 30 °C for the indicated time (0, 1, 3, 7, and 15 min). The digestion was stopped by boiling in SDS-PAGE sample buffer. Samples were subjected to urea-SDS-PAGE and stained by CBB. Expression of the Components of Atg8 System in E. coli— Previous studies of the Atg8 system revealed that the C-terminal glycine of Atg8p is covalently conjugated to an amino group of PE through ubiquitination-like reactions requiring Atg7p and Atg3p, functioning as E1- and E2-like enzymes, respectively (7Ichimura Y. Kirisako T. Takao T. Satomi Y. Shimonishi Y. Ishihara N. Mizushima N. Tanida I. Kominami E. Ohsumi M. Noda T. Ohsumi Y. Nature. 2000; 408: 488-492Crossref PubMed Scopus (1587) Google Scholar). We have not, however, excluded the possibility that other factor(s), such as an E3, is required for conjugation. To address this issue, we applied an E. coli expression system. For this purpose, expression plasmids encoding the components of the Atg8 system, Atg7p bearing a Myc epitope tag, Atg3p, and either the processed or unprocessed form of Atg8p, under the control of inducible arabinose promoter were constructed (Fig. 1A). E. coli BL21 (DE3) cells harboring these expression plasmids were cultured to logarithmic growth phase, then cultured for an additional 60 min in the presence of 0.2% arabinose. Total cell lysates from the E. coli were then subjected to SDS-PAGE containing 6 m urea (urea-SDS-PAGE). Subsequent immunoblot analyses with anti-Myc and anti-Atg3p antibodies detected Atg7p-Myc and Atg3p at the expected molecular masses of 78 and 36 kDa, respectively (Fig. 1B, lanes 1–3). Atg8pR117, the nascent, unprocessed form of Atg8p, was detected as a single band of ∼13.5 kDa, the predicted molecular mass; Atg8pG116, the processed form of Atg8p, migrated similarly to Atg8pR117 (Fig. 1B, lanes 1 and 2). Interestingly, only upon simultaneous expression of Atg7p-Myc, Atg3p, and Atg8pG116, an additional Atg8p band appeared (Fig. 1B, lane 3). This band, migrating faster than the Atg8pG116 form, was not observed in E. coli co-expressing the unprocessed Atg8pR117 form of Atg8p (Fig. 1B, lane 2). The faster migrating band is not an Atg8p degradation product, but a modified form, since only one band for Atg8p was observed by conventional SDS-PAGE (data not shown) (13Kirisako T. Ichimura Y. Okada H. Kabeya Y. Mizushima N. Yoshimori T. Ohsumi M. Takao T. Noda T. Ohsumi Y. J. Cell Biol. 2000; 151: 263-276Crossref PubMed Scopus (756) Google Scholar). Furthermore, the mobility of the modified Atg8p by urea-SDS-PAGE corresponded to that observed for Atg8p-PE derived from yeast cells (Fig. 1B, lanes 3 and 6). During Atg8p lipidation, the C-terminal glycine of Atg8p first links to Cys507 of Atg7p, then is transferred to Cys234 of Atg3p through generation of a thioester bond (7Ichimura Y. Kirisako T. Takao T. Satomi Y. Shimonishi Y. Ishihara N. Mizushima N. Tanida I. Kominami E. Ohsumi M. Noda T. Ohsumi Y. Nature. 2000; 408: 488-492Crossref PubMed Scopus (1587) Google Scholar). The mutant proteins Atg7pC507S-Myc and Atg3pC234S possess serine residues at the active site cysteines. Expression of the Atg7pC507S-Myc or Atg3pC234S mutants in E. coli in place of the wild-type Atg7p-Myc or Atg3p, respectively, abolished the appearance of the modified Atg8p form (Fig. 1C, lanes 5 and 6). Instead, expression of Atg3pC234S generated a novel 50-kDa band, detectable with both anti-Atg8p and anti-Atg3p antibodies (data not shown and Fig. 1C, lane 6). Appearance of the band was resistant to high concentrations of dithiothreitol (100 mm DTT) and dependent upon the Atg8p C-terminal glycine and active Atg7p (Fig. 1C, lanes 7 and 8). These results suggest that this species is an Atg8p-Atg3p ester conjugate formed between the C-terminal glycine (Gly116) of Atg8p and the Atg3pC234S serine residue (Fig. 1C, lane 6). We concluded that sequential enzymatic reactions occur in E. coli cells in a manner similar to that observed in yeast cells. Thus, these co-expression experiments indicate that the E1 and E2 enzymes, Atg7p and Atg3p, are sufficient for the modification of the processed form of Atg8p. PE Is the Target of Atg8p in E. coli—The above results prompted us to attempt the complete in vitro reconstitution of Atg8p conjugation using purified Atg proteins and liposomes. Plasmids encoding GST fusions of each of the Atg proteins were constructed using the pGEX4T-1 vector, to produce GST-Atg7-Myc, GST-Atg3, and GST-Atg8G116, then introduced into the BL21 (DE3) E. coli strain. The recombinant proteins were induced by IPTG and purified as described under “Experimental Procedures.” Purified Atg proteins exhibited high purity, as demonstrated by Coomassie Brilliant Blue (CBB) staining in SDS-PAGE (Fig. 2A, lanes 3–5). Liposomes were prepared using E. coli total phospholipids, as described under “Experimental Procedures.” After mixing the purified Atg7p-Myc, Atg3p, and Atg8pG116 with E. coli phospholipid-containing liposomes, the mixture was incubated at 30 °C in the presence of an ATP regeneration system. The reaction products were then subjected to urea-SDS-PAGE and visualized by CBB staining. The modified form of Atg8p was generated in this in vitro reaction in an ATP-dependent manner, as determined by the presence of the faster migrating, Atg8p-specific band (Fig. 2A, lanes 6 and 7). E. coli membrane phospholipids consist primarily of PE, phosphatidylglycerol (PG), cardiolipin (CL), and phosphatidic acid (PA). PE is the major glycerophospholipid (∼75% of total lipids). E. coli PE is produced exclusively from phosphatidylserine (PS) via decarboxylation. An E. coli strain, GN10, possesses a null mutant of the pssA gene that encodes PS synthase; thus, this strain completely lacks PE (22Saha S.K. Nishijima S. Matsuzaki H. Shibuya I. Matsumoto K. Biosci. Biotechnol. Biochem. 1996; 60: 111-116Crossref PubMed Scopus (44) Google Scholar). When this in vitro reaction was performed using liposomes made from the total lipids of the GN10 strain, the modified Atg8p could not be detected (Fig. 2A, lane 8). Liposomes were generated from a mixture of 30% GN10 total lipids and 70% purified E. coli PE; when these liposomes were subjected to in vitro reaction with purified Atg proteins, we observed the successful modification of Atg8p at levels similar to those seen with wild-type E. coli liposomes (Fig. 2A, lanes 6 and 9). These results clearly demonstrate that PE is the target of Atg8p modification. We also investigated Atg8p-PE formation using liposomes made from pure phospholipids. Atg8p-PE conjugation could be reproduced with the liposomes composed of 70% dioleoylphosphatidylethanolamine (DOPE) and 30% 1-palmitoyl-2-oleoylphoshatidylcholine (POPC) (Fig. 2B). Taking together, we concluded that the PE-containing liposome is sufficient for conjugation to Atg8p; biological membranes containing additional components do not appear to be necessary. Characterization of in Vitro Atg8p Lipidation—While in vitro conjugation studies of ubiquitin-like proteins, such as SUMO have been performed, conjugation reactions in the absence of E3 have always demonstrated low efficiencies. Such reactions were improved by the addition of excess amounts of E1 and E2 to SUMO (27Takahashi Y. Kahyo T. Toh E.A. Yasuda H. Kikuchi Y. J. Biol. Chem. 2001; 276: 48973-48977Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). To examine the efficiency of in vitro Atg8p-PE conjugation, we titrated the amounts of Atg8pG116 and PE against fixed amounts of Atg7p-Myc and Atg3p. The results clearly indicated that the amount of Atg8p-PE was augmented with increasing Atg8p and PE ranging from a 5-fold to a 20-fold molar excess of these targets to Atg7p-Myc and Atg3p (F

Autophagy is a catabolic process that delivers cytoplasmic material to the lysosome for degradation. The mechanisms regulating autophagosome formation and size remain unclear. Here, we show that autophagosome formation was triggered by the overexpression of a dominant-negative inactive mutant of Myotubularin-related phosphatase 3 (MTMR3). Mutant MTMR3 partially localized to autophagosomes, and PtdIns3P and two autophagy-related PtdIns3P-binding proteins, GFP-DFCP1 and GFP-WIPI-1alpha (WIPI49/Atg18), accumulated at sites of autophagosome formation. Knock-down of MTMR3 increased autophagosome formation, and overexpression of wild-type MTMR3 led to significantly smaller nascent autophagosomes and a net reduction in autophagic activity. These results indicate that autophagy initiation depends on the balance between PI 3-kinase and PI 3-phosphatase activity. Local levels of PtdIns3P at the site of autophagosome formation determine autophagy initiation and the size of the autophagosome membrane structure.

Salmonella develops into resident bacteria in epithelial cells, and the autophagic machinery (Atg) is thought to play an important role in this process. In this paper, we show that an autophagosome-like double-membrane structure surrounds the Salmonella still residing within the Salmonella-containing vacuole (SCV). This double membrane is defective in Atg9L1- and FAK family-interacting protein of 200 kDa (FIP200)-deficient cells. Atg9L1 and FIP200 are important for autophagy-specific recruitment of the phosphatidylinositol 3-kinase (PI3K) complex. However, in the absence of Atg9L1, FIP200, and the PI3K complex, LC3 and its E3-like enzyme, the Atg16L complex, are still recruited to Salmonella. We propose that the LC3 system is recruited through a mechanism that is independent of isolation membrane generation.

Autophagy is a catabolic cellular process involving dynamic membrane rearrangement. Here, we review the understanding of autophagy, focusing on the late stages of the process, from the closing of the autophagosome to fusion with the lysosome. We propose the Reverse fusion model, for the closing autophagosome. In this model, autophagosome closure proceeds in a topologically similar but reverse order to membrane fusion during the escape of influenza virus from the endosome. This dynamic process is thought to be directly catalyzed by LC3, an ubiquitin-like molecule. Further, we discuss the dynamics of the Atg16L complex in relation to the LC3 localization in these processes. Finally, the molecular mechanisms involved in the delivery of autophagosomes to the lysosome and fusion are introduced. Several key events exist in each step and seem to be coordinated to faithfully conduct the autophagic process.

The measurement of autophagic flux is critical in understanding the regulation of autophagy. The Pho8Delta60 assay employs a very sensitive enzymatic assay that provides a high signal-to-noise ratio and allows for precise quantification of autophagic flow in yeast. Pho8, alkaline phosphatase, is a resident vacuolar enzyme that is delivered to the vacuole membrane through a portion of the secretory pathway. The assay utilizes a genetically engineered version of Pho8 that lacks the N-terminal transmembrane domain that allows for translocation into the endoplasmic reticulum. Accordingly, Pho8Delta60 remains in the cytosol and is delivered to the vacuole only through autophagy. Once in the vacuole lumen, the C-terminal propeptide is proteolytically removed, which results in activation. Thus, the alkaline phosphatase activity reflects the amount of the cytosol delivered to the vacuole through nonspecific autophagy.

The endocytic and autophagic pathways are involved in the membrane trafficking of exogenous and endogenous materials to lysosomes. However, the mechanisms that regulate these pathways are largely unknown. We previously reported that Rubicon, a Beclin 1-binding protein, negatively regulates both the autophagic and endocytic pathways by unidentified mechanisms. In this study, we performed database searches to identify potential Rubicon homologues that share the common C-terminal domain, termed the RH domain. One of them, PLEKHM1, the causative gene of osteopetrosis, also suppresses endocytic transport but not autophagosome maturation. Rubicon and PLEKHM1 specifically and directly interact with Rab7 via their RH domain, and this interaction is critical for their function. Furthermore, we show that Rubicon but not PLEKHM1 uniquely regulates membrane trafficking via simultaneously binding both Rab7 and PI3-kinase.

Highly efficient coupling of photons from nanoemitters into single-mode optical fibers is demonstrated using tapered fibers. 7.4 +/- 1.2 % of the total emitted photons from single CdSe/ZnS nanocrystals were coupled into a 300-nm-diameter tapered fiber. The dependence of the coupling efficiency on the taper diameter was investigated and the coupling efficiency was found to increase exponentially with decreasing diameter. This method is very promising for nanoparticle sensing and single-photon sources.

Vps34, the sole PtdIns 3‐kinase in yeast, is essential for autophagy. Here, we show that the lipid‐kinase activity of Vps34 is required for autophagy, implying an essential role of its product PtdIns(3) P . The protein‐kinase activity of Vps15, a regulatory subunit of the PtdIns 3‐kinase complex, is also required for efficient autophagy. We monitored the distribution of PtdIns(3) P in living cells using a specific indicator, the 2xFYVE domain derived from mammalian Hrs. PtdIns(3) P was abundant at endosomes and on the vacuolar membrane during logarithmic growth phase. Under starvation conditions, we observed massive transport of PtdIns(3) P into the vacuole. This accumulation was dependent on the membrane dynamics of autophagy. Notably, PtdIns(3) P was highly enriched and delivered into the vacuole as a component of autophagosome membranes but not as a cargo enclosed within them, implying direct involvement of this phosphoinositide in autophagosome formation. We also found a possible enrichment of PtdIns(3) P on the inner autophagosomal membrane compared to the outer membrane. Based on these results we discuss the function of PtdIns(3) P in autophagy.

Autophagy is an intracellular degradation pathway conserved in eukaryotes. Among core autophagy-related (Atg) proteins, mammalian Atg9A is the sole multi-spanning transmembrane protein, and both of its N- and C-terminal domains are exposed to the cytoplasm. It is known that Atg9A travels through the trans-Golgi network (TGN) and the endosomal system under nutrient-rich conditions, and transiently localizes to the autophagosome upon autophagy induction. However, the significance of Atg9A trafficking for autophagosome formation remains elusive. Here, we identified sorting motifs in the N-terminal cytosolic stretch of Atg9A that interact with the adaptor protein AP-2. Atg9A with mutations in the sorting motifs could not execute autophagy and was abnormally accumulated at the recycling endosomes. The combination of defects in autophagy and Atg9A accumulation in the recycling endosomes was also found upon the knockdown of TRAPPC8, a specific subunit of the TRAPPIII complex. These results show directly that the trafficking of Atg9A through the recycling endosomes is an essential step for autophagosome formation.

Bone marrow stromal cells (BMSCs) have been studied as effective transplants for the treatment of spinal cord injury (SCI). Our previous study showed that BMSCs infused into the cerebrospinal fluid (CSF) exhibited distinct effects on the recovery of acute SCI. The present study examined the effects of BMSCs in sub-acute SCI (2 weeks post-injury) by transplanting them directly into the lesion. The spinal cord was crush-injured at the Th8-9 level in rats, and 2 weeks later, cultured BMSCs (5 × 105) derived from GFP-transgenic rats of the same strain were transplanted into the lesion. Tissue repair and nerve regeneration were examined by immunohistochemistry and electron microscopy. GFP-labeled BMSCs survived as cell assemblies in the spinal cord for 1-2 weeks after transplantation. The dorsal side of BMSC assemblies in the spinal cord usually showed an expanded GFAP-negative, astrocyte-devoid area, in which extracellular matrices including collagen fibrils were deposited. Numerous regenerating axons associated with Schwann cells grew out through such astrocyte-devoid extracellular matrices. Ascending (CGRP-containing) and descending (5HT- and TH-containing) axons were included in these regenerating axons. Regenerated axons were myelinated by Schwann cells beyond 2 weeks post-transplantation. Cavity formation was reduced in the cell transplantation group. Locomotory behavior assessed by the BBB scale improved to 9.8 points in the cell transplantation group, while it was to 5.5-5.7 in the control. BMSC transplantation into lesions of advanced SCI has markedly beneficial effects on tissue repair and axonal outgrowth, leading to improved locomotion in rats.

The composition of the anodic passive oxide film on iron in neutral solution has been investigated by cathodic reduction, chemical analysis and ellipsometry. The cathodic reduction using a borate solution of pH 6·35 containing arsenic trioxide as inhibitor estimates iron in the film to be all iron (III), indicating that no magnetite layer is present. Oxygen in the film is estimated from the ellipsometric thickness to be in excess of the stoichiometric ferric oxide, suggesting the presence of bound water. The average composition is represented as Fe2O3.0·4H2O, in which hydrogen may be replaced partly with iron-ion vacancy. The anodic oxide film is composed of an inner anhydrous ferric oxide layer, which thickens with the potential and an outer layer of hydrous ferric oxide whose thickness depends on the condition of passivation and environment. The anodic oxide film formed in the oxygen-potential region has also been measured by cathodic reduction, and it is found that the film retains nearly constant thickness above a critical potential where transpassive dissolution begins to occur. La composition des films passifs d'oxyde anodique engendrés sur le fer dans une solution neutre a été étudiée par réduction cathodique, analyse chimique et éllipsométrie. La réduction cathodique avec une solution au borate de pH 6,35 contenant du trioxyde d'arsenic comme inhibiteur, prouve que le fer présent dans le film est entièrement sous forme de fer (III), ce qui indique qu'il n'y a aucune couche de magnétite. A partir de l'épaisseur éllipsométrique, on estime que l'oxygène présent dans le film est en excès par rapport à l'oxyde ferrique stochiométrique, ce qui suggère la présence d'eau liée. La composition moyenne est représentée par la formule Fe2O3.0,4 H2O, dans laquelle l'hydrogène peut être substitué partiellement par une lacune ion-fer. Le film d'oxyde anodique est composé d'une couche d'oxyde ferrique anhydre interne, qui épaissit avec le potentiel et d'une couche externe d'oxyde ferrique hydraté dont l'épaisseur dépend des conditions de passivation et de l'environnement. Le film d'oxyde anodique formé dans la région du potentiel de l'oxygène a aussi été mesuré par réduction cathodique et on trouve que le film conserve une épaisseur presque constante au-dessus d'un potentiel critique où une dissolution transpassive commence à se manifester. Die Zusammensetzung anodischer, passiver Oxydfilme auf Eisen in neutralen Lösungen ist mittels kathodischer Reduktion, chemischer Analyse und Ellipsenmessung untersucht worden. Die kathodische Reduktion, welche eine borsaure Lösung von pH 6,35 mit einem Arsentrioxyd Gehalt als Inhibitor enthält, führt zu der Schätzung, dass das Eisen in dem Film völlig Eisen (III) ist und zeigt an, dass keine magnetische Schicht vorhanden ist. Sauerstoffgehalt wird in dem Film nach Ellipsenmessungstärke höher als das stöchiometrische Eisenoxyd geschätzt und lässt auf Vorhandensein von gebundenem Wasser schliessen. Die durchschnittliche Zusammensetzung wird als Fe2O3.0,4 H2O gegeben, in welcher Wasserstoff teilweise mit Eisenionen Leerstellen ersetzt werden kann. Der anodische Oxydfilm setzt sich aus einer inneren, anhydrischen Eisenoxydschicht zusammen, welche mit der Spannung dicker wird, und einer Aussenschicht von wasserhaltigem Eisenoxyd, dessen Stärk von den Umständen der Passivierung und Umgebung abhängt. Per anodische Oxydfilm, der in der Sauerstoff-Potential Region gebildet wurde, ist auch mit kathodischer Reduktion gemessen worden, und es wurde gefunden, dass der Film über ein kritisches Potential hinaus, wo transpassive Lösung beginnt, fast konstante Stärke beibehält.

This paper describes the effect of solution pH on the passive film on iron which was investigated by ellipsometry and by cathodic reduction combined with chemical analysis. Anodic two-step passivation was employed to exclude anodic deposition of ferrous ion, resulting from anodic oxidation of iron. The film in acid solution is almost anhydrous oxide, whereas the film in neutral and alkaline solution has an outer hydrated oxide layer, whose thickness decreases with decreasing pH. The hydrated outer layer depends also on the anion present in solution. The transition layer model of the film is proposed to explain the results.

Autophagy is an intracellular protein-degradation process that is conserved across eukaryotes including yeast and humans. Under nutrient starvation conditions, intracellular proteins are transported to lysosomes and vacuoles via membranous structures known as autophagosomes, and are degraded. The various steps of autophagy are regulated by the target of rapamycin complex 1 (TORC1/mTORC1). In this review, a history of this regulation and recent advances in such regulation both in yeast and mammals will be discussed. Recently, the mechanism of autophagy initiation in yeast has been deduced. The autophagy-related gene 13 (Atg13) and the unc-51 like autophagy activating kinase 1 (Ulk1) are the most crucial substrates of TORC1 in autophagy, and by its dephosphorylation, autophagosome formation is initiated. Phosphorylation/dephosphorylation of Atg13 is regulated spatially inside the cell. Another TORC1-dependent regulation lies in the expression of autophagy genes and vacuolar/lysosomal hydrolases. Several transcriptional and post-transcriptional regulations are controlled by TORC1, which affects autophagy activity in yeast and mammals.

We present a fiber-coupled diamond-based single photon system. Single nanodiamonds containing nitrogen vacancy defect centers are deposited on a tapered fiber of 273 nanometer in diameter providing a record-high number of 689,000 single photons per second from a defect center in a single-mode fiber. The system can be cooled to cryogenic temperatures and coupled evanescently to other nanophotonic structures, such as microresonators. The system is suitable for integrated quantum transmission experiments, two-photon interference, quantum-random-number generation and nano-magnetometry.

STING is essential for control of infections and for tumor immunosurveillance, but it can also drive pathological inflammation. STING resides on the endoplasmic reticulum (ER) and traffics following stimulation to the ERGIC/Golgi, where signaling occurs. Although STING ER exit is the rate-limiting step in STING signaling, the mechanism that drives this process is not understood. Here we identify STEEP as a positive regulator of STING signaling. STEEP was associated with STING and promoted trafficking from the ER. This was mediated through stimulation of phosphatidylinositol-3-phosphate (PtdIns(3)P) production and ER membrane curvature formation, thus inducing COPII-mediated ER-to-Golgi trafficking of STING. Depletion of STEEP impaired STING-driven gene expression in response to virus infection in brain tissue and in cells from patients with STING-associated diseases. Interestingly, STING gain-of-function mutants from patients interacted strongly with STEEP, leading to increased ER PtdIns(3)P levels and membrane curvature. Thus, STEEP enables STING signaling by promoting ER exit. STING ER exit is the rate-limiting step in STING signaling, but the mechanism that drives this process is not understood. Paludan and colleagues identify CxORF56, called STEEP here, as a positive regulator of STING signaling.

Mineralization is the most fundamental process in vertebrates. It is predominantly mediated by osteoblasts, which secrete mineral precursors, most likely through matrix vesicles (MVs). These vesicular structures are calcium and phosphate rich and contain organic material such as acidic proteins. However, it remains largely unknown how intracellular MVs are transported and secreted. Here, we use scanning electron-assisted dielectric microscopy and super-resolution microscopy for assessing live osteoblasts in mineralizing conditions at a nanolevel resolution. We found that the calcium-containing vesicles were multivesicular bodies containing MVs. They were transported via lysosome and secreted by exocytosis. Thus, we present proof that the lysosome transports amorphous calcium phosphate within mineralizing osteoblasts.

We have isolated 14 apg mutants defective in autophagy in yeast Saccharomyces cerevisiae (Tsukada and Ohsumi, 1993). Among them, APG1 encodes a novel Ser/Thr protein kinase whose kinase activity is essential for autophagy. In the course of searching for genes that genetically interact with APG1, we found that overexpression of APG1 under control of the GAL1 promoter suppressed the autophagy-defective phenotype of apg13-1 mutant. Cloning and sequencing analysis showed that the APG13 gene encodes a novel hydrophilic protein of 738 amino acid residues. APG13 gene is constitutively expressed bot not starvation-inducible. Though dispensable for cell proliferation, APG13 is important for maintenance of cell viability under starvation conditions. apg13 disruptants were defective in autophagy like apg13-1 mutants. Morphological and biochemical investigation showed that a defect in autophagy of Δapg13 was also suppressed by APG1 overexpression. These results imply genetic interaction between APG1 and APG13.

Synapsin I is one of the major synaptic vesicle-associated proteins. Previous experiments implicated its crucial role in synaptogenesis and transmitter release. To better define the role of synapsin I in vivo, we used gene targeting to disrupt the murine synapsin I gene. Mutant mice lacking synapsin I appeared to develop normally and did not have gross anatomical abnormalities. However, when we examined the presynaptic structure of the hippocampal CA3 field in detail, we found that the sizes of mossy fiber giant terminals were significantly smaller, the number of synaptic vesicles became reduced, and the presynaptic structures altered, although the mossy fiber long-term potentiation remained intact. These results suggest significant contribution of synapsin I to the formation and maintenance of the presynaptic structure.

Autophagy is a survival mechanism necessary for eukaryotic cells to overcome nutritionally challenged environments. When autophagy is triggered, cells degrade nonselectively engulfed cytosolic proteins and free ribosomes that are evenly distributed throughout the cytoplasm. The resulting pool of free amino acids is used to sustain processes crucial for survival. Here we characterize an autophagic degradation of the endoplasmic reticulum (ER) under starvation conditions in addition to cytosolic protein degradation. Golgi membrane protein was not engulfed by the autophagosome under the same conditions, indicating that the uptake of ER by autophagosome was the specific event. Although the ER exists in a network structure that is mutually connected and resides predominantly around the nucleus and beneath the plasma membrane, most of autophagosome engulfed ER. The extent of the ER uptake by autophagy was nearly identical to that of the soluble cytosolic proteins. This phenomenon was explained by the appearance of fragmented ER membrane structures in almost all autophagosomes. Furthermore, ER dynamism is required for this process: ER uptake by autophagosomes occurs in an actin‐dependent manner.

Protein conjugation, such as ubiquitination, is the process by which the C-terminal glycine of a small modifier protein is covalently attached to target protein(s) through sequential reactions with an activating enzyme and conjugating enzymes. Here we report on a novel protein conjugation system in yeast. A newly identified ubiquitin relatedmodifier, Urm1 is a 99-amino acid protein terminated with glycine-glycine. Urm1 is conjugated to target proteins, which requires the C-terminal glycine of Urm1. At the first step of this reaction, Urm1 forms a thioester with a novel E1-like protein, Uba4. Δ<i>urm1</i> and Δ<i>uba4</i> cells showed a temperature-sensitive growth phenotype. Urm1 and Uba4 show similarity to prokaryotic proteins essential for molybdopterin and thiamin biosynthesis, although the Urm1 system is not involved in these pathways. This is the fifth conjugation system in yeast, following ubiquitin, Smt3, Rub1, and Apg12, but it is unique in respect to relation to prokaryotic enzyme systems. This fact may provide an important clue regarding evolution of protein conjugation systems in eukaryotic cells.

Autophagy is a degradative process in which cytoplasmic components are non-selectively sequestered by double-membrane structures, termed autophagosomes, and transported to the vacuole. We have identified and characterized a novel protein Apg2p essential for autophagy in yeast. Biochemical and fluorescence microscopic analyses indicate that Apg2p functions at the step of autophagosome formation. Apg2p localizes to some membranous structure distinct from any known organelle. Using fluorescent protein-tagged Apg2p, we showed that Apg2p localizes to a dot structure close to the vacuole, where Apg8p also exists, but not on autophagosomes unlike Apg8p. This punctate localization of Apg2p depends on the function of Apg1p kinase, phosphatidylinositol 3-kinase complex and Apg9p. Apg2pG83E, encoded by anapg2-2 allele, shows a severely reduced activity of autophagy and a dispersed localization in the cytoplasm. Overexpression of the mutant Apg2p lessens the defect in autophagy. These results suggest that the dot structure is physiologically important. Apg2p and Apg8p are independently recruited to the structure but coordinately function there to form the autophagosome. Autophagy is a degradative process in which cytoplasmic components are non-selectively sequestered by double-membrane structures, termed autophagosomes, and transported to the vacuole. We have identified and characterized a novel protein Apg2p essential for autophagy in yeast. Biochemical and fluorescence microscopic analyses indicate that Apg2p functions at the step of autophagosome formation. Apg2p localizes to some membranous structure distinct from any known organelle. Using fluorescent protein-tagged Apg2p, we showed that Apg2p localizes to a dot structure close to the vacuole, where Apg8p also exists, but not on autophagosomes unlike Apg8p. This punctate localization of Apg2p depends on the function of Apg1p kinase, phosphatidylinositol 3-kinase complex and Apg9p. Apg2pG83E, encoded by anapg2-2 allele, shows a severely reduced activity of autophagy and a dispersed localization in the cytoplasm. Overexpression of the mutant Apg2p lessens the defect in autophagy. These results suggest that the dot structure is physiologically important. Apg2p and Apg8p are independently recruited to the structure but coordinately function there to form the autophagosome. cytoplasm to vacuole targeting Apg, autophagy alcohol dehydrogenase alkaline phosphatase aminopeptidase I cyan fluorescent protein N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide green fluorescent protein mature aminopeptidase I open reading frame phosphatidylinositol 3-kinase proteinase A precursor aminopeptidase I yellow fluorescent protein 1,4-piperazinediethanesulfonic acid synthetic medium synthetic minimal medium lacking nitrogen phenylmethylsulfonyl fluoride kilobase(s) polymerase chain reaction polyacrylamide gel electrophoresis low speed supernatant high speed supernatant low speed pellet high speed pellet The lysosome/vacuole is a central organelle for macromolecular turnover in eukaryotic cells, where various hydrolytic enzymes reside (1Jones E.W. Webb G.C. Hiller M.A. Pringle J.R. Broach J.R. Jones E.W. The Molecular and Cellular Biology of the Yeast Saccharomyces: Cell Cycle and Cell Biology. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1997: 363-470Google Scholar, 2Bryant N.J. Stevens T.H. Microbiol. Mol. Biol. Rev. 1998; 62: 230-247Crossref PubMed Google Scholar). These enzymes are transported to the vacuole via the secretory or the cytoplasm-to-vacuole targeting (Cvt)1 pathway. On the other hand, their substrates to be degraded are delivered from outside of the cells and the plasma membrane through the endocytic pathway or from the cytoplasm by autophagy. Macroautophagy is a cellular mechanism for bulk degradation and recycling of cytoplasmic components, which may be important for cellular remodeling during development and differentiation (3Klionsky D.J. Ohsumi Y. Annu. Rev. Cell Dev. Biol. 1999; 15: 1-32Crossref PubMed Scopus (389) Google Scholar). Genetic and morphological studies revealed an interesting fact that the biosynthetic Cvt pathway shares the overlapping mechanistic features with autophagy despite the differences in their cellular functions (4Harding T.M. Hefner-Gravink A. Thumm M. Klionsky D.J. J. Biol. Chem. 1996; 271: 17621-17624Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 5Scott S.V. Hefner-Gravink A. Morano K.A. Noda T. Ohsumi Y. Klionsky D.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12304-12308Crossref PubMed Scopus (213) Google Scholar, 6Baba M. Osumi M. Scott S.V. Klionsky D.J. Ohsumi Y. J. Cell Biol. 1997; 139: 1687-1695Crossref PubMed Scopus (277) Google Scholar, 7Scott S.V. Baba M. Ohsumi Y. Klionsky D.J. J. Cell Biol. 1997; 138: 37-44Crossref PubMed Scopus (141) Google Scholar). Macroautophagy is a dynamic process involving changes in membrane topology. During the autophagic process, cytoplasmic components including macromolecules and organelles are enwrapped by an isolation membrane to form a double membrane-bound structure, termed the autophagosome. Subsequently, its outer membrane fuses to the membrane of the lysosome/vacuole to release a single membrane vesicle into the lumen. Finally, this single membrane structure, termed the autophagic body, is degraded in a protease-dependent manner (8Takeshige K. Baba M. Tsuboi S. Noda T. Ohsumi Y. J. Cell Biol. 1992; 119: 301-311Crossref PubMed Scopus (949) Google Scholar, 9Baba M. Takeshige K. Baba N. Ohsumi Y. J. Cell Biol. 1994; 124: 903-913Crossref PubMed Scopus (402) Google Scholar, 10Baba M. Osumi M. Ohsumi Y. Cell Struct. Funct. 1995; 20: 465-471Crossref PubMed Scopus (128) Google Scholar). The process of autophagosome formation should require several events such as the supply of lipids or membranes to the site of formation and expansion of the isolation membrane. Many efforts have been made to understand the molecular mechanism of autophagy. In those processes, a lot of proteins essential for autophagy, such as Apg, Aut, and Cvt proteins, have been identified and characterized (11Kim J. Klionsky D.J. Annu. Rev. Biochem. 2000; 69: 303-342Crossref PubMed Scopus (320) Google Scholar). We have isolated the APG genes and characterized their gene products, and most of the Apg proteins are now classified into several groups by their functions. The Apg1p kinase complex is comprised of Apg1p, Apg13p, and Apg17p, and the enhancement of its kinase activity is necessary for the induction of autophagy, which is controlled by Apg13p and Apg17p (12Kamada Y. Funakoshi T. Shintani T. Nagano K. Ohsumi M. Ohsumi Y. J. Cell Biol. 2000; 150: 1507-1513Crossref PubMed Scopus (903) Google Scholar, 13Scott S.V. Nice 3rd, D.C. Nau J.J. Weisman L.S. Kamada Y. Keizer-Gunnink I. Funakoshi T. Veenhuis M. Ohsumi Y. Klionsky D.J. J. Biol. Chem. 2000; 275: 25840-25849Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). Apg6p/Vps30p and Apg14p are constituents of the autophagy-specific PI 3-kinase complex together with Vps34p and Vps15p (14Kihara A. Noda T. Ishihara N. Ohsumi Y. J. Cell Biol. 2001; 152: 519-530Crossref PubMed Scopus (800) Google Scholar). The autophagy/Cvt pathway requires two ubiquitination-like systems. The first ubiquitin-like protein, Apg12p, is conjugated to Apg5p via Apg7p (E1) and Apg10p (E2), and the conjugation facilitates the binding of Apg5p to Apg16p (15Mizushima N. Noda T. Yoshimori T. Tanaka Y. Ishii T. George M.D. Klionsky D.J. Ohsumi M. Ohsumi Y. Nature. 1998; 395: 395-398Crossref PubMed Scopus (1269) Google Scholar, 16Mizushima N. Noda T. Ohsumi Y. EMBO J. 1999; 18: 3888-3896Crossref PubMed Scopus (338) Google Scholar, 17Shintani T. Mizushima N. Ogawa Y. Matsuura A. Noda T. Ohsumi Y. EMBO J. 1999; 18: 5234-5241Crossref PubMed Scopus (234) Google Scholar, 18Tanida I. Mizushima N. Kiyooka M. Ohsumi M. Ueno T. Ohsumi Y. Kominami E. Mol. Biol. Cell. 1999; 10: 1367-1379Crossref PubMed Scopus (324) Google Scholar). Apg8p/Aut7p is the second ubiquitin-like protein of which the exposed C-terminal glycine is covalently attached to the amino group of phosphatidylethanolamine. This process is catalyzed by the Apg4p/Aut2p protease, Apg7p (E1), and Apg3p/Aut1p (E2) (19Kirisako T. Ichimura Y. Okada H. Kabeya Y. Mizushima N. Yoshimori T. Ohsumi M. Takao T. Noda T. Ohsumi Y. J. Cell Biol. 2000; 151: 263-276Crossref PubMed Scopus (731) Google Scholar, 20Ichimura Y. Kirisako T. Takao T. Satomi Y. Shimonishi Y. Ishihara N. Mizushima N. Tanida I. Kominami E. Ohsumi M. Noda T. Ohsumi Y. Nature. 2000; 408: 488-492Crossref PubMed Scopus (1489) Google Scholar). All these Apg proteins are suggested to function in autophagosome formation, and most of them are associated with certain membranes (13Scott S.V. Nice 3rd, D.C. Nau J.J. Weisman L.S. Kamada Y. Keizer-Gunnink I. Funakoshi T. Veenhuis M. Ohsumi Y. Klionsky D.J. J. Biol. Chem. 2000; 275: 25840-25849Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar,14Kihara A. Noda T. Ishihara N. Ohsumi Y. J. Cell Biol. 2001; 152: 519-530Crossref PubMed Scopus (800) Google Scholar, 21George M.D. Baba M. Scott S.V. Mizushima N. Garrison B.S. Ohsumi Y. Klionsky D.J. Mol. Biol. Cell. 2000; 11: 969-982Crossref PubMed Scopus (75) Google Scholar, 22Huang W.P. Scott S.V. Kim J. Klionsky D.J. J. Biol. Chem. 2000; 275: 5845-5851Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 24Kim J. Huang W.P. Klionsky D.J. J. Cell Biol. 2001; 152: 51-64Crossref PubMed Scopus (186) Google Scholar). It was also reported that Apg8p is a potential tracer of the autophagic process. Immuno-EM study revealed that Apg8p localizes to the autophagosomes, the autophagic bodies, and the isolation membranes near the vacuole (23Kirisako T. Baba M. Ishihara N. Miyazawa K. Ohsumi M. Yoshimori T. Noda T. Ohsumi Y. J. Cell Biol. 1999; 147: 435-446Crossref PubMed Scopus (712) Google Scholar). Fluorescence microscopic analysis showed that GFP-fused Apg8p/Aut7p localizes to the punctate structures proximal to the vacuole in addition to autophagic bodies (24Kim J. Huang W.P. Klionsky D.J. J. Cell Biol. 2001; 152: 51-64Crossref PubMed Scopus (186) Google Scholar). This punctate localization of GFP-Apg8p/Aut7p needs the Apg12p-Apg5p conjugation and Apg8p lipidation system, suggesting that the structure is physiologically important. As described above, in the past few years the molecular characterizations of Apg proteins have proceeded and allowed their classification into several functional groups. For the further understanding of the molecular mechanism of autophagy, it becomes important to elucidate how these proteins participate in autophagosome formation. However, the APG2 gene has not yet been identified and its identification is essential for an overall understanding of autophagy. In this study, we report the cloning and characterization of Apg2p. By fluorescence microscopic analysis, Apg2p co-localizes with Apg8p to the dot structure close to the vacuole. Its localization is perturbed in some apg mutants and by its own point mutation. These suggest that the structure is crucial for autophagy. The yeast strains used in this study are listed in Table I. Yeast cells are grown in YPD medium (1% yeast extract, 2% peptone, 2% glucose), synthetic complete (SC) medium containing nutritional supplements, or SCD medium (0.67% yeast nitrogen base without amino acids, 0.5% casamino acid, and 2% glucose) supplemented with 0.002% adenine sulfate, 0.002% uracil, and 0.002% tryptophan if necessary. For nitrogen starvation, SD(-N) medium (0.17% yeast nitrogen base without ammonium sulfate and amino acids and 2% glucose) was used. Standard genetic manipulations were performed as described by Adams et al. (25Adams A. Gottschling D.E. Kaiser C.A. Stearns T. Methods in Yeast Genetics, A Cold Spring Harbor Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1998Google Scholar). DNA manipulations were performed using standard methods (26Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar).Table IYeast strains used in this studyStrainsGenotypeSourceX2180–1AMATaSUC2 mal mel gal2 CUP1Yeast Genetic Stock CenterMT2X2180; apg2–1Tsukada and Ohsumi (31Tsukada M. Ohsumi Y. FEBS Lett. 1993; 333: 169-174Crossref PubMed Scopus (1390) Google Scholar)MT82X2180; apg2–2Tsukada and Ohsumi (31Tsukada M. Ohsumi Y. FEBS Lett. 1993; 333: 169-174Crossref PubMed Scopus (1390) Google Scholar)YTS19MATa/MATα ade2/ADE2 ura3/ura3 leu2/leu2 his3/his3 trp1/trp1 apg2–1/apg2–1This studyYW5–1BMATa ura3 leu2 trp1Y. WadaYTS20YW5–1B; Δapg2∷LEU2This studyYIT701YW5–1B; Δapg7∷LEU2Tanida et al. (18Tanida I. Mizushima N. Kiyooka M. Ohsumi M. Ueno T. Ohsumi Y. Kominami E. Mol. Biol. Cell. 1999; 10: 1367-1379Crossref PubMed Scopus (324) Google Scholar)YNM102YW5–1B; Δypt7∷LEU2N. MizushimaKA311AMATa ura3 leu2 his3 trp1Irie et al. (40Irie K. Takase M. Lee K.S. Levin D.E. Araki H. Matsumoto K. Oshima Y. Mol. Cell. Biol. 1993; 13: 3076-3083Crossref PubMed Scopus (259) Google Scholar)YYK100MATaura3 leu2 his3 trp1 apg2–1This studyYTS21KA311A; Δapg2∷HIS3This studyYTS27KA311A; Δapg2∷HIS3Δapg1∷LEU2This studyYTS28KA311A; Δapg2∷HIS3Δapg5∷HIS3This studyYTS29KA311A; Δapg2∷HIS3Δapg6∷LEU2This studyYTS30KA311A; Δapg2∷HIS3Δapg8∷TRP1This studyYTS31KA311A; Δapg2∷HIS3Δapg9∷TRP1This studyYTS32KA311A; Δapg2∷HIS3Δapg14∷LEU2This studyYTS33KA311A; Δapg2∷HIS3Δapg16∷LEU2This studyTN125MATa ade2 ura3 leu2 his3 trp1 lys2 PHO8∷pho8Δ60Noda et al.(30Noda T. Ohsumi Y. J. Biol. Chem. 1998; 273: 3963-3966Abstract Full Text Full Text PDF PubMed Scopus (1031) Google Scholar)YTS24TN125;PHO8∷pho8Δ60Δapg2∷LEU2This studySEY6210MATα ura3 leu2 his3 trp1 lys2 suc2-Δ9Abeliovich et al. (41Abeliovich H. Darsow T. Emr S.D. EMBO J. 1999; 18: 6005-6016Crossref PubMed Scopus (106) Google Scholar)GYS104SEY6210; Δapg2∷HIS3This studyKVY4SEY6210; Δypt7∷LEU2Kihara et al. (14Kihara A. Noda T. Ishihara N. Ohsumi Y. J. Cell Biol. 2001; 152: 519-530Crossref PubMed Scopus (800) Google Scholar)YAK2SEY6210; Δapg2∷HIS3Δypt7∷LEU2This study Open table in a new tab The APG2gene was cloned by complementing the sporulation-negative phenotype of the apg2-1 diploid strain YTS19 (apg2-1/apg2-1 ADE2/ade2 ura3/ura3) on the basis of random spore analysis as described by Adams et al. (25Adams A. Gottschling D.E. Kaiser C.A. Stearns T. Methods in Yeast Genetics, A Cold Spring Harbor Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1998Google Scholar). YTS19 cells were transformed with a YEp24-based yeast genomic library, spread and grown on SC-Ura plates at 30 °C for 3 days. About 60,000 transformants on 3 plates were collected and pooled as a frozen stock (∼1 ml). Ten microliters of the cell stock was spread on a YPD plate and incubated at 30 °C for 12 h. Then cells were collected from the plate, washed with sterile water three times, and resuspended in 2.5 ml of sporulation medium (1% potassium acetate, 0.025% glucose). After an 8-day incubation at 23 °C, sporulated culture was treated with 1 mg/ml Zymolyase 100T (Seikagaku kogyo) at 30 °C for 1 h and sonicated moderately to kill non-spore cells and disrupt asci to scatter the spores. One-hundred microliters of the spore suspension was then spread onto SC-Ura plates and incubated at 30 °C for 5 days. The red colonies derived from the ade2 ascospores were picked to exclude the surviving ADE2/ade2 diploid cells (white), and then checked their accumulation of autophagic bodies in SD(-N) containing 1 mm phenylmethylsulfonyl fluoride (PMSF). Plasmids were recovered from the positive clones and sequenced with the pBR322 oligonucleotides. Partial sequences were analyzed withSaccharomyces Genome Data base (genome-www.stanford.edu/Saccharomyces/) and the plasmids were found to contain one ORF, YNL242w. The 5.8-kb SphI-KpnI fragment containing the entire YNL242w ORF was subcloned into pUC18 to generate pTS101. The 2.0-kb XbaI-PstILEU2 fragment from pJJ282 (27Jones J.S. Prakash L. Yeast. 1990; 6: 363-366Crossref PubMed Scopus (327) Google Scholar) was ligated toXbaI-PstI digested pTS101 to generate pTS104. The 1.75-kb SmaI-HincII HIS3 fragment from pJJ215 (27Jones J.S. Prakash L. Yeast. 1990; 6: 363-366Crossref PubMed Scopus (327) Google Scholar) was ligated to the NcoI-BamHI digested and blunted pTS101 to generate pTS105. The 4.4-kbSphI-KpnI fragment from pTS105 was used for transformation of KA311A and the 4.05-kbSphI-KpnI fragment from pTS104 was for transformation of YW5-1B and TN125. The disruption of theAPG2 gene was verified by PCR. The 5.8-kbSphI-KpnI fragment containing APG2gene was subcloned into YCplac33 and YEp352 to generate pTS102 and pTS103, respectively. The DNA fragments encoding GFP-tagged Apg2 proteins were constructed as follows. The 1.4-kbSphI-EcoRV fragment from pTS101 was subcloned into pUC18 to generate pTS108. The BamHI site was introduced just after the initiation codon of the APG2 gene on pTS108 using a QuikChangeTM Site-directed Mutagenesis Kit (Stratagene) and the following primers: 5′-TTGATTTCGATACAATGGGATCCGCATTTTGGTTACCTCA-3′ and 5′-TGAGGTAACCAAAATGCGGATCCCATTGTATCGAAATCAA-3′, to generate pTS109. The DNA fragment encoding GFP (S65T) with BamHI site on both sites was then ligated to the BamHI site of pTS109 to generate pTS110. The 5.05-kb BglII-KpnI fragment from pTS101 was then ligated into the BglII-KpnI site of pTS110 to generate pTS111. Finally, pTS112 (GFP-APG2on YCplac33) was constructed by subcloning the 6.5-kbSphI-KpnI fragment of GFP-APG2 into YCplac33. pTS114 (YFP-APG2 on YCplac33) was constructed by the same procedure with GFP-APG2 using YFP fragments. For the construction of pTS119 (apg2-2 on YCplac33) and pTS120 (apg2-2 on YEp352), the 5.8-kbSphI-KpnI fragment containing theapg2-2 allele was amplified from MT82 genomic DNA by PCR and then cloned to YCplac33 and YEp352. The mutation site was verified by DNA sequencing analysis. Site-directed mutagenesis with primers 5′-CGGTGTGGAAATCGATGAGTCTGGTTTAAG-3′ and 5′-CTTAAACCAGACTCATCGATTTCCACACCG-3′ was used to change the glycine at position 83 in APG2 to a glutamate (QuikChangeTMSite-directed Mutagenesis Kit, Stratagene) to generate pTS121 (GFP-apg2–2 CEN). Antibody to Apg2p was prepared against the recombinant protein corresponding to the 824–1952 amino acid residues of Apg2p (Apg2p-C). To construct the plasmid for bacterial expression of Apg2p-C, a DNA fragment encoding Apg2p-C was amplified by PCR using following primers: 5′-AGGCAGATCTTCTTTCAAAGGCGAATACAC-3′and 5′-GTCTGCAAAAATTTTTAAGATCTCGAATCAGTCCGATTGG-3′. The resulting PCR product was digested with BglII and then ligated into theBamHI site of pET15b (Novagen, Madison, WI) to generate pTS130. Escherichia coli BL21(DE3) cells transformed with pTS130 were grown up to A 600 = 0.5 and then incubated in Luria- Bertani medium containing 0.5 mmisopropyl-1-thio-β-d-galactoside for expression of Apg2p-C. Because the recombinant Apg2p-C was obtained as an inclusion body, it was purified by washing with 1% Triton X-100 and subsequent SDS-PAGE. The gel containing Apg2p-C was stained by 0.0025% Coomassie Brilliant Blue G-250 and the protein band was excised, and eluted by soaking in Elution buffer (100 mm Tris-HCl, pH 6.8, 0.05% SDS) at 37 °C for 12 h. The concentration of the purified protein was estimated by a gel assay with bovine serum albumin as a standard. Standard procedure was used to generate antisera in female Japanese White rabbits. Antisera against aminopeptidase I (API) and Pep12p were provided by Dr. Klionsky (University of Michigan, Ann Arbor, MI). Antibodies of alkaline phosphatase (ALP), alcohol dehydrogenase (ADH), carboxypeptidase Y, proteinase A, Kex2p, and Sec12p were prepared by our laboratory (28Noda T. Matsuura A. Wada Y. Ohsumi Y. Biochem. Biophys. Res. Commun. 1995; 210: 126-132Crossref PubMed Scopus (295) Google Scholar, 29Noda T. Kim J. Huang W.P. Baba M. Tokunaga C. Ohsumi Y. Klionsky D.J. J. Cell Biol. 2000; 148: 465-480Crossref PubMed Scopus (304) Google Scholar). Yeast cells were grown in YPD and then incubated in SD(-N) for starvation conditions, and converted to spheroplasts as described previously (29Noda T. Kim J. Huang W.P. Baba M. Tokunaga C. Ohsumi Y. Klionsky D.J. J. Cell Biol. 2000; 148: 465-480Crossref PubMed Scopus (304) Google Scholar). The spheroplasts were suspended in PSM200 buffer (20 mmPIPES-KOH, pH 6.8, 0.2 m sorbitol, 5 mmMgCl2 and 1 × protease inhibitor mixture (Complete, EDTA-free; Roche Molecular Biochemicals)) with or without 0.5% Triton X-100 at a density of 40 A 600/ml and lysed by extrusion through a polycarbonate filter with 3-µm diameter pores. The filter effluent was centrifuged at 500 × g for 2 min to remove cell debris. For fractionation, small amounts of the supernatant were withdrawn as a total cell lysate (Total), and the remaining sample was then centrifuged at 5,000 × g for 5 min to generate the supernatant (S5) and the pellet (P5) fractions. For the protection assay, the P5 fraction equivalent to 32A 600 cells was suspended in 400 µl of PSM200 buffer, and divided into four aliquots (100 µl). Each aliquot was diluted 2-fold in 100 µg/ml proteinase K, 100 µg/ml proteinase K plus 0.5% Triton X-100, 0.5% Triton X-100, or distilled water. The samples were incubated for 30 min on ice, and then 200 µl of 20% trichloroacetic acid was added to terminate reactions. After centrifugation and two washes with cold acetone, the samples were dissolved in 200 µl of SDS loading buffer, and were subjected to immunoblot analysis with anti-API antibody. Yeast cells were grown in YPD or incubated in SD(-N), and then converted to spheroplasts. The spheroplasts were lysed in Lysis buffer (20 mm PIPES-KOH, pH 6.8, 0.2 m sorbitol, 50 mm sodium acetate, 1 mm EDTA, 1 mmPMSF, and 4 × protease inhibitor mixture) by extrusion through a polycarbonate filter with 3-µm diameter pores. After removing cell debris during a low-speed spin (500 × g) for 5 min, the supernatant (Total) was centrifuged at 13,000 × gfor 15 min to separate into low-speed supernatant (LSS) and pellet (LSP) fractions. The LSS fraction was then centrifuged at 100,000 × g for 1 h to obtain high-speed supernatant (HSS) and pellet (HSP) fractions. The total membrane fraction was prepared by centrifugation of cell lysate at 100,000 × g for 30 min. This fraction (1 ml) was layered onto an Optiprep (NYCOMED PHARMA AS, Oslo, Norway) step gradient in Lysis buffer (0.5 ml of 50%, 1 ml of 40%, 1 ml of 30%, 1.5 ml of 25%, 2 ml of 20%, 2 ml of 15%, and 1.5 ml of 10% w/v Optiprep) and centrifuged at 174,000 × g for 16 h at 4 °C in a PS40T rotor (Hitachi). Fourteen fractions were collected from the top of gradient and examined by immunoblot analysis. The total membrane fraction was suspended in lysis buffer and treated with 2% Triton X-100 (in Lysis buffer), 1 m NaCl (in Lysis buffer), 0.1 m Na2CO3 (pH 11.5 in distilled water), 6 m urea (in Lysis buffer), or control Lysis buffer for 20 min on ice. The samples were centrifuged at 100,000 × g for 30 min to separate into the supernatant and pellet fractions. The fractions were probed by immunoblot analysis with anti-Apg2p, anti-ALP, and anti-Kex2p antibodies. Fluorescence microscopic analysis was performed with a DeltaVision microscope system (Applied Precision, Issaquah, WA). The cells expressing the fluorescent protein-fused Apg2p or Apg8p were grown to midlog phase in SCD medium, and then observed as growing cells. For induction of autophagy, cells were further treated with 0.2 µg/ml rapamycin in SCD medium at 30 °C. For FM4-64 (Molecular Probes, Eugene, OR) staining, cells were labeled in SCD medium containing 0.5 µg/ml FM4-64 at 30 °C for 15 min. After washing with medium, cells were incubated in the medium at 30 °C for 30 min and subjected to microscopic analysis. For measurement of autophagic activity, the ALP assay was performed as described previously (30Noda T. Ohsumi Y. J. Biol. Chem. 1998; 273: 3963-3966Abstract Full Text Full Text PDF PubMed Scopus (1031) Google Scholar). Immunoblot analysis was performed as described previously (17Shintani T. Mizushima N. Ogawa Y. Matsuura A. Noda T. Ohsumi Y. EMBO J. 1999; 18: 5234-5241Crossref PubMed Scopus (234) Google Scholar). We isolated and characterized anapg2-1 mutant (31Tsukada M. Ohsumi Y. FEBS Lett. 1993; 333: 169-174Crossref PubMed Scopus (1390) Google Scholar). Wild-type cells accumulated autophagic bodies in their vacuoles in a nitrogen-starvation medium containing 1 mm PMSF, while not in the apg2-1 mutant cells (Fig. 1 A). The mutation severely caused loss of viability under starvation conditions (Fig.1 C). Precursor aminopeptidase I (proAPI) is transported to the vacuole by the Cvt pathway and processed to a mature form in aPEP4-dependent manner (7Scott S.V. Baba M. Ohsumi Y. Klionsky D.J. J. Cell Biol. 1997; 138: 37-44Crossref PubMed Scopus (141) Google Scholar). The apg2-1mutant strain also showed the defect in API maturation in both rich and starvation media, whereas the maturation of other vacuolar enzymes, carboxypeptidase Y and proteinase A, was normal (Fig. 1 B), indicating that Apg2p functions in the Cvt pathway but not in the Vps pathway. All of apg mutants show a defect in sporulation (31Tsukada M. Ohsumi Y. FEBS Lett. 1993; 333: 169-174Crossref PubMed Scopus (1390) Google Scholar). We used this phenotype for cloning of the APG2 gene. YEp24-based yeast genomic library was introduced into theapg2-1/apg2-1 diploid strain (ADE2/ade2) and the transformants were subjected to sporulation conditions. After a procedure that kills non-spore cells, the spores were grown on SC-Ura plates. The ade2 ascospores show red pigment, so that they can be distinguished from the white colonies including theADE2/ade2 or ADE2 cells. Therefore, the red colonies were picked to exclude the surviving diploid cells from the screening. More than 500 red colonies were obtained and 16 clones of them were checked for restoration of autophagy. Twelve clones accumulated autophagic bodies in their vacuoles in SD(-N) containing 1 mm PMSF. Finally, two kinds of plasmids were yielded from those clones, containing 8.6- and 8.5-kb genomic fragments, respectively. Sequencing and data base analyses revealed that both DNA fragments contained only one entire ORF, YNL242w. It was reported that a Δynl242w/Δynl242w homozygous diploid cell was unable to sporulate (32Saiz J.E. Santos M.A. Vazquez de Aldana C.R. Revuelta J.L. Yeast. 1999; 15: 155-164Crossref PubMed Scopus (7) Google Scholar), corresponding with our observation for the apg2-1 mutant. The 5.8-kbSphI-KpnI fragment containing the entire ORF was subcloned to YCplac33 and introduced into the apg2-1 cell. A single copy of YNL242w was sufficient to complement the mutation (Fig.1). Next, to determine whether YNL242w was the authentic APG2gene, this ORF was disrupted by replacing with a LEU2 orHIS3 gene. The disruptants exhibited the same phenotypes as the apg2-1 mutant for autophagy, activation of proAPI, and viability under starvation conditions (Fig. 1). A diploid cell obtained by crossing apg2-1 and Δynl242wcells was also defective in autophagy (data not shown). We, therefore, concluded that YNL242w is the authentic APG2 gene. APG2 is a novel gene and encodes a hydrophilic protein of 1,592 amino acids with a predicted molecular mass of 178 kDa. The amino acid sequence of Apg2p provided no insight into its function. A BLAST search identified proteins closely related to Apg2p in human (KIAA0404), Drosophila melanogaster (CG1241),Caenorhabditis elegans (M03A8.2), andSchizosaccharomyces pombe (SPBC31E1.01c). Their functions are not characterized yet, but are expected to be involved in autophagy, as is the case with Apg8p and Apg12p (33Kabeya Y. Mizushima N. Ueno T. Yamamoto A. Kirisako T. Noda T. Kominami E. Ohsumi Y. Yoshimori T. EMBO J. 2000; 19: 5720-5728Crossref PubMed Scopus (5395) Google Scholar, 34Mizushima N. Yamamoto A. Hatano M. Kobayashi Y. Kabeya Y. Suzuki K. Tokuhisa T. Ohsumi Y. Yoshimori T. J. Cell Biol. 2001; 152: 657-667Crossref PubMed Scopus (1153) Google Scholar). We generated polyclonal antibody against the C-terminal half of Apg2 protein produced bacterially. As shown in Fig.2 A, the affinity-purified anti-Apg2p antibody specifically recognized a 180-kDa protein in crude yeast extracts, which corresponds well to the predicted molecular mass of Apg2p. This band was not detected in the strain deleted forAPG2, and therefore represents Apg2p. The expression level of Apg2p was unaffected by starvation or treatment with the immunosuppressant drug rapamycin, a specific inhibitor of Tor, which mimics starvation and induces autophagy (data not shown), indicating that Apg2p was constitutively expressed in both growing and starvation conditions. Using this antibody for immunoblot analysis, we surveyed the expression of Apg2 protein in the original 13 apg2 mutants. Among them, only one strain, apg2-2 mutant, expressed Apg2p with full size. Sequence analysis revealed that apg2-2 contains a single amino acid change from glycine to glutamate at the 83rd amino acid of Apg2p. We cloned the mutant APG2 gene,apg2-2, and introduced it into theΔapg2 strain. The Δapg2strain showed a complete defect in API transport under both growing and starvation conditions. Single copy apg2-2 did not complement the defect under growing conditions, however, under starvation conditions proAPI was partially matured (Fig. 2 C, column 3). This partial maturation was much enhanced when apg2-2 was expressed via a multicopy plasmid (Fig. 2, column 5). Thus, the mutation does not cause complete loss of Apg2p function. Autophagic activities can be measured by monitoring autophagy-dependent processing of the cytosolic form o

In this study, we propose a process framework for understanding the evolution of intraindustry firm heterogeneity, drawing on a longitudinal study of the development of the cellular telephone servi...

An improved method for the detection of nitric oxide radicals (NO. in cultures of activated macrophages was developed, involving a nitric oxide radical scavenger, 2-(4-carboxyphenyl)-4,4,5,5- tetramethylimidazoline-3-oxide-1-oxyl (carboxy PTIO) and Griess reagent. A murine macrophage-like cell line, J774.1, was activated with interferon-gamma (IFN-gamma) and bacterial lipopolysaccharide (LPS), which induced the production and secretion of NO2- into the culture supernatant. Addition of carboxy PTIO to the activated macrophages increased the amount of NO2- to 4- to 5-fold without cell damages, probably because carboxy PTIO rapidly reacted with NO. to form NO2-, which was finally assayed by the Griess reaction.

T. Noda, H. Ogawa, N. Noma and Y. Shirota, J. Mater. Chem., 1999, 9, 2177 DOI: 10.1039/A902800E

A single nucleotide polymorphism in Atg16L1, an autophagy-related gene (ATG), is a risk factor for Crohn disease, a major form of chronic inflammatory bowel disease. However, it is still unknown how the Atg16L1 variant contributes to disease development. The Atg16L1 protein possesses a C-terminal WD repeat domain whose function is entirely unknown, and the Crohn disease-associated mutation (T300A) is within this domain. To elucidate the function of the WD repeat domain, we established an experimental system in which a WD repeat domain mutant of Atg16L1 is stably expressed in Atg16L1-deficient mouse embryonic fibroblasts. Using the system, we show that the Atg16L1 complex forms a dimeric complex and that the total Atg16L1 protein level is strictly maintained, possibly by the ubiquitin proteasome system. Furthermore, we show that an Atg16L1 WD repeat domain deletion and the T300A mutant have little impact on canonical autophagy and autophagy against Salmonella enterica serovar Typhimurium. Therefore, we propose that Atg16L1 T300A is differentially involved in Crohn disease and canonical autophagy. A single nucleotide polymorphism in Atg16L1, an autophagy-related gene (ATG), is a risk factor for Crohn disease, a major form of chronic inflammatory bowel disease. However, it is still unknown how the Atg16L1 variant contributes to disease development. The Atg16L1 protein possesses a C-terminal WD repeat domain whose function is entirely unknown, and the Crohn disease-associated mutation (T300A) is within this domain. To elucidate the function of the WD repeat domain, we established an experimental system in which a WD repeat domain mutant of Atg16L1 is stably expressed in Atg16L1-deficient mouse embryonic fibroblasts. Using the system, we show that the Atg16L1 complex forms a dimeric complex and that the total Atg16L1 protein level is strictly maintained, possibly by the ubiquitin proteasome system. Furthermore, we show that an Atg16L1 WD repeat domain deletion and the T300A mutant have little impact on canonical autophagy and autophagy against Salmonella enterica serovar Typhimurium. Therefore, we propose that Atg16L1 T300A is differentially involved in Crohn disease and canonical autophagy.

Autophagy is a unique form of membrane trafficking, which delivers macromolecules and organelles from the cytoplasm to lysosomes for degradation. This fundamental and ubiquitous process in eukaryotic cells is mediated by the double-membrane-bound structures called autophagosomes, which transiently emerge in the cytoplasm. The recent remarkable explosion of knowledge of autophagy has revealed its multiple roles, especially in mammals; in addition to its basic role in turnover and reuse of cellular constituents, the process unexpectedly functions in elimination of invading bacteria and antigen presentation. Analysis of mammalian homologs of the autophagy-related (Atg) proteins identified in yeast has shed light on not only the common molecular mechanisms underlying autophagosome formation, but also specialized mechanisms that are related to the diverse functions and complex regulation of autophagy in higher organisms.

Group A streptococcus (GAS; Streptococcus pyogenes) is a common pathogen that invades non-phagocytic human cells via endocytosis. Once taken up by cells, it escapes from the endocytic pathway to the cytoplasm, but here it is contained within a membrane-bound structure termed GAS-containing autophagosome-like vacuoles (GcAVs). The autophagosome marker GFP-LC3 associates with GcAVs, and other components of the autophagosomal pathway are involved in GcAV formation. However, the mechanistic relationship between GcAV and canonical autophagy is largely unknown. Here, we morphologically analyzed GcAV formation in detail. Initially, a small, GFP-LC3-positive GcAV sequesters each streptococcal chain, and these then coalesce into a single, large GcAV. Expression of a dominant-negative form of Rab7 or RNAi-mediated knockdown of Rab7 prevented the initial formation of small GcAV structures. Our results demonstrate that mechanisms of GcAV formation includes not only the common machinery of autophagy, but also Rab7 as an additional component, which is dispensable in canonical autophagosome formation.

In autophagy, cytoplasmic substrates are targeted for degradation in the lysosome via membrane structures called autophagosomes. The formation of the autophagosome is the primary regulatory point for autophagy activity, and PI3P plays a central role in this process. In this review, we will discuss the role of PI3P in autophagosome formation from three different perspectives: PI3-kinase, PI3-binding proteins, and PI3-phosphatase. Recent developments in this field suggest that the local PI3P concentration is dynamically regulated during autophagy, and that this molecule is critical to the proper control of autophagy.

Ellipsometric measurements were made during galvanostatic reduction of the passivefilms formed on Fe in a sodium borate buffer solution. Galvanostatic reduction in optical thickness of the film takes place in two successive stages. In the first stage solid-state reduction of Fe2O3 to lower oxide as well as reductive dissolution to Fe2+ occurs in the film, and this lower oxide transformed from the original oxide film is further reduced to metallic Fe in the second stage. Then, it appears that the passive film is a single layer of Fe2O3 rather than a bi-layer of Fe3O4, and Fe2O3. The composition of the film estimated from the results of ellipsometry, coulometry and chemical analysis of Fe2+ reductively dissolved from the film is represented as Fe2O3. O·39H2O. On a réalisé des mesures ellipsométriques lors de la réduction intensiostatique d'une pellicule passive formée sur Fe en solution tampon de borate de Na. La réduction intensiostatique de l'épaisseur optique de la pellicule a lieu en deux temps: il se produit d'abord au sein de la pellicule une réduction de Fe2O3 solide en oxyde inférieur ainsi que sa dissolution réductrice en Fe2+; ensuite l'oxyde inférieur est réduit jusqu'à l'état métallique. Il apparaît ainsi que la pellicule passive estune mono-couche d'oxyde ferrique plutôt qu'une bi-couche de magnétite et d'oxyde ferrique. La composition de cette pellicule, déduite des résultats d'analyses coulométriques, ellipsométriques et chimiques de Fe2+ provenant de sa dissolution réductrice, est représentée par Fe23. −0.39 H2O. Ellipsometrische Messungen wurden während der galvanostatischen Reduktionvon Passivschichten durchgeführt die auf Fe in Natriumborat-Lösungen erzeugt worden waren. Die galvanostatische Reduktion der Schichten findet in zwei aufeinander folgenden Stufen der Abnahme der optisch meßbaren Dicke statt. In der ersten Stufe tritt sowohl Reduktion von Fe2O3 zu niederen Oxiden als auch reduktive Auflösung unter Bildung von Fe2+ auf. In der zweiten Stufe wird das dabei gebildete niedere Eisenoxid zu metallischem Eisen reduziert. Es hat den Anschein daß die Passivschicht einheitlich aus Fe2O3 und nicht als Doppelschicht aus Fe3O4 + Fe2O3 aufgebaut ist. Die Zusammensetzung der Schicht ergibt sich aus dem ellipsometrischen coulorimetrischen und analytischen Ergebnissen zu Fe2O3. O,39 H2O.

Autophagy is a bulk protein-degradation process that is regulated by many factors. In this study, we quantitatively assessed the contribution of each essential yeast gene to autophagy. Of the contributing factors that we identified, we focused on the TRAPPIII complex, which was recently shown to act as a guanine-nucleotide exchange factor for the Rab small GTPase Ypt1. Autophagy is defective in the TRAPPIII mutant under nutrient-rich conditions (Cvt pathway), but starvation-induced autophagy is only partially affected. Here, we show that TRAPPIII functions at the Golgi complex to receive general retrograde vesicle traffic from early endosomes. Cargo proteins in this TRAPPIII-dependent pathway include Atg9, a transmembrane protein that is essential for autophagy, and Snc1, a SNARE unrelated to autophagy. When cells were starved, further disruption of vesicle movement from late endosomes to the Golgi caused defects in Atg9 trafficking and autophagy. Thus, TRAPPIII-dependent sorting pathways provide Atg9 reservoirs for pre-autophagosomal structure and phagophore assembly sites under nutrient-rich conditions, whereas the late endosome-to-Golgi pathway is added to these reservoirs when nutrients are limited. This clarification of the role of TRAPPIII elucidates how general membrane traffic contributes to autophagy.

In response to amino acid supply, mTORC1, a master regulator of cell growth, is recruited to the lysosome and activated by the small GTPase Rheb. However, the intracellular localization of Rheb is controversial. In this study, we showed that a significant portion of Rheb is localized on the Golgi but not on the lysosome. GFP-Rheb could activate mTORC1, even when forced to exclusively localize to the Golgi. Likewise, artificial recruitment of mTORC1 to the Golgi allowed its activation. Accordingly, the Golgi was in contact with the lysosome at an newly discovered area of the cell that we term the Golgi-lysosome contact site (GLCS). The number of GLCSs increased in response to amino acid supply, whereas GLCS perturbation suppressed mTORC1 activation. These results suggest that inter-organelle communication between the Golgi and lysosome is important for mTORC1 regulation and the Golgi-localized Rheb may activate mTORC1 at GLCSs.

Macroautophagy is an intracellular degradation system that involves the de novo formation of membrane structures called autophagosomes, although the detailed process by which membrane lipids are supplied during autophagosome formation is yet to be elucidated. Macroautophagy is thought to be associated with canonical membrane trafficking, but several mechanistic details are still missing. In this review, the current understanding and potential mechanisms by which membrane trafficking participates in macroautophagy are described, with a focus on the enigma of the membrane protein Atg9, for which the proximal mechanisms determining its movement are disputable, despite its key role in autophagosome formation.

Autophagy is an intracellular degradation process that delivers cytosolic material to lysosomes and vacuoles. To investigate the mechanisms that regulate autophagy, we performed a genome-wide screen using a yeast deletion-mutant collection, and found that Npr2 and Npr3 mutants were defective in autophagy. Their mammalian homologs, NPRL2 and NPRL3, were also involved in regulation of autophagy. Npr2-Npr3 function upstream of Gtr1-Gtr2, homologs of the mammalian RRAG GTPase complex, which is crucial for TORC1 regulation. Both npr2∆ mutants and a GTP-bound Gtr1 mutant suppressed autophagy and increased Tor1 vacuole localization. Furthermore, Gtr2 binds to the TORC1 subunit Kog1. A GDP-bound Gtr1 mutant induced autophagy even under nutrient-rich conditions, and this effect was dependent on the direct binding of Gtr2 to Kog1. These results revealed that 2 molecular mechanisms, Npr2-Npr3-dependent GTP hydrolysis of Gtr1 and direct binding of Gtr2 to Kog1, are involved in TORC1 inactivation and autophagic induction.

TORC1 regulates cellular growth, metabolism, and autophagy by integrating various signals, including nutrient availability, through the small GTPases RagA/B/C/D in mammals and Gtr1/2 in budding yeast. Rag/Gtr is anchored to the lysosomal/vacuolar membrane by the scaffold protein complex Ragulator/Ego. Here we show that Ego consists of Ego1 and Ego3, and novel subunit Ego2. The ∆ego2 mutant exhibited only partial defects both in Gtr1-dependent TORC1 activation and Gtr1 localization on the vacuole. Ego1/2/3, Gtr1/2, and Tor1/Tco89 were colocalized on the vacuole and associated puncta. When Gtr1 was in its GTP-bound form and TORC1 was active, these proteins were preferentially localized on the vacuolar membrane, whereas when Gtr1 was in its GDP-bound form, they were mostly localized on the puncta. The localization of TORC1 to puncta was further facilitated by direct binding to Gtr2, which is involved in suppression of TORC1 activity. Thus regulation of TORC1 activity through Gtr1/Gtr2 is tightly coupled to the dynamic relocation of these proteins.

The cerebral cortex is organized in vertical columns that contain neurons with similar functions. The cellular micro-architecture of such columns is an essential determinant of brain dynamics and cortical information processing. However, a detailed understanding of columns is incomplete, even in the best studied cortical regions, and mostly restricted to the upper cortical layers. Here, we developed a two-photon Ca2+-imaging-based method for the serial functional mapping of all pyramidal layers of the mouse primary auditory cortex at single-neuron resolution in individual animals. We demonstrate that the best frequency-responsive neurons are organized in all-layers-crossing narrow columns, with fuzzy boundaries and a bandwidth of about one octave. This micro-architecture is, in many ways, different from what has been reported before, indicating the region and stimulus specificity of functional cortical columns in vivo.

The color of light emitted by organic electroluminescent devices can be tuned between light blue and orange in the family of materials presented here simply by varying the conjugation length ( n = 1 to 4, see Figure) of the oligothiophene moiety. The synthesis of the amorphous molecular materials and the optical behavior of single‐layer electroluminescent devices based on them—see also the cover—are described. magnified image

Autophagy is a starvation response in eukaryotes by which the cell delivers cytoplasmic components to the vacuole for degradation, and is mediated by a double membrane structure called the autophagosome. We have previously proposed that the specific combination of COPII like components, including Sec24p, is required for autophagy (Ishihara, N. et al. (2001) Mol. Biol. Cell, 12: 3690-3702). The autophagic defect in sec24 deleted mutant cells was, however, suppressed upon the recovery of its secretory flow by the overexpression of its homologue, Sfb2p. We have also reported that the autophagic defect is not observed in sec13 and sec31 mutants, a phenomenon that can be explained by the fact that starvation stress suppresses the secretory defect of these mutants. These observations indicate that the active flow in the early secretory pathway plays an important role in autophagy; that is, autophagy proceeds in the presence, but not in the absence of the early secretory flow. Both autophagy and its closely related cytoplasm to vacuole-targeting (Cvt) pathway occur through a pre-autophagosomal structure (PAS), and since the PAS and the functional Cvt pathway exist in all sec mutants, the early secretory pathway must be involved specifically in autophagy, subsequent to PAS formation.

The association behavior of random copolymers of sodium 2-(acrylamido)-2-methylpropanesulfonate (AMPS) and dodecyl methacrylate (DMA) with its content (fDMA) varying from 1 to 15 mol % was investigated by fluorescence and quasielastic light scattering (QELS) techniques in 0.1 M NaCl aqueous solutions. The association behavior of the copolymers was found to depend strongly on fDMA. When fDMA < 3 mol %, some polymer chains exist as unimers with a hydrodynamic radius (Rh) of about 5 nm while some polymers form multipolymer aggregates with Rh ≈ 100 nm, arising from "hydrophobic cross-linking" by the interpolymer association of dodecyl groups. When fDMA is increased to 9 mol %, interpolymer hydrophobe associations lead to the formation of a multipolymer micelle with Rh ≈ 10−13 nm. The aggregation number (Nagg) of dodecyl groups in a micelle core was estimated to be about 195. When fDMA is further increased to 15 mol %, Rh for the multipolymer micelle markedly increases to 70−180 nm, the size increasing with increasing polymer concentration, whereas Nagg remains constant at about 158 independent of the polymer concentration. On the basis of the results from the fluorescence and QELS measurements, a unicore micelle model was proposed for the copolymer with fDMA = 9 mol %, and a bridged micelle model was proposed for the copolymer with fDMA = 15 mol %.

Abstract BACKGROUND The adrenal steroids dehydroepiandrosterone and androstenediones are converted into active androgen testosterone in prostatic tissues. Different 17β‐hydroxysteroid dehydrogenase (17βHSD) isozymes are characterized by either oxidation or reduction reactions. These redox reactions represent an important step in both biosynthesis and metabolism of androgens. This study presents the differential expression of 17βHSD isozyme genes in cancerous and noncancerous prostate tissues of in vivo samples. METHODS Thirty‐four fresh specimens of transrectal prostatic needle biopsy were obtained; 11 were pathologically diagnosed as adenocarcinoma and 23 as without malignancy. The gene expression levels of five isozymes (type 1–5) of 17βHSD were evaluated. The quantification of gene expression was assessed by means of the real‐time polymerase chain reaction. RESULTS The expression levels of the type 3 17βHSD gene with malignancy were significantly higher than those in prostatic tissues without malignancy, and those of type 2 17βHSD with malignancy were significantly lower than those in nonmalignant tissues. There were no significant differences in 17βHSD type 1, type 4, and type 5 gene expression in cancerous and noncancerous tissues. CONCLUSION Our results suggest that 17βHSD type 2 and type 3 play an important role in the conversion of adrenal steroids into potential androgens in prostate cancer tissue. Prostate 53: 154–159, 2002. © 2002 Wiley‐Liss, Inc.

An IR absorption measurement of SiO2/Si nanowires made by thermal evaporation was conducted. In comparison with SiO2 nanoparticles, enhancement absorption of SiO2/Si nanowires around 1130 cm−1 was observed. This enhancement was considered to result from: (1) the interface effect of the open structure of chainlike SiO2/Si nanowires; (2) the vibration of an interstitial oxygen atom in a silicon single-crystalline core of nanowire; and. The longitudinal optical (LO) modes of Si–O–Si stretching in an amorphous SiO2 outer shell of SiO2/Si nanowires were also discussed.

A combined experimental/numerical methodology is developed to fully consolidate pure ultrafine WC powder under a current-control mode.Three applied currents, 1900, 2100 and 2700 A, and a constant pressure of 20 MPa were employed as process conditions.The developed spark plasma sintering (SPS) finite-element model includes a moving-mesh technique to account for the contact resistance change due to sintering shrinkage and punch sliding.The effects of the heating rate on the microstructure and hardness were investigated in detail along the sample radius from both experimental and modeling points of view.The maximum hardness (2700 HV10) was achieved for a current of 1900 A at the core sample, while the maximum densification was achieved for 2100 and 2700 A. A direct relationship between the compact microstructure and both the sintering temperature and the heating rate was established.

The origin and source of autophagosomal membranes are long-standing questions. By electron microscopy, we show that the endoplasmic reticulum (ER) associates with early autophagic structures called isolation membranes (IM) or phagophores in mammalian culture cells. Overexpression of a mutant of Atg4B, which causes defects in autophagosome formation, caused accumulation of ER-IM complexes. Electron tomography revealed the ER-IM complex as a subdomain of the ER forming a cradle encircling the IM, and showed that both ER and isolation membranes are interconnected.

Analysis of permeation rates of D2 gas at 10−4 to 10−1 Pa through 304 and 304L stainless steel membranes (51 μm and 127 μm thick) at 650 to 1050 K revealed that the chemisorption rate constant, κ1, permeability and diffusion coefficient were 5.0 × 1015 + 2.2 × 1026exp(−2.1 × 104T−1K) moleculesD2 · cm−2 · sec−1. Pa−1, 1.4 × 1016 exp(−7700T−1K) molecules D2 · cm−2 · sec−1 · Pa−12, and 3.5 × 10−4exp-(−5100T−1K) cm2s−1, respectively. The value of κ1 indicates two chemisorption sites (one unactivated). The scaling laws and the transition between the high and low pressure asymptotes were clarified by developing a dimensionless form of permeation equation applicable for low D2 activities. Following gas permeation experiments, permeation rates resulting from bombardment with 10 μA, 15 keV deuteron beams were measured. A high transient permeation rate was observed which was attributed to a surface impedance to reemission which decreased under bombardment. L'analyse des vitesses de permeation du gaz D2 de 10−4 à 10−1 Pa à travers des membranes d'aciers inoxydables 304 et 304 L de 51 μm et 127 μm à 650 et 1050 K a révélé que la constante de vitesse de chimisorption κ1, la perméabilité et le coefficient de diffusion étaient respectivement 5 × 1015 + 2,2 × 1026 exp.(2,1 × 104K−1K) molécules D2 · cm−1 · sec−1 · Pa−1, 1,4 × 1016 exp(−7.700−1K) molécules D2 · cm−2 · sec−1 · Pa−12, 3,5 × 10−4exp(−5100−1K) cm2 · sec−1. La valeur de κ1 indique l'existence de deux sites de chimisorption, dont l'un n'est pas activé. Les lois de proportionalité et la transition entre les asymptotes à haute et faible pressions ont été clarifiées en développant une forme sans dimension d'une équation de permeation applicable aux faibles activités de D2. En suivant des expériences de perméation de gaz, les vitesses de perméation résultant d'un bombardement avec des faisceaux de deutérons de 10 μA sous 15 keV ont été mesurées. Une vitesse de perméation transitoire élevée a été observée, celle-ci étant attribuée à une impédance superficielle à la réémission qui diminuait sous bombardement. Die Analyse der Durchlässigkeitsgeschwindigkeit von Deuteriumgas durch Membrane aus den rostfreien Stählen 304 und 304L (51 und 127 μm dick) zwischen 10−4 und 10−1 Pa sowie zwischen 650 und 1050 K ergibt eine Geschwindigkeitskonstante für die Chemisorption κ1 = 5,0 · 1015 + 2,2 · 1026exp(−2,1 · 104/T)D2-Moleküle/cm2 · s · Pa, einen Durchlässigkeits-koeffizienten 1,4 · 1016 exp(−7700/T) D2-Moleküle/cm2 · s · Pa12 und einen Diffusionskoeffizienten 3,5 · 10−4 exp(−5100/T) cm2/s, T in K. Der Wert von κ1 weist auf zwei Chemisorptionsplätze hin (einen unaktivierten). Die Geschwindigkeitsgesetze und der Übergang der Asymptoten von hohem zu niedrigem Druck werden aufgeklärt durch Aufstellung einer dimensionslosen Durchlässigkeitsgleichung, die bei niedrigen D2-Aktivitäten anwendbar ist. Nach den Experimenten zur Gasdurchlässigkeit wurde die Durchlässigkeitsgeschwindigkeit gemessen, die sich durch Bestrahlung mit 15 keV-Deuteronenstrahlen bei 10 μA ergibt. Es wurde ein hoher Transient der Durchlässigkeitsgeschwindigkeit beobachtet, die auf einem Widerstand an der Oberfläche durch Reemission beruht, welche während der Bestrahlung abnimmt.

ABSTRACT Hepatitis C virus (HCV) is a major cause of chronic liver diseases. A high risk of chronicity is the major concern of HCV infection, since chronic HCV infection often leads to liver cirrhosis and hepatocellular carcinoma. Infection with the HCV genotype 1 in particular is considered a clinical risk factor for the development of hepatocellular carcinoma, although the molecular mechanisms of the pathogenesis are largely unknown. Autophagy is involved in the degradation of cellular organelles and the elimination of invasive microorganisms. In addition, disruption of autophagy often leads to several protein deposition diseases. Although recent reports suggest that HCV exploits the autophagy pathway for viral propagation, the biological significance of the autophagy to the life cycle of HCV is still uncertain. Here, we show that replication of HCV RNA induces autophagy to inhibit cell death. Cells harboring an HCV replicon RNA of genotype 1b strain Con1 but not of genotype 2a strain JFH1 exhibited an incomplete acidification of the autolysosome due to a lysosomal defect, leading to the enhanced secretion of immature cathepsin B. The suppression of autophagy in the Con1 HCV replicon cells induced severe cytoplasmic vacuolation and cell death. These results suggest that HCV harnesses autophagy to circumvent the harmful vacuole formation and to maintain a persistent infection. These findings reveal a unique survival strategy of HCV and provide new insights into the genotype-specific pathogenicity of HCV.

Since its discovery nearly four decades ago, sequential microelectrode mapping using hundreds of recording sites has been able to reveal a precise tonotopic organization of the auditory cortex. Despite concerns regarding the effects that anesthesia might have on neuronal responses to tones, anesthesia was essential for these experiments because such dense mapping was elaborate and time-consuming. Here, taking an 'all-at-once' approach, we investigated how isoflurane modifies spatiotemporal activities by using a dense microelectrode array. The array covered the entire auditory cortex in rats, including the core and belt cortices. By comparing neuronal activity in the awake state with activity under isoflurane anesthesia, we made four observations. First, isoflurane anesthesia did not modify the tonotopic topography within the auditory cortex. Second, in terms of general response properties, isoflurane anesthesia decreased the number of active single units and increased their response onset latency. Third, in terms of tuning properties, isoflurane anesthesia shifted the response threshold without changing the shape of the frequency response area and decreased the response quality. Fourth, in terms of population activities, isoflurane anesthesia increased the noise correlations in discharges and phase synchrony in local field potential (LFP) oscillations, suggesting that the anesthesia made neuronal activities redundant at both single-unit and LFP levels. Thus, while isoflurane anesthesia had little effect on the tonotopic topography, its profound effects on neuronal activities decreased the encoding capacity of the auditory cortex.

TORC1 is a central regulator of cell growth in response to amino acids. The role of the evolutionarily conserved Gtr/Rag pathway in the regulation of TORC1 is well-established. Recent genetic studies suggest that an additional regulatory pathway, depending on the activity of Pib2, plays a role in TORC1 activation independently of the Gtr/Rag pathway. However, the interplay between the Pib2 pathway and the Gtr/Rag pathway remains unclear. In this study, we show that Pib2 and Gtr/Ego form distinct complexes with TORC1 in a mutually exclusive manner, implying dedicated functional relationships between TORC1 and Pib2 or Gtr/Rag in response to specific amino acids. Furthermore, simultaneous depletion of Pib2 and the Gtr/Ego system abolishes TORC1 activity and completely compromises the vacuolar localization of TORC1. Thus, the amino acid-dependent activation of TORC1 is achieved through the Pib2 and Gtr/Ego pathways alone. Finally, we show that glutamine induces a dose-dependent increase in Pib2-TORC1 complex formation, and that glutamine binds directly to the Pib2 complex. These data provide strong preliminary evidence for Pib2 functioning as a putative glutamine sensor in the regulation of TORC1.

After the SARS-CoV-2 omicron variants replaced the delta variant (B.1.617.2), omicron subvariants, including BQ.1.1 and XBB, emerged and became the dominant strain worldwide. Although the omicron subvariants are more immunoevasive than earlier variants,1Imai M Ito M Kiso M et al.Efficacy of antiviral agents against omicron subvariants BQ.1.1 and XBB.N Engl J Med. 2023; 388: 89-91Crossref PubMed Scopus (74) Google Scholar, 2Uraki R Ito M Furusawa Y et al.Humoral immune evasion of the omicron subvariants BQ.1.1 and XBB.Lancet Infect Dis. 2023; 23: 30-32Summary Full Text Full Text PDF PubMed Scopus (50) Google Scholar their virological characteristics, such as replicative capacity in respiratory organs during pyrexia,3Herder V Dee K Wojtus JK et al.Elevated temperature inhibits SARS-CoV-2 replication in respiratory epithelium independently of IFN-mediated innate immune defenses.PLoS Biol. 2021; 19e3001065 Crossref Scopus (12) Google Scholar are not fully understood. We compared the replicative capacity of B.1.617.2, BA.5, and BQ.1.1 during pyrexia by using human alveolar epithelial cells (AECs) in an air–liquid interface culture, which were generated from induced pluripotent stem cells. Human AECs were infected with 1 × 104 50% tissue culture infectious dose (TCID50) of B.1.617.2, BA.5, and BQ.1.1, and incubated at two different temperatures, 37°C (normal human body temperature) and 40°C (elevated human body temperature during illness). Samples were collected daily from the apical surface of the AECs up to 4 days post infection (dpi) for viral titration. All three variants had similar growth kinetics on human AECs at 37°C, reaching peak titres of 107·5–108·5 TCID50/mL at 2 dpi (appendix p 2). Notably, although the viral titres of B.1.617.2 at 2 dpi were 10 times lower at 40°C (106·5 TCID50/mL) than at 37°C (108·2 TCID50/mL), viral titres of BA.5 were 1000 times lower at 40°C (104·6 TCID50/mL) than at 37°C (107·5 TCID50/mL), and BQ.1.1 was unable to replicate at the higher temperature in human AECs (appendix p 2). In Vero E6 cells expressing TMPRSS2 (VeroE6/TMPRSS2), the three SARS-CoV-2 variants had similar replication kinetics at 37°C to those in human AECs at the same temperature, with peak titres at 2 dpi (appendix p 1). Virus-infected VeroE6/TMPRSS2 cells grown at 37°C were dead by 3 dpi due to virus growth. At 40°C in VeroE6/TMPRSS2 cells, the titre of B.1.617.2 was again 10 times lower than that at 37°C (appendix p 1). The virus titres for BA.5 and BQ.1.1 were also substantially reduced at 40°C compared with titres at 37°C, showing that the replicative capacities of BA.5 and BQ.1.1 were restricted at the higher temperature. Our data show that omicron variants—especially BQ.1.1—cannot replicate efficiently at high temperatures, unlike B.1.617.2. Because pyrexia is one of the most common symptoms in patients with SARS-CoV-2 infection (including the omicron variant), elevated body temperature during the illness might substantially restrict BA.5 and BQ.1.1 replication in the lungs and could have an important role in limiting disease severity caused by the omicron variants. Thus, BA.5 and BQ.1.1 show lower pathogenicity than B.1.617.2. Further study is needed to reveal the determinants responsible for the temperature sensitivity of SARS-CoV-2 variants, which could lead to a better understanding of viral pathogenesis. YM designed and did infection experiments and data analysis. ST and SG generated human AECs derived from induced pluripotent stem cells. PJH, TN, and YK obtained funding, conceived the study, and wrote the draft, with all other authors providing editorial comments. YK is supported by grants from the Center for Research on Influenza Pathogenesis (HHSN272201400008C) and from the Center for Research on Influenza Pathogenesis and Transmission (75N93021C00014), funded by the National Institute of Allergy and Infectious Disease; by a Research Program on Emerging and Reemerging Infectious Diseases (JP21fk0108552 and JP21fk0108615), a Project Promoting Support for Drug Discovery (JP21nf0101632), the Japan Program for Infectious Diseases Research and Infrastructure (JP22wm0125002), and a grant (JP223fa627001) from the Japan Agency for Medical Research and Development; and has received unrelated funding support from Daiichi Sankyo Pharmaceutical, Toyama Chemical, Tauns Laboratories, Shionogi, Otsuka Pharmaceutical, KM Biologics, Kyoritsu Seiyaku, Shinya Corporation, and Fuji Rebio. TN is supported by the JST Core Research for Evolutional Science and Technology (JPMJCR20HA), the JSPS Core-to-Core Program A (JPJSCCA20190008), the Kansai Economic Federation (KANKEIREN), the Joint Research Project of the Institute of Medical Science at the University of Tokyo, and the Joint Usage/Research Center Program of the Institute for Life and Medical Sciences at Kyoto University. SG is supported by the Fight Corona Project funded by the COVID-19 Private Fund (to Shinya Yamanaka, CiRA, Kyoto University), has received unrelated funding support from Kyorin Pharmaceutical, and is a founder and shareholder of HiLung. All other authors declare no competing interests. We thank Susan Watson for scientific editing. We also thank Chiho Onishi, Koichi Igura, and Naoyuki Sone for technical assistance. Download .pdf (.57 MB) Help with pdf files Supplementary appendix

Target of rapamycin complex 1 (TORC1) is a master regulator that monitors the availability of various amino acids to promote cell growth in Saccharomyces cerevisiae. It is activated via two distinct upstream pathways: the Gtr pathway, which corresponds to mammalian Rag, and the Pib2 pathway. This study shows that Ser3 was phosphorylated exclusively in a Pib2-dependent manner. Using Ser3 as an indicator of TORC1 activity, together with the established TORC1 substrate Sch9, we investigated which pathways were employed by individual amino acids. Different amino acids exhibited different dependencies on the Gtr and Pib2 pathways. Cysteine was most dependent on the Pib2 pathway and increased the interaction between TORC1 and Pib2 in vivo and in vitro. Moreover, cysteine directly bound to Pib2 via W632 and F635, two critical residues in the T(ail) motif that are necessary to activate TORC1. These results indicate that Pib2 functions as a sensor for cysteine in TORC1 regulation.

Neural systems have evolved to process sensory stimuli in a way that allows for efficient and adaptive behavior in a complex environment. Recent technological advances enable us to investigate sensory processing in animal models by simultaneously recording the activity of large populations of neurons with single-cell resolution, yielding high-dimensional datasets. In this review, we discuss concepts and approaches for assessing the population-level representation of sensory stimuli in the form of a representational map. In such a map, not only are the identities of stimuli distinctly represented, but their relational similarity is also mapped onto the space of neuronal activity. We highlight example studies in which the structure of representational maps in the brain are estimated from recordings in humans as well as animals and compare their methodological approaches. Finally, we integrate these aspects and provide an outlook for how the concept of representational maps could be applied to various fields in basic and clinical neuroscience.

Pure Al powder was sintered by spark plasma sintering (SPS) process at various sintering temperatures and loading pressures. The density, electrical resistivity, tensile properties and microstructure of powder compacts were investigated. The powder compacts with the similar properties as base aluminum metal was obtained at sintering temperature above 873 K, loading pressure above 23.5 MPa. For the powder compacts with the similar density but with the large difference in the electrical resistivity and tensile properties, the interfaces between Al powder particles were investigated using high resolution transmission electron microscopy (HRTEM) and energy dispersive X-ray spectroscopy (EDS). Two types of interfaces, with metal/metal bonding and metal/oxide film layer/metal bonding, were observed in Al powder compacts. The properties of powder compacts were mainly subject to the behavior of oxide film between the powder particles.

Pure aluminum (Al) powder was sintered by means of a pulse electric current sintering (PECS) process at various pulse frequencies. The effects of pulse frequency on the density, electrical resistivity and tensile properties of the compacts were investigated. The results showed that the effects were not significant. From the activation energy calculation in the sintering process and scanning electron microscopy observation of compacts and transmission electron microscopy observation of interfaces between powder particles, the pulse frequency effects on PECS process and microstructure were not observed.

A novel family of photo‐and electroactive amorphous molecular materials containing an oligothiophene moiety linked to two triphenylamine moieties has been designed and synthesized (see Figure). Their glass‐forming properties, morphological changes, and molecualr and solid‐state properties are described. They are reported to exhibit multiredox behavior on electrochemical oxidation. magnified image

Neurospheres were obtained by culturing hippocampal cells from transgenic rat fetuses (E16) expressing green fluorescent protein (GFP). The neurosphere cells were injected into the cerebrospinal fluid (CSF) through the 4th ventricle of young rats (4 weeks old) that had been given a contusion injury at T8-9 of the spinal cord. The injected neural stem cells were transported through the CSF to the spinal cord, attached to the pial surface at the lesion, and invaded extensively into the spinal cord tissue as well as into the nerve roots. The grafted stem cells survived well in the host spinal cord for as long as 8 months after transplantation. Immunohistochemical study showed that many grafted stem cells had differentiated into astrocytes at 1-4 months, and some into oligodendrocytes at 8 months postoperatively. Immunoelectron microscopy showed that the grafted stem cells were well integrated into the host tissue, extending their processes around nerve fibers in the same manner as astrocytes. In addition, grafted stem cells within nerve roots closely surrounded myelinated fibers or were integrated into unmyelinated fiber bundles; those associated with myelinated fibers formed basal laminae on their free surface, whereas those associated with unmyelinated fibers were directly attached to axons and Schwann cells, indicating that grafted stem cells behaved like Schwann cells in the nerve roots.

Intracisternal granules (ICG) develop in the rough ER of hyperstimulated thyrotrophs or thyroid hormone-secreting cells of the anterior pituitary gland. To determine the fate of these granules, we carried out morphological and immunocytochemical studies on pituitaries of thyroxine-treated, thyroidectomized rats. Under these conditions the ER of thyrotrophs is dramatically dilated and contains abundant ICG; the latter contain beta subunits of thyrotrophic hormone (TSH-beta). Based on purely morphologic criteria, intermediates were identified that appeared to represent stages in the transformation of a part rough/part smooth ER cisterna into a lysosome. Using immunocytochemical and cytochemical markers, two major types of intermediates were distinguished: type 1 lacked ribosomes but were labeled with antibodies against both ER markers (PDI, KDEL, ER membrane proteins) and a lysosomal membrane marker, lgp120. They also were reactive for the lysosomal enzyme, acid phosphatase, by enzyme cytochemistry. Type 2 intermediates were weakly reactive for ER markers and contained both lgp120 and lysosomal enzymes (cathepsin D, acid phosphatase). Taken together these results suggest that in hyperstimulated thyrotrophs part rough/part smooth ER elements containing ICG lose their ribosomes, their membrane is modified, and they sequentially acquire a lysosome-type membrane and lysosomal enzymes. The findings are compatible with the conclusion that a pathway exists by which under certain conditions, secretory proteins present in the ER as well as ER membrane and content proteins can be degraded by direct conversion of ER cisternae into lysosomes.

Continuous ATRP of MMA was carried out in a flow tubular reactor with varying flow rate, temperature, and [monomer]/[initiator] ratios. Changing the flow rate directly relates to the reaction time. This process produces polymer continuously with the conversion increasing with decreasing flow rate. The molecular weight (relating to the flow rate) increases linearly with conversion which is also observed when the [monomer]/[initiator] ratio was changed. The effect of altering the reaction temperature was studied and the apparent activation energy of the propagation reaction of MMA in this system was calculated to be ∼56.9 kJ mol−1, close to the values reported previously. Preparation of diblock copolymers is also reported with varying comonomers and the conversion, and SEC results suggested that this continuous system is an excellent and facile way to have a continuous ATRP process.

It is essential to have a clear understanding of the present condition of cardiopulmonary resuscitation (CPR) training courses and the associated problems. The present study was performed to identify the current conditions of CPR training in Japanese high schools and the attitudes of students toward CPR.We distributed a questionnaire study to the students of 12 cooperating high schools regarding their willingness to perform CPR in 5 hypothetical scenarios of cardiopulmonary arrest: a stranger, a trauma patient, a child, an elderly person, and a relative. Between February and March 2006, a total of 3316 questionnaires were completed. Across all scenarios, only 27% of respondents from general high schools reported willingness to perform chest compression (CC) plus mouth-to-mouth ventilation (MMV), and 31% reported willingness to perform CC alone. Fifty-nine percent of students had previous CPR training, and only 35% were willing to perform CC plus MMV. Most of the respondents who reported that they would decline to perform full CPR, stated that poor knowledge and/or fear of incomplete performance of CPR were deciding factors.Japanese high school students are reluctant to perform CC plus MMV, despite having received training. The present educational system in Japan has limitations in encouraging high school students to perform CC plus MMV.