Hitoshi Nakatogawa

Autophagy is a fundamental function of eukaryotic cells and is well conserved from yeast to humans. The most remarkable feature of autophagy is the synthesis of double membrane-bound compartments that sequester materials to be degraded in lytic compartments, a process that seems to be mechanistically distinct from conventional membrane traffic. The discovery of autophagy in yeast and the genetic tractability of this organism have allowed us to identify genes that are responsible for this process, which has led to the explosive growth of this research field seen today. Analyses of autophagy-related (Atg) proteins have unveiled dynamic and diverse aspects of mechanisms that underlie membrane formation during autophagy.

Autophagy involves de novo formation of double membrane-bound structures called autophagosomes, which engulf material to be degraded in lytic compartments. Atg8 is a ubiquitin-like protein required for this process in Saccharomyces cerevisiae that can be conjugated to the lipid phosphatidylethanolamine by a ubiquitin-like system. Here, we show using an in vitro system that Atg8 mediates the tethering and hemifusion of membranes, which are evoked by the lipidation of the protein and reversibly modulated by the deconjugation enzyme Atg4. Mutational analyses suggest that membrane tethering and hemifusion observed in vitro represent an authentic function of Atg8 in autophagosome formation in vivo. In addition, electron microscopic analyses indicate that these functions of Atg8 are involved in the expansion of autophagosomal membranes. Our results provide further insights into the mechanisms underlying the unique membrane dynamics of autophagy and also indicate the functional versatility of ubiquitin-like proteins.

Translation of SecM stalls unless its N-terminal part is "pulled" by the protein export machinery. Here we show that the sequence motif FXXXXWIXXXXGIRAGP that includes a specific arrest point (Pro) causes elongation arrest within the ribosome. Mutations that bypass the elongation arrest were isolated in 23S rRNA and L22 r protein. Such suppressor mutations occurred at a few specific residues of these components, which all face the narrowest constriction of the ribosomal exit tunnel. Thus, we suggest that this region of the exit tunnel interacts with nascent translation products and functions as a discriminating gate.

Autophagy is a non-selective bulk degradation process in which isolation membranes enclose a portion of cytoplasm to form double-membrane vesicles, called autophagosomes, and deliver their inner constituents to the lytic compartments. Recent studies have also shed light on another mode of autophagy that selectively degrades various targets. Yeast Atg8 and its mammalian homologue LC3 are ubiquitin-like modifiers that are localized on isolation membranes and play crucial roles in the formation of autophagosomes. These proteins are also involved in selective incorporation of specific cargo molecules into autophagosomes, in which Atg8 and LC3 interact with Atg19 and p62, receptor proteins for vacuolar enzymes and disease-related protein aggregates, respectively. Using X-ray crystallography and NMR, we herein report the structural basis for Atg8-Atg19 and LC3-p62 interactions. Remarkably, Atg8 and LC3 were shown to interact with Atg19 and p62, respectively, in a quite similar manner: they recognized the side-chains of Trp and Leu in a four-amino acid motif, WXXL, in Atg19 and p62 using hydrophobic pockets conserved among Atg8 homologues. Together with mutational analyses, our results show the fundamental mechanism that allows Atg8 homologues, in association with WXXL-containing proteins, to capture specific cargo molecules, thereby endowing isolation membranes and/or their assembly machineries with target selectivity.

In autophagy, the autophagosome, a transient organelle specialized for the sequestration and lysosomal or vacuolar transport of cellular constituents, is formed via unique membrane dynamics. This process requires concerted actions of a distinctive set of proteins named Atg (autophagy-related). Atg proteins include two ubiquitin-like proteins, Atg12 and Atg8 [LC3 (light-chain 3) and GABARAP (γ-aminobutyric acid receptor-associated protein) in mammals]. Sequential reactions by the E1 enzyme Atg7 and the E2 enzyme Atg10 conjugate Atg12 to the lysine residue in Atg5, and the resulting Atg12-Atg5 conjugate forms a complex with Atg16. On the other hand, Atg8 is first processed at the C-terminus by Atg4, which is related to ubiquitin-processing/deconjugating enzymes. Atg8 is then activated by Atg7 (shared with Atg12) and, via the E2 enzyme Atg3, finally conjugated to the amino group of the lipid PE (phosphatidylethanolamine). The Atg12-Atg5-Atg16 complex acts as an E3 enzyme for the conjugation reaction of Atg8; it enhances the E2 activity of Atg3 and specifies the site of Atg8-PE production to be autophagy-related membranes. Atg8-PE is suggested to be involved in autophagosome formation at multiple steps, including membrane expansion and closure. Moreover, Atg4 cleaves Atg8-PE to liberate Atg8 from membranes for reuse, and this reaction can also regulate autophagosome formation. Thus these two ubiquitin-like systems are intimately involved in driving the biogenesis of the autophagosomal membrane.

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.

Significance A central event during autophagy is the biogenesis of double-membrane vesicles called autophagosomes, which sequester various intracellular materials for degradation in lysosomes/vacuoles. Recent studies have suggested the involvement of the endoplasmic reticulum (ER) in autophagosome formation, and that pre-autophagosomal membranes contact with the ER. However, the mechanistic basis of these contacts has remained unknown. Here we describe two membrane-binding domains responsible for autophagosome formation in the autophagy-related protein Atg2, which localizes to the pre-autophagosomal membrane–ER contact sites in yeast cells. Our data suggest that the amphipathic helix in the C-terminal region of Atg2 targets the protein to pre-autophagosomal membranes, whereas the N-terminal region of the same molecule associates with the ER, tethering these membranes together to mediate membrane expansion during autophagosome formation.

Atg8 is a ubiquitin-like protein required for autophagy in the budding yeast Saccharomyces cerevisiae. A ubiquitin-like system mediates the conjugation of the C terminus of Atg8 to the lipid phosphatidylethanolamine (PE), and this conjugate (Atg8-PE) plays a crucial role in autophagosome formation at the phagophore assembly site/pre-autophagosomal structure (PAS). The cysteine protease Atg4 processes the C terminus of newly synthesized Atg8 and also delipidates Atg8 to release the protein from membranes. While the former is a prerequisite for lipidation of Atg8, the significance of the latter in autophagy has remained unclear. Here, we show that autophagosome formation is significantly retarded in cells deficient for Atg4-mediated delipidation of Atg8. We find that Atg8-PE accumulates on various organelle membranes including the vacuole, the endosome and the ER in these cells, which depletes unlipidated Atg8 and thereby attenuates its localization to the PAS. Our results suggest that the Atg8-PE that accumulates on organelle membranes is erroneously produced by lipidation system components independently of the normal autophagic process. It is also suggested that delipidation of Atg8 by Atg4 on different organelle membranes promotes autophagosome formation. Considered together with other results, we propose that Atg4 acts to compensate for the intrinsic defect in the lipidation system; it recycles Atg8-PE generated on inappropriate membranes to maintain a reservoir of unlipidated Atg8 that is required for autophagosome formation at the PAS.

The product of the Escherichia coli secM gene (secretion monitor, formerly gene X), upstream of secA, is involved in secretion-responsive control of SecA translation. In wild-type cells, SecM is rapidly degraded by the periplasmic tail-specific protease. It is also subject to a transient translation pause at a position close to the C terminus. The elongation arrest was strikingly prolonged when translocation of SecM was impaired. SRP was not required for this arrest. Instead, the nascent SecM product itself may participate, as the arrest was diminished when it incorporated a proline analog, azetidine. We propose that cytosolically localized nascent SecM undergoes self-translation arrest, thereby enhancing translation of secA through an altered secondary structure of the secM-secA messenger RNA.

<h2>Summary</h2> E1 enzymes activate ubiquitin-like proteins and transfer them to cognate E2 enzymes. Atg7, a noncanonical E1, activates two ubiquitin-like proteins, Atg8 and Atg12, and plays a crucial role in autophagy. Here, we report crystal structures of full-length Atg7 and its C-terminal domain bound to Atg8 and MgATP, as well as a solution structure of Atg8 bound to the extreme C-terminal domain (ECTD) of Atg7. The unique N-terminal domain (NTD) of Atg7 is responsible for Atg3 (E2) binding, whereas its C-terminal domain is comprised of a homodimeric adenylation domain (AD) and ECTD. The structural and biochemical data demonstrate that Atg8 is initially recognized by the C-terminal tail of ECTD and is then transferred to an AD, where the Atg8 C terminus is attacked by the catalytic cysteine to form a thioester bond. Atg8 is then transferred via a <i>trans</i> mechanism to the Atg3 bound to the NTD of the opposite protomer within a dimer.

The arrest sequence, FXXXXWIXXXXGIRAGP, of E. coli SecM interacts with the ribosomal exit tunnel, thereby interfering with translation elongation. Here, we studied this elongation arrest in vitro using purified translation components. While a simplest scenario would be that elongation is arrested beyond Pro166, the last arrest-essential amino acid, and that the Pro166 codon is positioned at the P site of the ribosomal peptidyl transferase center (PTC), our toeprint analyses revealed that the ribosome actually stalls when the Pro166 codon is positioned at the A site. Northern hybridization identification of the polypeptide bound tRNA and mass determination showed that the last amino acid of the arrested peptidyl-tRNA is Gly165, which is only inefficiently transferred to Pro166. Also, puromycin does not effectively release the arrested peptidyl-tRNA under the conditions of A site occupancy by Pro166-tRNA. These results reveal that secM-encoded Pro166-tRNA functions as a nonpolypeptide element in fulfilling SecM's role as a secretion monitor.

Autophagy-related degradation selective for mitochondria (mitophagy) is an evolutionarily conserved process that is thought to be critical for mitochondrial quality and quantity control. In budding yeast, autophagy-related protein 32 (Atg32) is inserted into the outer membrane of mitochondria with its N- and C-terminal domains exposed to the cytosol and mitochondrial intermembrane space, respectively, and plays an essential role in mitophagy. Atg32 interacts with Atg8, a ubiquitin-like protein localized to the autophagosome, and Atg11, a scaffold protein required for selective autophagy-related pathways, although the significance of these interactions remains elusive. In addition, whether Atg32 is the sole protein necessary and sufficient for initiation of autophagosome formation has not been addressed. Here we show that the Atg32 IMS domain is dispensable for mitophagy. Notably, when anchored to peroxisomes, the Atg32 cytosol domain promoted autophagy-dependent peroxisome degradation, suggesting that Atg32 contains a module compatible for other organelle autophagy. X-ray crystallography reveals that the Atg32 Atg8 family-interacting motif peptide binds Atg8 in a conserved manner. Mutations in this binding interface impair association of Atg32 with the free form of Atg8 and mitophagy. Moreover, Atg32 variants, which do not stably interact with Atg11, are strongly defective in mitochondrial degradation. Finally, we demonstrate that Atg32 forms a complex with Atg8 and Atg11 prior to and independent of isolation membrane generation and subsequent autophagosome formation. Taken together, our data implicate Atg32 as a bipartite platform recruiting Atg8 and Atg11 to the mitochondrial surface and forming an initiator complex crucial for mitophagy. Autophagy-related degradation selective for mitochondria (mitophagy) is an evolutionarily conserved process that is thought to be critical for mitochondrial quality and quantity control. In budding yeast, autophagy-related protein 32 (Atg32) is inserted into the outer membrane of mitochondria with its N- and C-terminal domains exposed to the cytosol and mitochondrial intermembrane space, respectively, and plays an essential role in mitophagy. Atg32 interacts with Atg8, a ubiquitin-like protein localized to the autophagosome, and Atg11, a scaffold protein required for selective autophagy-related pathways, although the significance of these interactions remains elusive. In addition, whether Atg32 is the sole protein necessary and sufficient for initiation of autophagosome formation has not been addressed. Here we show that the Atg32 IMS domain is dispensable for mitophagy. Notably, when anchored to peroxisomes, the Atg32 cytosol domain promoted autophagy-dependent peroxisome degradation, suggesting that Atg32 contains a module compatible for other organelle autophagy. X-ray crystallography reveals that the Atg32 Atg8 family-interacting motif peptide binds Atg8 in a conserved manner. Mutations in this binding interface impair association of Atg32 with the free form of Atg8 and mitophagy. Moreover, Atg32 variants, which do not stably interact with Atg11, are strongly defective in mitochondrial degradation. Finally, we demonstrate that Atg32 forms a complex with Atg8 and Atg11 prior to and independent of isolation membrane generation and subsequent autophagosome formation. Taken together, our data implicate Atg32 as a bipartite platform recruiting Atg8 and Atg11 to the mitochondrial surface and forming an initiator complex crucial for mitophagy.

Atg16 interacts with the Atg12-Atg5 protein conjugate through its N-terminal domain and self-assembles through its coiled-coil domain (CCD). Formation of the Atg12-Atg5.Atg16 complex is essential for autophagy, the bulk degradation process conserved among most eukaryotes. Here, we report the crystal structures of full-length Saccharomyces cerevisiae Atg16 at 2.8 A resolution and its CCD at 2.5 A resolution. The CCD and full-length Atg16 each exhibit an extended alpha-helix, 90 and 130 A, respectively, and form a parallel coiled-coil dimer in the crystals. Although the apparent molecular weight of Atg16 observed by gel-filtration chromatography suggests that Atg16 is tetrameric, an analytical ultracentrifugation study showed Atg16 as a dimer in solution, consistent with the crystal structure. Evolutionary conserved surface residues clustered at the C-terminal half of Atg16 CCD were shown to be crucial for autophagy. These results will give a structural basis for understanding the molecular functions and significance of Atg16 in autophagy.

In selective autophagy, degradation targets are specifically recognized, sequestered by the autophagosome, and transported into the lysosome or vacuole. Previous studies delineated the molecular basis by which the autophagy machinery recognizes those targets, but the regulation of this process is still poorly understood. In this paper, we find that the highly conserved multifunctional kinase Hrr25 regulates two distinct selective autophagy–related pathways in Saccharomyces cerevisiae. Hrr25 is responsible for the phosphorylation of two receptor proteins: Atg19, which recognizes the assembly of vacuolar enzymes in the cytoplasm-to-vacuole targeting pathway, and Atg36, which recognizes superfluous peroxisomes in pexophagy. Hrr25-mediated phosphorylation enhances the interactions of these receptors with the common adaptor Atg11, which recruits the core autophagy-related proteins that mediate the formation of the autophagosomal membrane. Thus, this study introduces regulation of selective autophagy as a new role of Hrr25 and, together with other recent studies, reveals that different selective autophagy–related pathways are regulated by a uniform mechanism: phosphoregulation of the receptor–adaptor interaction.

In autophagy, a cup-shaped membrane called the isolation membrane is formed, expanded, and sealed to complete a double membrane-bound vesicle called the autophagosome that encapsulates cellular constituents to be transported to and degraded in the lysosome/vacuole. The formation of the autophagosome requires autophagy-related (Atg) proteins. Atg8 is a ubiquitin-like protein that localizes to the isolation membrane; a subpopulation of this protein remains inside the autophagosome and is transported to the lysosome/vacuole. In the budding yeast <i>Saccharomyces cerevisiae,</i> Atg1 is a serine/threonine kinase that functions in the initial step of autophagosome formation and is also efficiently transported to the vacuole via autophagy. Here, we explore the mechanism and significance of this autophagic transport of Atg1. In selective types of autophagy, receptor proteins recognize degradation targets and also interact with Atg8, via the Atg8 family interacting motif (AIM), to link the targets to the isolation membrane. We find that Atg1 contains an AIM and directly interacts with Atg8. Mutations in the AIM disrupt this interaction and abolish vacuolar transport of Atg1. These results suggest that Atg1 associates with the isolation membrane by binding to Atg8, resulting in its incorporation into the autophagosome. We also show that mutations in the Atg1 AIM cause a significant defect in autophagy, without affecting the functions of Atg1 implicated in triggering autophagosome formation. We propose that in addition to its essential function in the initial stage, Atg1 also associates with the isolation membrane to promote its maturation into the autophagosome.

A hallmark of autophagy is the de novo formation of double-membrane vesicles called autophagosomes, which sequester various cellular constituents for degradation in lysosomes or vacuoles. The membrane dynamics underlying the biogenesis of autophagosomes, including the origin of the autophagosomal membrane, are still elusive. Although previous studies suggested that COPII vesicles are closely associated with autophagosome biogenesis, it remains unclear whether these vesicles serve as a source of the autophagosomal membrane. Using a recently developed COPII vesicle-labeling system in fluorescence and immunoelectron microscopy in the budding yeast Saccharomyces cerevisiae, we show that the transmembrane cargo Axl2 is loaded into COPII vesicles in the ER. Axl2 is then transferred to autophagosome intermediates, ultimately becoming part of autophagosomal membranes. This study provides a definitive answer to a long-standing, fundamental question regarding the mechanisms of autophagosome formation by implicating COPII vesicles as a membrane source for autophagosomes.

Abstract The endoplasmic reticulum (ER) is selectively degraded by autophagy (ER-phagy) through proteins called ER-phagy receptors. In Saccharomyces cerevisiae , Atg40 acts as an ER-phagy receptor to sequester ER fragments into autophagosomes by binding Atg8 on forming autophagosomal membranes. During ER-phagy, parts of the ER are morphologically rearranged, fragmented, and loaded into autophagosomes, but the mechanism remains poorly understood. Here we find that Atg40 molecules assemble in the ER membrane concurrently with autophagosome formation via multivalent interaction with Atg8. Atg8-mediated super-assembly of Atg40 generates highly-curved ER regions, depending on its reticulon-like domain, and supports packing of these regions into autophagosomes. Moreover, tight binding of Atg40 to Atg8 is achieved by a short helix C-terminal to the Atg8-family interacting motif, and this feature is also observed for mammalian ER-phagy receptors. Thus, this study significantly advances our understanding of the mechanisms of ER-phagy and also provides insights into organelle fragmentation in selective autophagy of other organelles.

Eukaryotic cells adequately control the mass and functions of organelles in various situations. Autophagy, an intracellular degradation system, largely contributes to this organelle control by degrading the excess or defective portions of organelles. The endoplasmic reticulum (ER) is an organelle with distinct structural domains associated with specific functions. The ER dynamically changes its mass, components, and shape in response to metabolic, developmental, or proteotoxic cues to maintain or regulate its functions. Therefore, elaborate mechanisms are required for proper degradation of the ER. Here, we review our current knowledge on diverse mechanisms underlying selective autophagy of the ER, which enable efficient degradation of specific ER subdomains according to different demands of cells.

In selective autophagy of the nucleus (hereafter nucleophagy), nucleus-derived double-membrane vesicles (NDVs) are formed, sequestered within autophagosomes, and delivered to lysosomes or vacuoles for degradation. In Saccharomyces cerevisiae, the nuclear envelope (NE) protein Atg39 acts as a nucleophagy receptor, which interacts with Atg8 to target NDVs to the forming autophagosomal membranes. In this study, we revealed that Atg39 is anchored to the outer nuclear membrane via its transmembrane domain and also associated with the inner nuclear membrane via membrane-binding amphipathic helices (APHs) in its perinuclear space region, thereby linking these membranes. We also revealed that autophagosome formation-coupled Atg39 crowding causes the NE to protrude toward the cytoplasm, and the tips of the protrusions are pinched off to generate NDVs. The APHs of Atg39 are crucial for Atg39 crowding in the NE and subsequent NE protrusion. These findings suggest that the nucleophagy receptor Atg39 plays pivotal roles in NE deformation during the generation of NDVs to be degraded by nucleophagy.

The Atg8 family of ubiquitin-like proteins play pivotal roles in autophagy and other processes involving vesicle fusion and transport where the lysosome/vacuole is the end station. Nuclear roles of Atg8 proteins are also emerging. Here, we review the structural and functional features of Atg8 family proteins and their protein-protein interaction modes in model organisms such as yeast, Arabidopsis, C. elegans and Drosophila to humans. Although varying in number of homologs, from one in yeast to seven in humans, and more than ten in some plants, there is a strong evolutionary conservation of structural features and interaction modes. The most prominent interaction mode is between the LC3 interacting region (LIR), also called Atg8 interacting motif (AIM), binding to the LIR docking site (LDS) in Atg8 homologs. There are variants of these motifs like “half-LIRs” and helical LIRs. We discuss details of the binding modes and how selectivity is achieved as well as the role of multivalent LIR-LDS interactions in selective autophagy. A number of LIR-LDS interactions are known to be regulated by phosphorylation. New methods to predict LIR motifs in proteins have emerged that will aid in discovery and analyses. There are also other interaction surfaces than the LDS becoming known where we presently lack detailed structural information, like the N-terminal arm region and the UIM-docking site (UDS). More interaction modes are likely to be discovered in future studies.

The autophagy-related protein 8 (Atg8) conjugation system is essential for the formation of double-membrane vesicles called autophagosomes during autophagy, a bulk degradation process conserved among most eukaryotes. It is also important in yeast for recognizing target vacuolar enzymes through the receptor protein Atg19 during the cytoplasm-to-vacuole targeting (Cvt) pathway, a selective type of autophagy. Atg3 is an E2-like enzyme that conjugates Atg8 with phosphatidylethanolamine. Here, we show that Atg3 directly interacts with Atg8 through the WEDL sequence, which is distinct from canonical interaction between E2 and ubiquitin-like modifiers. Moreover, NMR experiments suggest that the mode of interaction between Atg8 and Atg3 is quite similar to that between Atg8/LC3 and the Atg8 family interacting motif (AIM) conserved in autophagic receptors, such as Atg19 and p62. Thus, the WEDL sequence in Atg3 is a canonical AIM. In vitro analyses showed that Atg3 AIM is crucial for the transfer of Atg8 from the Atg8∼Atg3 thioester intermediate to phosphatidylethanolamine but not for the formation of the intermediate. Intriguingly, in vivo experiments showed that it is necessary for the Cvt pathway but not for starvation-induced autophagy. Atg3 AIM attenuated the inhibitory effect of Atg19 on Atg8 lipidation in vitro, suggesting that Atg3 AIM may be important for the lipidation of Atg19-bound Atg8 during the Cvt pathway.

Autophagy targets various intracellular components ranging from proteins and nucleic acids to organelles for their degradation in lysosomes or vacuoles. In selective types of autophagy, receptor proteins play central roles in target selection. These proteins bind or localize to specific targets, and also interact with Atg8 family proteins on forming autophagosomal membranes, leading to the efficient sequestration of the targets by the membranes. Our recent study revealed that yeast cells actively degrade the endoplasmic reticulum (ER) and even part of the nucleus via selective autophagy under nitrogen-deprived conditions. We identified novel receptors, Atg39 and Atg40, specific to these pathways. Here, we summarize our findings on 'reticulophagy' (or 'ER-phagy') and 'nucleophagy', and discuss key issues that remain to be solved in future studies.

Autophagy is a cellular degradation pathway involving the shape transformation of lipid bilayers. During the onset of autophagy, the water-soluble protein Atg8 binds covalently to phosphatdylethanolamines (PEs) in the membrane in an ubiquitin-like reaction coupled to ATP hydrolysis. We reconstituted the Atg8 conjugation system in giant and nm-sized vesicles with a minimal set of enzymes and observed that formation of Atg8-PE on giant vesicles can cause substantial tubulation of membranes even in the absence of Atg12-Atg5-Atg16. Our findings show that ubiquitin-like processes can actively change properties of lipid membranes and that membrane crowding by proteins can be dynamically regulated in cells. Furthermore we provide evidence for curvature sorting of Atg8-PE. Curvature generation and sorting are directly linked to organelle shapes and, thus, to biological function. Our results suggest that a positive feedback exists between the ubiquitin-like reaction and the membrane curvature, which is important for dynamic shape changes of cell membranes, such as those involved in the formation of autophagosomes.

In autophagy, Atg proteins organize the pre-autophagosomal structure (PAS) to initiate autophagosome formation. Previous studies in yeast revealed that the autophagy-related E3 complex Atg12-Atg5-Atg16 is recruited to the PAS via Atg16 interaction with Atg21, which binds phosphatidylinositol 3-phosphate (PI3P) produced at the PAS, to stimulate conjugation of the ubiquitin-like protein Atg8 to phosphatidylethanolamine. Here, we discover a novel mechanism for the PAS targeting of Atg12-Atg5-Atg16, which is mediated by the interaction of Atg12 with the Atg1 kinase complex that serves as a scaffold for PAS organization. While autophagy is partially defective without one of these mechanisms, cells lacking both completely lose the PAS localization of Atg12-Atg5-Atg16 and show no autophagic activity. As with the PI3P-dependent mechanism, Atg12-Atg5-Atg16 recruited via the Atg12-dependent mechanism stimulates Atg8 lipidation, but also has the specific function of facilitating PAS scaffold assembly. Thus, this study significantly advances our understanding of the nucleation step in autophagosome formation.

The mechanisms underlying turnover of the nuclear pore complex (NPC) and the component nucleoporins (Nups) are still poorly understood. In this study, we found that the budding yeast Saccharomyces cerevisiae triggers NPC degradation by autophagy upon the inactivation of Tor kinase complex 1. This degradation largely depends on the selective autophagy-specific factor Atg11 and the autophagy receptor–binding ability of Atg8, suggesting that the NPC is degraded via receptor-dependent selective autophagy. Immunoelectron microscopy revealed that NPCs embedded in nuclear envelope–derived double-membrane vesicles are sequestered within autophagosomes. At least two pathways are involved in NPC degradation: Atg39-dependent nucleophagy (selective autophagy of the nucleus) and a pathway involving an unknown receptor. In addition, we found the interaction between Nup159 and Atg8 via the Atg8-family interacting motif is important for degradation of this nucleoporin not assembled into the NPC. Thus, this study provides the first evidence for autophagic degradation of the NPC and Nups, which we term “NPC-phagy” and “nucleoporinophagy.”

Mitophagy plays an important role in mitochondrial quality control. In yeast, phosphorylation of the mitophagy receptor Atg32 by casein kinase 2 (CK2) upon induction of mitophagy is a prerequisite for interaction of Atg32 with Atg11 (an adaptor protein for selective autophagy) and following delivery of mitochondria to the vacuole for degradation. Because CK2 is constitutively active, Atg32 phosphorylation must be precisely regulated to prevent unrequired mitophagy. We found that the PP2A (protein phosphatase 2A)-like protein phosphatase Ppg1 was essential for dephosphorylation of Atg32 and inhibited mitophagy. We identified the Far complex proteins, Far3, Far7, Far8, Far9, Far10, and Far11, as Ppg1-binding proteins. Deletion of Ppg1 or Far proteins accelerated mitophagy. Deletion of a cytoplasmic region (amino acid residues 151-200) of Atg32 caused the same phenotypes as in ppg1Δ cells, which suggested that dephosphorylation of Atg32 by Ppg1 required this region. Therefore, Ppg1 and the Far complex cooperatively dephosphorylate Atg32 to prevent excessive mitophagy.

Eukaryotic cells have evolved different modes of autophagy, including macroautophagy and microautophagy, to deliver their own components to lysosomes or vacuoles for degradation. While an increasing body of research has established that autophagy plays pivotal roles for the maintenance and regulation of various cellular constituents, recent studies have begun to reveal that parts of the nucleus, for example, nucleus-derived vesicles and nuclear proteins, also become targets of autophagic degradation in different physiological or pathological contexts, including nutrient deprivation, defective nuclear pore complex (NPC) assembly, DNA damage, cellular senescence, and oncogenic insults. Here, we overview our current knowledge on the mechanisms and physiological roles of these 'nucleophagy' pathways and discuss their possible interplays and remaining issues.

SecA, the protein translocation ATPase of E. coli is subject to secretion-defect-response control. SecM (secretion monitor) encoded by the 5' region of the secM-secA mRNA is involved in this regulation. SecM translation is subject to transient elongation arrest at Pro166, which is prolonged when export of the nascent SecM is blocked. An "arrest sequence", FXXXXWIXXXXGIRAGP, was identified at a carboxy-terminal region of SecM that interacts with the ribosomal exit tunnel. Presumably, the stalled ribosome disrupts the secondary structure of the secM-secA mRNA such that the Shine-Dalgarno sequence for translation of secA is exposed. Mutation studies established that the SecM elongation arrest is required for the viability of E. coli as well as for constitutive (in secretion-proficient cells) and upregulated (in secretion compromised cells) expression of SecA. Furthermore, evidence suggests that elongation-arresting SecM has a role of upregulating the functionality of newly synthesized SecA molecules, presumably by bringing the mRNA to the vicinity of the membrane/Sec translocation apparatus. These results are discussed in relation to the versatile nature of SecA in its localization and structure.

The SecM protein of Escherichia coli contains an arrest sequence (F(150)XXXXWIXXXXGIRAGP(166)), which interacts with the ribosomal exit tunnel to halt translation elongation beyond Pro-166. This inhibition is reversed by active export of the nascent SecM chain. Here, we studied the physiological roles of SecM. Arrest-alleviating mutations in the arrest sequence reduced the expression of secA, a downstream gene on the same mRNA. Among such mutations, the arrest-abolishing P166A substitution mutation on the chromosomal secM gene proved lethal unless the mutant cells are complemented with excess SecA. Whereas secretion defect due either to azide addition, a secY mutation, or low temperature leads to up-regulated SecA biosynthesis, this regulation was lost by a secM mutation, which synergistically retarded growth of cells with lowered secretion activity. Finally, an arrest-alleviating rRNA mutation affecting the constricted part of the exit tunnel lowered the basal level of SecA as well as its secretion defect-induced up-regulation. Thus, the arrest sequence of SecM has at least two roles in SecA translation. First, the transient elongation arrest in normal cells is required for the synthesis of SecA at levels sufficient to support cell growth. Second, the prolonged SecM elongation arrest under conditions of unfavorable protein secretion is required for the enhanced expression of SecA to cope with such conditions.

Yeast Atg8 and its mammalian homolog LC3 are ubiquitin-like proteins involved in autophagy, a primary pathway for degradation of cytosolic constituents in vacuoles/lysosomes. Whereas the lipid phosphatidylethanolamine (PE) was identified as the sole in vivo target of their conjugation reactions, in vitro studies showed that the same system can mediate the conjugation of these proteins with phosphatidylserine as efficiently as with PE. Here, we show that, in contrast to PE conjugation, the in vitro phosphatidylserine conjugation of Atg8 is markedly suppressed at physiological pH. Furthermore, the addition of acidic phospholipids to liposomes also results in the preferential formation of the Atg8-PE conjugate. We have successfully captured authentic thioester intermediates, allowing us to elucidate which step in the conjugation reaction is affected by these changes in pH and membrane lipid composition. We propose that these factors contribute to the selective formation of Atg8-PE in the cell. Yeast Atg8 and its mammalian homolog LC3 are ubiquitin-like proteins involved in autophagy, a primary pathway for degradation of cytosolic constituents in vacuoles/lysosomes. Whereas the lipid phosphatidylethanolamine (PE) was identified as the sole in vivo target of their conjugation reactions, in vitro studies showed that the same system can mediate the conjugation of these proteins with phosphatidylserine as efficiently as with PE. Here, we show that, in contrast to PE conjugation, the in vitro phosphatidylserine conjugation of Atg8 is markedly suppressed at physiological pH. Furthermore, the addition of acidic phospholipids to liposomes also results in the preferential formation of the Atg8-PE conjugate. We have successfully captured authentic thioester intermediates, allowing us to elucidate which step in the conjugation reaction is affected by these changes in pH and membrane lipid composition. We propose that these factors contribute to the selective formation of Atg8-PE in the cell.

Article5 October 2015free access Phospholipid methylation controls Atg32-mediated mitophagy and Atg8 recycling Kaori Sakakibara Kaori Sakakibara Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan Search for more papers by this author Akinori Eiyama Akinori Eiyama Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan Search for more papers by this author Sho W Suzuki Sho W Suzuki Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan Frontier Research Center, Tokyo Institute of Technology, Kanagawa, Japan Search for more papers by this author Machiko Sakoh-Nakatogawa Machiko Sakoh-Nakatogawa Frontier Research Center, Tokyo Institute of Technology, Kanagawa, Japan Search for more papers by this author Nobuaki Okumura Nobuaki Okumura Institute for Protein Research, Osaka University, Osaka, Japan Search for more papers by this author Motohiro Tani Motohiro Tani Department of Chemistry, Kyushu University, Fukuoka, Japan Search for more papers by this author Ayako Hashimoto Ayako Hashimoto Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan Search for more papers by this author Sachiyo Nagumo Sachiyo Nagumo Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan Search for more papers by this author Noriko Kondo-Okamoto Noriko Kondo-Okamoto Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan Search for more papers by this author Chika Kondo-Kakuta Chika Kondo-Kakuta Frontier Research Center, Tokyo Institute of Technology, Kanagawa, Japan Search for more papers by this author Eri Asai Eri Asai Frontier Research Center, Tokyo Institute of Technology, Kanagawa, Japan Search for more papers by this author Hiromi Kirisako Hiromi Kirisako Frontier Research Center, Tokyo Institute of Technology, Kanagawa, Japan Search for more papers by this author Hitoshi Nakatogawa Hitoshi Nakatogawa Frontier Research Center, Tokyo Institute of Technology, Kanagawa, Japan Search for more papers by this author Osamu Kuge Osamu Kuge Department of Chemistry, Kyushu University, Fukuoka, Japan Search for more papers by this author Toshifumi Takao Toshifumi Takao Institute for Protein Research, Osaka University, Osaka, Japan Search for more papers by this author Yoshinori Ohsumi Yoshinori Ohsumi Frontier Research Center, Tokyo Institute of Technology, Kanagawa, Japan Search for more papers by this author Koji Okamoto Corresponding Author Koji Okamoto Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan Search for more papers by this author Kaori Sakakibara Kaori Sakakibara Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan Search for more papers by this author Akinori Eiyama Akinori Eiyama Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan Search for more papers by this author Sho W Suzuki Sho W Suzuki Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan Frontier Research Center, Tokyo Institute of Technology, Kanagawa, Japan Search for more papers by this author Machiko Sakoh-Nakatogawa Machiko Sakoh-Nakatogawa Frontier Research Center, Tokyo Institute of Technology, Kanagawa, Japan Search for more papers by this author Nobuaki Okumura Nobuaki Okumura Institute for Protein Research, Osaka University, Osaka, Japan Search for more papers by this author Motohiro Tani Motohiro Tani Department of Chemistry, Kyushu University, Fukuoka, Japan Search for more papers by this author Ayako Hashimoto Ayako Hashimoto Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan Search for more papers by this author Sachiyo Nagumo Sachiyo Nagumo Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan Search for more papers by this author Noriko Kondo-Okamoto Noriko Kondo-Okamoto Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan Search for more papers by this author Chika Kondo-Kakuta Chika Kondo-Kakuta Frontier Research Center, Tokyo Institute of Technology, Kanagawa, Japan Search for more papers by this author Eri Asai Eri Asai Frontier Research Center, Tokyo Institute of Technology, Kanagawa, Japan Search for more papers by this author Hiromi Kirisako Hiromi Kirisako Frontier Research Center, Tokyo Institute of Technology, Kanagawa, Japan Search for more papers by this author Hitoshi Nakatogawa Hitoshi Nakatogawa Frontier Research Center, Tokyo Institute of Technology, Kanagawa, Japan Search for more papers by this author Osamu Kuge Osamu Kuge Department of Chemistry, Kyushu University, Fukuoka, Japan Search for more papers by this author Toshifumi Takao Toshifumi Takao Institute for Protein Research, Osaka University, Osaka, Japan Search for more papers by this author Yoshinori Ohsumi Yoshinori Ohsumi Frontier Research Center, Tokyo Institute of Technology, Kanagawa, Japan Search for more papers by this author Koji Okamoto Corresponding Author Koji Okamoto Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan Search for more papers by this author Author Information Kaori Sakakibara1, Akinori Eiyama1,‡, Sho W Suzuki1,2,‡, Machiko Sakoh-Nakatogawa2,‡, Nobuaki Okumura3,‡, Motohiro Tani4,‡, Ayako Hashimoto1, Sachiyo Nagumo1, Noriko Kondo-Okamoto1, Chika Kondo-Kakuta2, Eri Asai2, Hiromi Kirisako2, Hitoshi Nakatogawa2, Osamu Kuge4, Toshifumi Takao3, Yoshinori Ohsumi2 and Koji Okamoto 1 1Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan 2Frontier Research Center, Tokyo Institute of Technology, Kanagawa, Japan 3Institute for Protein Research, Osaka University, Osaka, Japan 4Department of Chemistry, Kyushu University, Fukuoka, Japan ‡These authors contributed equally to this work *Corresponding author. Tel: +81 6 6879 7970; E-mail: [email protected] The EMBO Journal (2015)34:2703-2719https://doi.org/10.15252/embj.201591440 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 Abstract Degradation of mitochondria via selective autophagy, termed mitophagy, contributes to mitochondrial quality and quantity control whose defects have been implicated in oxidative phosphorylation deficiency, aberrant cell differentiation, and neurodegeneration. How mitophagy is regulated in response to cellular physiology remains obscure. Here, we show that mitophagy in yeast is linked to the phospholipid biosynthesis pathway for conversion of phosphatidylethanolamine to phosphatidylcholine by the two methyltransferases Cho2 and Opi3. Under mitophagy-inducing conditions, cells lacking Opi3 exhibit retardation of Cho2 repression that causes an anomalous increase in glutathione levels, leading to suppression of Atg32, a mitochondria-anchored protein essential for mitophagy. In addition, loss of Opi3 results in accumulation of phosphatidylmonomethylethanolamine (PMME) and, surprisingly, generation of Atg8–PMME, a mitophagy-incompetent lipid conjugate of the autophagy-related ubiquitin-like modifier. Amelioration of Atg32 expression and attenuation of Atg8–PMME conjugation markedly rescue mitophagy in opi3-null cells. We propose that proper regulation of phospholipid methylation is crucial for Atg32-mediated mitophagy. Synopsis The phosphatidylethanolamine (PE) methyltransferase Cho2 is repressed in yeast under non-fermentable conditions, causing a decrease in the glutathione (GSH) levels and subsequent induction of the mitophagy protein Atg32. Loss of phospholipid methyltransferase Opi3 delays Cho2 repression. Prolonged Cho2 expression causes accumulation of its product phosphatidylmonomethylethanolamine (PMME) and increases the GSH levels, leading to suppression of Atg32 induction and disruption of mitophagy. Atg8, known to be PE-linked during autophagosome formation, is aberrantly conjugated to PMME in opi3-null cells. Atg8–PMME cannot be efficiently delipidated by the cysteine protease Atg4, thereby attenuating Atg8 recycling and thus mitophagy. Introduction Mitochondria act as dynamic, multitasking platforms that constantly remodel their shape and activity in order to adjust their metabolism and signaling for cell homeostasis, death, and differentiation (Kasahara & Scorrano, 2014; Labbe et al, 2014; Mishra & Chan, 2014). The energy-converting organelles properly change their quantity in response to diverse intra- and extracellular cues (Nunnari & Suomalainen, 2012). When cells need high levels of ATP, mitochondrial biogenesis is strongly activated to replicate pre-existing organelles. Conversely, a decrease in cellular energy demands or hypoxia leads to suppression of their proliferation and induction of their turnover. Since mitochondria also generate reactive oxygen species from the respiratory chain, their quality must be maintained by safeguarding against oxidative stress or eliminating damaged organelles (Rugarli & Langer, 2012; Youle & van der Bliek, 2012). Numerous studies in the last decade have established that mitochondrial degradation depends on autophagy, a membrane trafficking process in which newly generated cup-shaped structures, called isolation membranes, sequester cytoplasmic constituents as cargoes and expand to form autophagosomes, double membrane-bound vesicles that ultimately fuse with lysosomes (or vacuoles in yeast) to digest the cargoes by hydrolytic enzymes for recycling (Mizushima, 2011; Weidberg et al, 2011). Importantly, autophagy can mediate degradation of mitochondria in a highly selective manner, thereby contributing to mitochondrial quality and quantity control (Okamoto, 2014; Rogov et al, 2014; Stolz et al, 2014). Defects in this catabolic process, termed mitophagy, are associated with mitochondrial dysfunction and a myriad of disorders including aberrant hematopoiesis and neuronal degeneration, underscoring its physiological relevance (Ney, 2011; Hirota et al, 2012; Narendra et al, 2012). Selectivity of mitophagy is determined by various landmark molecules on the surface of mitochondria that recruit core autophagy-related (Atg) proteins essential for autophagosome biogenesis. Despite the diversity of landmark molecules, the basic principles underlying mitophagy are conserved among eukaryotes (Youle & Narendra, 2011; Liu et al, 2014). There are two major types of mitophagy, receptor- and ubiquitin-mediated processes (Okamoto, 2014). In the budding yeast Saccharomyces cerevisiae, mitophagy requires Atg32, a single-pass membrane protein anchored to mitochondria (Kanki et al, 2009; Okamoto et al, 2009). Loss of Atg32 results in mitochondrial genome instability during prolonged starvation and chronological lifespan shortening under calorie restriction, highlighting the biological significance of mitophagy (Kurihara et al, 2012; Richard et al, 2013). Atg32 serves as a receptor that interacts with Atg8, a phosphatidylethanolamine (PE)-conjugated ubiquitin-like modifier necessary for formation of autophagosomes, and Atg11, a selective autophagy-specific scaffold required for assembly of core Atg proteins (Kanki & Klionsky, 2008; Kanki et al, 2009; Okamoto et al, 2009). The cytosolically exposed N-terminal domain of Atg32 is both necessary and sufficient for these protein–protein interactions and mitophagy (Aoki et al, 2011; Kondo-Okamoto et al, 2012). Phosphorylation of Atg32 stabilizes Atg32–Atg11 interaction, which is a crucial step for promoting degradation of mitochondria (Aoki et al, 2011; Kondo-Okamoto et al, 2012; Kanki et al, 2013). In addition to this post-translational modification, induction of the ATG32 gene expression is a key determinant for mitophagy efficiency (Okamoto et al, 2009; Aihara et al, 2014). Indeed, the Atg32 protein levels increase to facilitate mitophagy under non-fermentable and nitrogen-depleted conditions (Okamoto et al, 2009; Aoki et al, 2011; Eiyama et al, 2013). Although oxidative stress seems to trigger upregulation of Atg32 in respiring cells (Okamoto et al, 2009), how this signaling event is established remains largely unknown. Emerging evidence reveals that the receptor-mediated mitophagy involves an evolutionarily conserved mode of protein–protein interactions between receptor molecules and Atg8 family members including LC3, GABARAP, and GATE-16 (Noda et al, 2010; Birgisdottir et al, 2013). In mammals, NIX, BNIP3, and FUNDC1 are mitochondria-anchored receptors that interact with these Atg8 homologs and promote mitophagy in a manner similar to Atg32-mediated process (Liu et al, 2014). Notably, Atg32–Atg8 interaction contributes to mitophagy, implying the relevance of its evolutionary conservation (Kondo-Okamoto et al, 2012). How recruitment of Atg8 to the mitochondrial surface assists formation of autophagosomes surrounding mitochondria has not been defined. In addition, whether lipidation of Atg8, a prerequisite step for acting on the autophagosomal membrane, is regulated upon mitophagy remains to be investigated. Here, we have uncovered a previously unappreciated link between phospholipid biosynthesis and mitophagy. Loss of Opi3, an endoplasmic reticulum (ER)-localized phospholipid methyltransferase acting in biosynthesis of phosphatidylcholine (PC) from PE (Henry et al, 2012), impairs Atg32 expression and Atg8 lipidation, synergistically leading to strong mitophagy deficiencies. These defects result from aberrantly elevated synthesis of the major antioxidant glutathione (GSH), and atypical conjugation of Atg8 to phosphatidylmonomethylethanolamine (PMME). Thus, our findings demonstrate that mitophagy induction is regulated in concert with phospholipid methylation and implicate that PE-to-PC biosynthesis disorders may raise the risk of disrupting autophagosome biogenesis. Results Opi3 is required for efficient mitophagy To elucidate how Opi3 functions in autophagy-dependent degradation of mitochondria, we examined opi3-null mutants by microscopy and Western blotting. Cells expressing mitochondrial matrix-targeted GFP (mito-GFP) and vacuole-anchored mCherry (Vph1–mCherry) were grown to stationary phase in medium containing non-fermentable carbon source and observed using fluorescence microscopy. Since GFP is quite resistant against vacuolar proteases, its fluorescence can be retained in the vacuole. As described previously (Okamoto et al, 2009), wild-type cells displayed vacuole-localized green signals, indicating transport of mitochondria to the vacuole in a manner dependent on the mitophagy receptor Atg32 (Fig 1A). By contrast, mito-GFP was barely overlapped with Vph1–mCherry in cells lacking Opi3 (Fig 1A). Next, processing of mitochondrial matrix-localized dihydrofolate reductase–mCherry (mito-DHFR–mCherry) was monitored for cells grown under the same conditions. Upon mitophagy, mitochondria are degraded, and mito-DHFR–mCherry is processed to become a free mCherry in the vacuole (Kondo-Okamoto et al, 2012). Consistent with our microscopic observations, loss of Opi3 resulted in a strong reduction in mitochondrial degradation (31% compared with wild-type cells) (Fig 1B and C), which could be effectively rescued by reintroduction of the OPI3 gene into the null mutant (Fig EV1A and B). We also found the higher protein levels of Por1 and Mge1, authentic mitochondrial outer membrane and matrix proteins, respectively, in cells lacking Opi3 compared to those in wild-type cells under prolonged respiratory growth, further confirming the mitophagy defects (Fig 1D and E). Figure 1. Cells lacking Opi3 exhibit severe mitophagy defects Representative images of mitochondria-targeted GFP (mito-GFP) and vacuole-targeted mCherry (Vph1–mCherry) patterns. Wild-type, atg32Δ, and opi3Δ cells were grown in glycerol medium (Gly) for 72 h and observed under a fluorescence microscope. Scale bar, 2 μm. Wild-type, atg32Δ, and opi3Δ cells expressing mito-DHFR–mCherry (depicted by arrowhead) were cultured as in (A). Cells were harvested at the indicated time points and subjected to Western blotting. Generation of free mCherry (depicted by arrow) indicated transport of mitochondria to the vacuole. Pgk1 was monitored as a loading control. The amounts of free mCherry detected in (B) were quantified at the indicated time points. The signal intensity value of free mCherry in wild-type cells at 72 h was set to 100%. Data represent the averages of three experiments, with error bars indicating standard deviations. Authentic mitochondrial proteins in wild-type and opi3Δ cells cultured as in (B) were analyzed by Western blotting. Por1 is an outer membrane protein. Mge1 and Ssc1 are matrix proteins. Pgk1 was monitored as a loading control. The Por1 protein levels analyzed in (D) were quantified at the indicated time points. The signal intensity values in wild-type and opi3Δ cells at 24 h were set to 100%. Data represent the averages of three experiments, with error bars indicating standard deviations. Representative images of mitochondria-targeted mCherry (mito-mCherry) patterns. ypt7Δ, ypt7Δ atg7Δ, and ypt7Δ opi3Δ cells grown in glycerol medium (Gly) for 72 h were shifted to fresh dextrose (Dex) medium for 3 h and observed as in (A). Mitophagosomes are depicted by arrows. Scale bar, 2 μm. A schematic diagram of the phospholipid methylation pathway in the yeast S. cerevisiae. Cho2 and Opi3 are methyltransferases to synthesize phosphatidylcholine (PC) from phosphatidylethanolamine (PE). PMME, phosphatidylmonomethylethanolamine; PDME, phosphatidyldimethylethanolamine. Wild-type, opi3Δ, cho2Δ, and opi3Δ cho2Δ cells expressing mito-DHFR–mCherry were cultured and analyzed as in (A) and (B), respectively. Pgk1 was monitored as a loading control. The amounts of free mCherry detected in (H) were quantified as in (C). The signal intensity value of free mCherry in wild-type cells at 72 h was set to 100%. Data represent the averages of three experiments, with error bars indicating standard deviations. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Loss of Opi3 leads to minor defects in bulk autophagy, pexophagy, and the Cvt pathway Reintroduction of an OPI3 gene into the opi3-null mutant rescues mitophagy. Wild-type, opi3Δ, and opi3Δ cells with a chromosomally reintroduced OPI3 gene expressing mito-DHFR–mCherry (depicted by arrowhead) were grown in glycerol medium (Gly). Cells were harvested at the indicated time points and subjected to Western blotting. Generation of free mCherry (depicted by arrow) indicated transport of mitochondria to the vacuole. The cytosolic protein Pgk1 was monitored as a loading control. The amounts of free mCherry detected in (A) were quantified at the indicated time points. The signal intensity value of free mCherry in wild-type cells at the 72-h time point was set to 100%. Data represent the averages of three experiments, with error bars indicating standard deviations. Deletion of the OPI3 gene does not affect the Cvt pathway under fermentable conditions. Wild-type, opi3Δ, cho2Δ, opi3Δ cho2Δ, and atg7Δ cells expressing mito-DHFR–mCherry were grown in dextrose medium (Dex) and harvested at mid-log phase. The vacuolar aminopeptidase Ape1 is synthesized as a precursor (p) in the cytosol, transported to the vacuole via the Cvt pathway, and processed into a mature form (m) in the vacuolar lumen. Pgk1 was monitored as a loading control. Pexophagy is only slightly reduced in starving cells lacking Opi3. Wild-type, atg1Δ, and opi3Δ cells expressing Pot1–mCherry (a peroxisome marker depicted by arrowhead) and Atg36–9Myc (a protein essential for pexophagy), and atg36Δ cells expressing Pot1–mCherry were grown in oleic acid medium, transferred to dextrose medium lacking nitrogen (−N), and harvested at the indicated time points. Generation of free mCherry (depicted by arrow) indicated transport of peroxisomes to the vacuole. Pgk1 was monitored as a loading control. The amounts of free mCherry detected in (D) were quantified at the indicated time points. The signal intensity value of free mCherry in wild-type cells at the 6-h time point was set to 100%. Data represent the averages of three experiments, with error bars indicating standard deviations. Opi3 is dispensable for autophagy. Wild-type, atg7Δ, and opi3Δ cells expressing Pho8Δ60 (a cytosolic alkaline phosphatase for monitoring autophagy activity) pre-grown in nutrient-rich dextrose medium (YPD) were incubated in synthetic dextrose medium lacking nitrogen (SD-N) for 6 h and subjected to alkaline phosphatase (ALP) assays. Pho8Δ60 is transported to the vacuole via autophagy and processed into an active form in the vacuolar lumen. Starvation-induced autophagy flux is normal in the absence of Opi3. Wild-type, opi3Δ, and atg7Δ cells expressing GFP–Atg8 (depicted by arrowhead) were grown to mid-log phase in rich dextrose medium, incubated for the indicated time points in starvation medium (−N), and subjected to Western blotting. Generation of free GFP (depicted by arrow) indicates transport of autophagosomes to the vacuole. Atg7 is an E1 enzyme essential for autophagy. Pgk1 was monitored as a loading control. The amounts of free GFP detected in (G) were quantified at the 4- and 12-h time points. The signal intensity value of free GFP in wild-type cells at the 12-h time point was set to 100%. Data represent the averages of three experiments, with error bars indicating standard deviations. Download figure Download PowerPoint It remains conceivable that sequestration of mitochondria occurs normally, whereas fusion of mitophagosomes to vacuoles is impaired in the opi3-null mutant. To test this possibility, we used a strain lacking Ypt7, a Rab family GTPase required for homotypic vacuole fusion. Deletion of the YPT7 gene blocks membrane vesicle fusion with the vacuole, resulting in accumulation of autophagosome-related structures in the cytosol. Under mitophagy-inducing conditions, cells lacking Ypt7 accumulate mitochondria-containing autophagic vesicles termed mitophagosomes. As described previously (Okamoto et al, 2009), when these cells were shifted to medium containing fermentable carbon source, mitochondrial fragments were fused to become tubules that could be morphologically distinguished from mitophagosomes (Fig 1F). Under the same conditions, mitochondrial vesicles were hardly seen in cells lacking Opi3 or Atg7 (Fig 1F). These observations suggest that mitophagosome formation is impaired in the absence of Opi3. Next, we investigated the cytoplasm-to-vacuole targeting (Cvt) pathway, peroxisome-specific autophagy (pexophagy), and non-selective autophagy in the opi3-null mutant. The Cvt pathway is a constitutively active, autophagy-related process selective for several vacuolar proteins such as Ape1, a vacuolar aminopeptidase. Ape1 is synthesized as a precursor in the cytosol and processed into a mature form in the vacuole. When vegetatively grown in medium containing fermentable carbon source, the opi3-null mutant exhibited wild-type-like Ape1 processing (Fig EV1C). For pexophagy, peroxisomal matrix-localized Pot1–mCherry was monitored in cells shifted from oleic acid medium to nitrogen starvation medium. Upon pexophagy, peroxisomes are degraded, and Pot1–mCherry is processed to become a free mCherry in the vacuole. We found a partial defect in peroxisomal degradation, which was much weaker than that in mitophagy, in cells lacking Opi3 (Fig EV1D and E). Finally, autophagic activity during nitrogen starvation was assessed with a well-established alkaline phosphatase assay. Even in the absence of Opi3, cytoplasmic constituents were non-selectively transported to the vacuole with near wild-type efficiency (Fig EV1F). We also monitored vacuolar protease-dependent processing of GFP–Atg8 and found wild-type-like autophagy flux in the opi3-null mutant under starved conditions (Fig EV1G and H). Collectively, these results support the idea that loss of Opi3 leads to dominant defects in degradation of mitochondria. Given the fact that Opi3 plays a critical role in PE-to-PC conversion (Henry et al, 2012), we initially hypothesized that altered phospholipid composition such as a decrease in PC content is the primary cause of mitophagy deficiencies in the opi3-null mutant. To test this idea, we monitored degradation of mitochondria in cells lacking Cho2, another ER-localized phospholipid methyltransferase acting upstream of Opi3, that are also defective in PE-to-PC conversion (Fig 1G) (Henry et al, 2012). Unexpectedly, mitochondria were substantially degraded in the cho2- and opi3 cho2-null mutants (95 and 74%, respectively, compared to wild-type cells) (Fig 1H and I). Thus, it seems unlikely that mitophagy depends on PC homeostasis. PMME and GSH are elevated in cells lacking Opi3 under mitophagy-inducing conditions To elucidate how single loss of Opi3 leads to severe defects in mitochondrial degradation, we first examined the expression profiles of Cho2 and Opi3. To our surprise, when shifted to medium containing non-fermentable carbon source, wild-type cells exhibited a marked reduction in the Cho2 protein levels (Fig 2A). Notably, this downregulation was delayed in opi3-null cells (Fig 2A). These results suggest that, in the absence of Opi3, Cho2 functions negatively affect mitophagy. Figure 2. PMME and GSH increase in opi3-null cells under mitophagy-inducing conditions Wild-type, opi3Δ, cho2Δ, and opi3Δ cho2Δ cells expressing mito-DHFR–mCherry were cultured and analyzed as in Fig 1B. Cho2-specific and Cho2-non-specific bands are depicted by arrow and asterisk, respectively. Pgk1 was monitored as a loading control. A schematic diagram of PC biosynthesis from PE in budding yeast. PE, phosphatidylethanolamine; PMME, phosphatidylmonomethylethanolamine; PDME, phosphatidyldimethylethanolamine; PC, phosphatidylcholine; SAM, S-adenosyl methionine; SAH, S-adenosyl homocysteine (see text for details). A schematic diagram of the transmethylation cycle and transsulfuration–GSH pathway. Hcy, homocysteine; BSO, buthionine sulfoximine (see text for details). One-dimensional thin-layer chromatography (1-D TLC) for wild-type, opi3Δ, cho2Δ, and opi3Δ cho2Δ cells. Cells were grown in synthetic media containing dextrose (Dex) or glycerol (Gly), or subjected to nitrogen starvation (−N) in the presence of 32Pi, and collected at the indicated time points. Total cellular phospholipids were then extracted in the organic phase and separated by 1-D TLC. CL, cardiolipin; PA, phosphatidic acid; PC, phosphatidylcholine; PDME, phosphatidyldimethyl-ethanolamine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PMME, phosphatidylmonomethylethanolamine; PS, phosphatidylserine. The radioactive spots of PMME and PC in (D) were quantified using densitometry. The signal intensity value of total phospholipids in each sample was set to 100%. Wild-type, opi3Δ, cho2Δ, and opi3Δ cho2Δ cells were grown in synthetic glycerol medium for 24 h, collected, and subjected to quantification of total cellular reduced glutathione (GSH). Data represent the averages of three experiments, with error bars indicating standard deviations. Wild-type, opi3Δ, cho2Δ, and opi3Δ cho2Δ cells expressing a chromosomally Myc-tagged Ctt1, a cytosolic catalase, were cultured and analyzed as in (A). Pgk1 was monitored as a loading control. The Ctt1–Myc protein levels analyzed in (F) were quantified at the indicated time points. The signal intensity values in wild-type cells at 24 h were set to 1.0. Data represent the averages of three experiments, with error bars indicating standard deviations. Download figure Download PowerPoint As Cho2 generates phosphatidylmonomethylethanolamine (PMME), one of the substrates for Opi3, it seems plausible that this mono-methylated phospholipid accumulates in the cell if Opi3 functions are compromised (Fig 2B). We also noticed previous studies suggesting an increased level of PMME in an opi3 mutant (Greenberg et al, 1982, 1983). To confirm a high PMME content in our opi3-null strain, we analyzed total cellular phospholipid composition by one-dimensional thin-layer chromatography (1-D TLC), a method that allows separation of phospholipids with high resolution and reproducibility (Vaden et al, 2005). Cells under fermentable, non-fermentable (mitophagy-inducing), and nitrogen starvation conditions were labeled with 32Pi and subjected to phospholipid extraction. Our assays revealed strongly increased levels of PMME in opi3-null cells under all the aforementioned conditions, which was not the case in wild-type, cho2-null, and opi3 cho2-null cells (Fig 2D and E). As expected, the PC contents were significantly reduced in opi3-, cho2-, and opi3 cho2-null cells (Fig 2D and E), but still partially maintained via the Kennedy pathway, an alternative process for PC biosynthesis using choline and phosphoethanolamine (Henry et al, 2012). Cho2 and Opi3 catalyze transfer of a methyl group from S-adenosyl methionine (SAM) to PE, PMME, and PDME (Tehlivets, 2011; Henry et al, 2012) (Fig 2B). Concomitantly, each reaction converts SAM to S-adenosyl homocysteine (SAH), a precursor for homocysteine (Hcy) biosynthesis (Tehlivets et al, 2013) (Fig 2B and C). Hcy can then be utilized to generate methionine (Met) and SAM in the methylation cycle or subjected to produce cysteine (Cys) and GSH, a principal antioxidant, in the transsulfuration–GSH pathway (Tehlivets et al, 2013) (Fig 2C). Hence, we hypothesized that loss of Opi3 results in dysregulation of Cho2-mediated SAH production, ultimately facilitating GSH biosynthesis (Fig 2B and C). The cellular GSH content was indeed elevated in opi3-null cells under mitophagy-inducing conditions (1.5- to 3-fold increase compared with wild-type, cho2-null, and opi3 cho2-null cells) (Fig 2F). In addition, we investigated the pro

The E2 enzyme Atg3 conjugates the ubiquitin‐like protein Atg8 to phosphatidylethanolamine (PE) to drive autophagosome formation in Saccharomyces cerevisiae . In this study, we show that Atg3 localizes to the pre‐autophagosomal structure (PAS) and the isolation membrane (IM), providing crucial evidence that Atg8‐PE conjugates are produced on these structures. We also find that mutations in the Atg8‐family interacting motif (AIM) of Atg3 significantly impairs the PAS/IM localization of Atg3, resulting in inefficient IM expansion. It is suggested that the AIM‐mediated PAS/IM localization of Atg3 facilitates membrane expansion in these structures probably by ensuring active production of Atg8‐PE on the membranes.

The ubiquitin-like protein Atg8, in its lipidated form, plays central roles in autophagy. Yet, remarkably, Atg8 also carries out lipidation-independent functions in non-autophagic processes. How Atg8 performs its moonlighting roles is unclear. Here we report that in the fission yeast Schizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae, the lipidation-independent roles of Atg8 in maintaining normal morphology and functions of the vacuole require its interaction with a vacuole membrane protein Hfl1 (homolog of human TMEM184 proteins). Crystal structures revealed that the Atg8-Hfl1 interaction is not mediated by the typical Atg8-family-interacting motif (AIM) that forms an intermolecular β-sheet with Atg8. Instead, the Atg8-binding regions in Hfl1 proteins adopt a helical conformation, thus representing a new type of AIMs (termed helical AIMs here). These results deepen our understanding of both the functional versatility of Atg8 and the mechanistic diversity of Atg8 binding.

In Saccharomyces cerevisiae , under nitrogen‐starvation conditions, the α‐mannosidase Ams1 is recognized by the autophagic receptor Atg34 and transported into the vacuole, where it functions as an active enzyme. In this study, we identified Hrr25 as the kinase that phosphorylates Atg34 under these conditions. Hrr25‐mediated phosphorylation does not affect the interaction of Atg34 with Ams1, but instead promotes Atg34 binding to the adaptor protein Atg11, which recruits the autophagy machinery to the Ams1–Atg34 complex, resulting in activation of the vacuolar transport of Ams1. Our findings reveal the regulatory mechanism of a biosynthetic pathway mediated by the autophagy machinery.

Selective autophagy mediates the degradation of various cargoes, including protein aggregates and organelles, thereby contributing to cellular homeostasis. Cargo receptors ensure selectivity by tethering specific cargo to lipidated Atg8 at the isolation membrane. However, little is known about the structural requirements underlying receptor-mediated cargo recognition. Here, we report structural, biochemical, and cell biological analysis of the major selective cargo protein in budding yeast, aminopeptidase I (Ape1), and its complex with the receptor Atg19. The Ape1 propeptide has a trimeric coiled-coil structure, which tethers dodecameric Ape1 bodies together to form large aggregates. Atg19 disassembles the propeptide trimer and forms a 2:1 heterotrimer, which not only blankets the Ape1 aggregates but also regulates their size. These receptor activities may promote elongation of the isolation membrane along the aggregate surface, enabling sequestration of the cargo with high specificity.

In macroautophagy, cellular components are sequestered within autophagosomes and transported to lysosomes/vacuoles for degradation. Although phosphatidylinositol 3-kinase complex I (PI3KCI) plays a pivotal role in the regulation of autophagosome biogenesis, little is known about how this complex localizes to the pre-autophagosomal structure (PAS). In Saccharomyces cerevisiae, PI3KCI is composed of PI3K Vps34 and conserved subunits Vps15, Vps30, Atg14, and Atg38. In this study, we discover that PI3KCI interacts with the vacuolar membrane anchor Vac8, the PAS scaffold Atg1 complex, and the pre-autophagosomal vesicle component Atg9 via the Atg14 C-terminal region, the Atg38 C-terminal region, and the Vps30 BARA domain, respectively. While the Atg14–Vac8 interaction is constitutive, the Atg38–Atg1 complex interaction and the Vps30–Atg9 interaction are enhanced upon macroautophagy induction depending on Atg1 kinase activity. These interactions cooperate to target PI3KCI to the PAS. These findings provide a molecular basis for PAS targeting of PI3KCI during autophagosome biogenesis.

In autophagy, a membrane cisterna called the isolation membrane expands, bends, becomes spherical, and closes to sequester cytoplasmic constituents into the resulting double-membrane vesicle autophagosome for lysosomal/vacuolar degradation. Here, we discover a mechanism that allows the isolation membrane to expand with a large opening to ensure non-selective cytoplasm sequestration within the autophagosome. A sorting nexin complex that localizes to the opening edge of the isolation membrane plays a critical role in this process. Without the complex, the isolation membrane expands with a small opening that prevents the entry of particles larger than about 25 nm, including ribosomes and proteasomes, although autophagosomes of nearly normal size eventually form. This study sheds light on membrane morphogenesis during autophagosome formation and selectivity in autophagic degradation.

"Arrest sequence" of Escherichia coli SecM interacts with the ribosomal exit tunnel and arrests its own translation elongation, which is released by cotranslational export of the nascent SecM chain. This property of SecM is essential for the basal and regulated expression of SecA. Here we report that SecM has an additional role of facilitating SecA activities. Systematic determinations of the SecA-abundance-protein export relationships of cells with different SecA contents revealed that SecA was less functional when SecM was absent from the upstream region of the secM-secA message, when SecM had the arrest-defective mutation, and also when SecM lacked the signal sequence. These results suggest that cotranslational targeting of nascent SecM to the translocon plays previously unrecognized roles of facilitating the formation of functional SecA molecules. Biosynthesis in the vicinity of the membrane and the Sec translocon will be beneficial for this multiconformation ATPase to adopt ready-to-function conformations.

Autophagy is a bulk degradation system highly conserved among eukaryotic cells and plays crucial roles in a wide range of physiological and pathological situations. Remarkably, this process involves two ubiquitin-like (Ubl) conjugation systems. Here, we describe two sodium dodecyl sulfate-polyacrylamide gel electrophoresis techniques to analyze these systems: one that allows separation of the Ubl protein Atg8 conjugated to the lipid phosphatidylethanolamine from its unlipidated form, and the other by which otherwise labile thioester intermediates between Atg8 and either the E1 enzyme Atg7 or the E2 enzyme Atg3 are stably preserved during electrophoresis, and thus easily detected by following protein visualization. Especially, the latter technique is also ubiquitously applicable for studies on conjugation reactions of ubiquitin (Ub) and other Ubl proteins.

During macroautophagy, cytoplasmic constituents are engulfed by autophagosomes. Lysosomes fuse with closed autophagosomes but not with unclosed intermediate structures. This is achieved in part by the late recruitment of the autophagosomal SNARE syntaxin 17 (STX17) to mature autophagosomes. However, how STX17 recognizes autophagosome maturation is not known. Here, we show that this temporally regulated recruitment of STX17 depends on the positively charged C-terminal region of STX17. Consistent with this finding, mature autophagosomes are more negatively charged compared with unclosed intermediate structures. This electrostatic maturation of autophagosomes is likely driven by the accumulation of phosphatidylinositol 4-phosphate (PI4P) in the autophagosomal membrane. Accordingly, dephosphorylation of autophagosomal PI4P prevents the association of STX17 to autophagosomes. Furthermore, molecular dynamics simulations support PI4P-dependent membrane insertion of the transmembrane helices of STX17. Based on these findings, we propose a model in which STX17 recruitment to mature autophagosomes is temporally regulated by a PI4P-driven change in the surface charge of autophagosomes.

SecA initiates protein translocation by interacting with ATP, preprotein, and the SecYEG membrane components. Under such conditions, it undergoes a conformational change characterized as membrane insertion, which is then followed by hydrolysis of ATP, enabling the release of the preprotein and deinsertion of SecA itself for the next cycle of reactions. Without ongoing translocation, the ATPase activity of SecA is kept very low. Previously, it was shown that the C-terminal 34-kDa domain of SecA interacts with the N-terminal 68-kDa ATPase domain to down-regulate the ATPase. Here, we show, using a deregulated SecA mutant, that the intrinsic ATPase activity is subject to dual inhibitory mechanisms. Thus, the proposed second ATP-binding domain down-regulates the ATPase activity executed by the primary ATPase domain. This regulation, within the N-terminal ATPase domain, operates independently of the C-terminal domain-mediated regulation. The absence of both the mechanisms resulted in a 50-fold elevation of translocation-uncoupled ATP hydrolysis. SecA initiates protein translocation by interacting with ATP, preprotein, and the SecYEG membrane components. Under such conditions, it undergoes a conformational change characterized as membrane insertion, which is then followed by hydrolysis of ATP, enabling the release of the preprotein and deinsertion of SecA itself for the next cycle of reactions. Without ongoing translocation, the ATPase activity of SecA is kept very low. Previously, it was shown that the C-terminal 34-kDa domain of SecA interacts with the N-terminal 68-kDa ATPase domain to down-regulate the ATPase. Here, we show, using a deregulated SecA mutant, that the intrinsic ATPase activity is subject to dual inhibitory mechanisms. Thus, the proposed second ATP-binding domain down-regulates the ATPase activity executed by the primary ATPase domain. This regulation, within the N-terminal ATPase domain, operates independently of the C-terminal domain-mediated regulation. The absence of both the mechanisms resulted in a 50-fold elevation of translocation-uncoupled ATP hydrolysis. intramolecular regulator of ATP hydrolysis C-terminal 34-kDa fragment Translocation of newly synthesized preproteins across theEscherichia coli cytoplasmic membrane is facilitated by the Sec translocase. The membrane-integrated SecYEG component provides a translocation pathway, and SecA drives the movement of the preprotein. SecA is a dimeric ATPase, containing 901 amino acid residues in each subunit (1Oliver D.B. Beckwith J. J. Bacteriol. 1982; 150: 686-691Crossref PubMed Google Scholar), which consists of a C-terminal 34 kDa domain and an N-terminal ATPase domain (68 kDa). The ATPase domain has two proposed ATP-binding sites (2Mitchell C. Oliver D. Mol. Microbiol. 1993; 10: 483-497Crossref PubMed Scopus (188) Google Scholar), the high affinity site (NBS I) and the low affinity site (NBS II). Whereas NBS I acts as the primary ATPase domain, the role of the NBS II region is less clear (2Mitchell C. Oliver D. Mol. Microbiol. 1993; 10: 483-497Crossref PubMed Scopus (188) Google Scholar, 3Economou A. Pogliano J.A. Beckwith J. Oliver D.B. Wickner W. Cell. 1995; 83: 1171-1181Abstract Full Text PDF PubMed Scopus (272) Google Scholar). The reaction cycle of SecA is accompanied by its striking conformational changes, in which the SecA-preprotein complex inserts into the membrane in response to ATP binding followed by deinsertion of SecA in response to ATP hydrolysis (3Economou A. Pogliano J.A. Beckwith J. Oliver D.B. Wickner W. Cell. 1995; 83: 1171-1181Abstract Full Text PDF PubMed Scopus (272) Google Scholar, 4Economou A. Wickner W. Cell. 1994; 78: 835-843Abstract Full Text PDF PubMed Scopus (483) Google Scholar). In this way, SecA seems to drive the movement of an ∼20-amino acid segment of preprotein into the membrane (5Schiebel E. Driessen A.J. Hartl F.U. Wickner W. Cell. 1991; 64: 927-939Abstract Full Text PDF PubMed Scopus (374) Google Scholar). The insertion of SecA was originally defined by thein vitro generation of a 30-kDa C-terminal fragment that was protected by membrane from proteolysis (4Economou A. Wickner W. Cell. 1994; 78: 835-843Abstract Full Text PDF PubMed Scopus (483) Google Scholar, 6Price A. Economou A. Duong F. Wickner W. J. Biol. Chem. 1996; 271: 31580-31584Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). It was shown later that some N-terminal portions of SecA insert as well, because they were also protected from an external protease (7Eichler J. Wickner W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5574-5581Crossref PubMed Scopus (73) Google Scholar) or accessible from the periplasmic side (8Ramamurthy V. Oliver D. J. Biol. Chem. 1997; 272: 23239-23246Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 9Kim Y.J. Rajapandi T. Oliver D. Cell. 1994; 78: 845-853Abstract Full Text PDF PubMed Scopus (146) Google Scholar). SecA exhibits three levels of ATPase activities (10Lill R. Dowhan W. Wickner W. Cell. 1990; 60: 271-280Abstract Full Text PDF PubMed Scopus (466) Google Scholar). Although its intrinsic activity is very low, it is activated significantly by membranes or anionic phospholipids; the latter activity is called “membrane ATPase.” In the presence of both a preprotein and membrane vesicles containing functional SecYEG complexes, ATPase activity is enhanced markedly. This activity, referred to as “translocation ATPase,” should result from the SecA reaction cycles outlined above. The present work was aimed at elucidating the mechanisms by which the intrinsic ATPase activity of SecA is kept extremely low. Previous studies (6Price A. Economou A. Duong F. Wickner W. J. Biol. Chem. 1996; 271: 31580-31584Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 11Song M. Kim H. J. Biochem. 1997; 122: 1010-1018Crossref PubMed Scopus (28) Google Scholar, 12Karamanou S. Vrontou E. Sianidis G. Baud C. Roos T. Kuhn A. Politou A.S. Economou A. Mol. Microbiol. 1999; 34: 1133-1145Crossref PubMed Scopus (118) Google Scholar) indicate that SecA ATPase is down-regulated by interdomain interactions between the C-terminal regulatory domain (34 kDa) and the N-terminal ATPase domain (68 kDa). A region in the C-terminal domain responsible for this interaction is called the intramolecular regulator of ATP hydrolysis (IRA)1 (12Karamanou S. Vrontou E. Sianidis G. Baud C. Roos T. Kuhn A. Politou A.S. Economou A. Mol. Microbiol. 1999; 34: 1133-1145Crossref PubMed Scopus (118) Google Scholar). We have identified secY mutations that do not sufficiently support the SecA functions (13Taura T. Yoshihisa T. Ito K. Biochimie (Paris ). 1997; 79: 517-521Crossref PubMed Scopus (30) Google Scholar, 14Matsumoto G. Yoshihisa T. Ito K. EMBO J. 1997; 16: 6384-6393Crossref PubMed Scopus (86) Google Scholar, 15Nakatogawa H. Mori H. Matsumoto G. Ito K. J. Biochem. 2000; 127: 1071-1079Crossref PubMed Scopus (7) Google Scholar). 2H. Mori and K. Ito, manuscript in preparation. Suppressor mutations in secA have been isolated using one of thesesecY mutations as the primary mutation (14Matsumoto G. Yoshihisa T. Ito K. EMBO J. 1997; 16: 6384-6393Crossref PubMed Scopus (86) Google Scholar, 16Matsumoto, G., Nakatogawa, H., Mori, H., and Ito, K. (2000) Genes Cells, in press.Google Scholar). Many of the SecA variants thus isolated proved to be “super-active” in that they suppressed a number of different sec mutations (16Matsumoto, G., Nakatogawa, H., Mori, H., and Ito, K. (2000) Genes Cells, in press.Google Scholar).2 Here, we characterized one such deregulated SecA variants biochemically. Our results show that, in addition to the IRA-mediated regulation, there is an independent regulatory mechanism within the N-terminal ATPase domain in which the NBS II region acts to down-regulate the ATPase activity of the NBS I region. Plasmid pKY173 carried wild-typesecA under the control of the lac promoter (14Matsumoto G. Yoshihisa T. Ito K. EMBO J. 1997; 16: 6384-6393Crossref PubMed Scopus (86) Google Scholar). pHM348 was a similar plasmid with the secA348mutation, causing an Asp-580 to Val alteration in SecA.2This mutant SecA protein is called SecAD580V in this paper. pNH14 encoded SecAD209N (with an Asp-209 to Asn alteration) and was constructed by site-directed mutagenesis (QuickChange mutagenesis kit, Stratagene) using the mutagenic primers 5′-GCACTATGCGCTGGTGAACGAAGTGGACTCC-3′ and its complementary strand (mutation to be introduced is underlined). pNH15 encoded SecAD580V-D209N (Asp-209 to Asn and Asp-580 to Val double mutant); an ∼800-base pair BglII-SphI segment of pHM348 was replaced by the corresponding fragment from pNH14. pNH11 encoded the N68 fragment of SecA in which the Leu-610 codon (CTG) was mutated to UAG; a BglII-MfeI segment of pKY173 was replaced by a product of polymerase chain reaction (template, pKY173; primers, 5′-AATGATTCGTAAAGATCTGCCGG-3′ and 5′-GCTTCAATTGGCTTCATACCCTATTTACGCATCATGCCGG-3′, mutation to be introduced is underlined) and BglII-MfeI digestion. pNH12 encoded SecAD580V-N68, which was constructed as above but based on pHM348. pNH13 encoded C34-His6; a polymerase chain reaction product using pKY173 as template and a set of primers, 5′-TATCCGGCATGCATCGTAAACTGGG-3′ and 5′-GCATGCTCTAGATTAATGATGATGATGATGATGTTGCAGGCGGCCATGGCACTG-3′, was digested with NsiI and XbaI and cloned into a derivative of pUC118, named pNH10, in which the first two codons oflacZα (ATGACC) were mutated to the NsiI recognition sequence (ATGCAT). Wild-type SecA was overproduced from pKY173 in strain GN45 (a MC4100 derivative carryingleu-82::Tn10 and F′ lacIQlacPL8 lacZ + Y + A +) as described previously (14Matsumoto G. Yoshihisa T. Ito K. EMBO J. 1997; 16: 6384-6393Crossref PubMed Scopus (86) Google Scholar). SecAD580V was similarly overproduced from pHM348 in a GN45 equivalent strain with the chromosomalsecA348 and secY205 mutations. SecAD209N and SecAD580V-D209N were overproduced from pNH13 and pNH14, respectively, in strain CK4706 (F-ΔlacU araD rpsL relA thi zab::Tn10 secA853–128) (17McFarland L. Francetic O. Kumamoto C.A. J. Bacteriol. 1993; 175: 2255-2262Crossref PubMed Google Scholar) harboring pSTD343 (a pACYC184-derived plasmid carryinglacI). 3Y. Akiyama, personal communication. Thus, the mutant forms of SecA were overproduced either in cells having the identical secA allele both on the chromosome and on plasmid or in the presence of a chromosomally encoded SecA variant that was easily distinguishable from the SecA species to be purified. SecA proteins were purified as described previously by Mitchell and Oliver (2Mitchell C. Oliver D. Mol. Microbiol. 1993; 10: 483-497Crossref PubMed Scopus (188) Google Scholar). N68 domains from wild-type SecA and from SecAD580V were overproduced from pNH11 and pNH12, respectively, in strain AD16 (Δpro-lac thi/F' lacIQ ZM15Y + pro + ) (18Kihara A. Akiyama Y. Ito K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4532-4536Crossref PubMed Scopus (212) Google Scholar) and purified essentially as described by Karamanou et al. (12Karamanou S. Vrontou E. Sianidis G. Baud C. Roos T. Kuhn A. Politou A.S. Economou A. Mol. Microbiol. 1999; 34: 1133-1145Crossref PubMed Scopus (118) Google Scholar), except that a phenyl-Superose column was used in place of phenyl-Sepharose and a MonoQ 5/5 column was used in place of Fast Flow Q-Sepharose. The His6-tagged C34 domain of SecA was overproduced from pNH13 in strain AD16 by culturing the cells in the presence of 1 mm isopropyl-1-thio-β-d-galactoside for 2 h. Cells were suspended in 20 mm Tris-HCl (pH 8.0) containing 0.5 m NaCl, 5 mm imidazole, 10 mm 2-mercaptoethanol, and 0.1 mmphenylmethylsulfonylfluoride and disrupted by sonication. The sample was then ultracentrifuged at 45,000 rpm for 30 min (Beckman, Ti-70 rotor), and supernatant was loaded onto a nickel-nitrilotriacetic acid-agarose column, which was washed with 50 mm Tris-HCl (pH 8.0), 60 mm imidazole, 0.5 m NaCl and then eluted with 50 mm Tris-HCl (pH 8.0), 1 mimidazole, 0.5 m NaCl. Intrinsic ATPase activity of SecA was assayed by means of the coupled enzyme reactions (19Tokuda H. Yamanaka M. Mizushima S. Biochem. Cell Biol. 1993; 195: 1415-1421Google Scholar). In this assay, ADP, the product of ATP hydrolysis, was recycled back to ATP by the coupled reactions of pyruvate kinase and lactate dehydrogenase, in which the accompanying NADH consumption was followed spectroscopically. The reaction at 37 °C was started by the addition of a purified preparation of SecA (2–10 μg) to 200 μl of reaction mixture that consisted of 50 mm Tris-HCl (pH 7.5), 2 mm MgSO4, 3 mmphosphoenolpyruvate, 0.25 mm NADH, 5 units of pyruvate kinase, 7.5 units of lactate dehydrogenase, and 1 mm (or other indicated concentrations) ATP. Absorbance at 340 nm due to NADH was monitored in real time. The rate of ATP hydrolysis was calculated from the linear phase of the decrease inA 340. We isolated a number of secA mutations as suppressors against cold-sensitive SecY defect caused by either the secY205(14Matsumoto G. Yoshihisa T. Ito K. EMBO J. 1997; 16: 6384-6393Crossref PubMed Scopus (86) Google Scholar, 15Nakatogawa H. Mori H. Matsumoto G. Ito K. J. Biochem. 2000; 127: 1071-1079Crossref PubMed Scopus (7) Google Scholar, 16Matsumoto, G., Nakatogawa, H., Mori, H., and Ito, K. (2000) Genes Cells, in press.Google Scholar) or the secY39 (20Baba T. Jacq A. Brickman E. Beckwith J. Taura T. Ueguchi C. Akiyama Y. Ito K. J. Bacteriol. 1990; 172: 7005-7010Crossref PubMed Google Scholar)2 mutation. Many of these mutations proved to be omnipotent in that they suppressed a number of different secY and other sec mutations. This class of mutant SecA proteins that we examined all possessed increased intrinsic and membrane ATPase activities; they are called super-active variants. The suppressor secAalterations were found to cluster within or adjacent to NBS II, the proposed low affinity ATP-binding domain of SecA. We characterized one such secA mutant, secA348, to understand the mechanisms of SecA regulation. This mutant had an amino acid alteration at Asp-580 (to Val), a hot spot for the super-active suppressor mutations (16Matsumoto, G., Nakatogawa, H., Mori, H., and Ito, K. (2000) Genes Cells, in press.Google Scholar).2 The wild-type and the mutant forms of SecA were overproduced and purified to characterize their enzymatic activities. Measurements of ATP hydrolysis in the presence of varying concentrations of the substrate (Fig. 1) showed that the V max of the intrinsic ATPase reaction was more than 10 times higher for SecAD580V than for the wild-type SecA (27.5 versus 2.2 nmol of ATP hydrolyzed/min/nmol of SecA-monomer). In contrast, the mutant enzyme had an ∼3-fold higher apparent K m value than the wild type (25.9 versus 8.4 μm). Thus, the mutational alteration only slightly affects the affinity for ATP but greatly enhances the rate of translocation-uncoupled ATP hydrolysis in the presence of sufficient concentrations of ATP. Although ATPase catalytic activity of SecA is supposed to be carried out by NBS I, the presence of the residue altered by thesecA348 mutation within NBS II raised a question of whether the mutationally enhanced ATPase activity was executed by NBS I or by NBS II itself. To examine this point, a known alteration in NBS I, Asp-209 to Asn (D209N), was combined with the D580V alteration. A previous study by Mitchell and Oliver (2Mitchell C. Oliver D. Mol. Microbiol. 1993; 10: 483-497Crossref PubMed Scopus (188) Google Scholar) showed that the D209N alteration inactivated the translocation ATPase activity but did not crucially affect the binding of the nucleotide. However, the intrinsic and membrane ATPase activities were unchanged or apparently enhanced, respectively, by this mutation (2Mitchell C. Oliver D. Mol. Microbiol. 1993; 10: 483-497Crossref PubMed Scopus (188) Google Scholar). The D209N form of SecA as well as the D580V-D209N double mutant form were purified, and their intrinsic ATPase activities were measured. As shown in Fig.2, the introduction of the D209N alteration into SecAD580V strikingly lowered the activity (Fig. 2, compare open circles and crosses). Now, the activity was identical with that of the D209N single mutant protein (Fig. 2, open triangles). Thus, the D580V effect was suppressed completely by the D209N amino acid change. As observed previously (2Mitchell C. Oliver D. Mol. Microbiol. 1993; 10: 483-497Crossref PubMed Scopus (188) Google Scholar), the intrinsic ATPase activity of the wild-type protein was no higher than that observed for the D209N mutant protein. These results indicates that the NBS I domain function is required for the enhanced ATPase activity observed in the SecAD580V mutant form of SecA with alteration in the NBS II region. The latter domain may have a regulatory role against the ATPase activity executed by the former domain. Previous studies show that the SecA ATPase is down-regulated by an intramolecular domain interaction (6Price A. Economou A. Duong F. Wickner W. J. Biol. Chem. 1996; 271: 31580-31584Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 11Song M. Kim H. J. Biochem. 1997; 122: 1010-1018Crossref PubMed Scopus (28) Google Scholar, 12Karamanou S. Vrontou E. Sianidis G. Baud C. Roos T. Kuhn A. Politou A.S. Economou A. Mol. Microbiol. 1999; 34: 1133-1145Crossref PubMed Scopus (118) Google Scholar), in which the C-terminal 34-kDa domain acts as a negative regulatory element, termed IRA (12Karamanou S. Vrontou E. Sianidis G. Baud C. Roos T. Kuhn A. Politou A.S. Economou A. Mol. Microbiol. 1999; 34: 1133-1145Crossref PubMed Scopus (118) Google Scholar). This regulation was reconstituted by combining separately the purified N-terminal 68-kDa domain and the C-terminal 34-kDa domain (12Karamanou S. Vrontou E. Sianidis G. Baud C. Roos T. Kuhn A. Politou A.S. Economou A. Mol. Microbiol. 1999; 34: 1133-1145Crossref PubMed Scopus (118) Google Scholar). Given this mechanism, two possibilities are conceivable for the mechanism responsible for the D580V enhancement of the ATPase activity. First, the NBS II region normally down-regulates the NBS I activity, and the mutation impairs this regulation. Second, the IRA action is mediated by its interaction with the NBS II region, leading to the inhibition of the NBS I ATPase activity, and the mutation abolishes the IRA-NBS II interaction. As reported by Price et al. (6Price A. Economou A. Duong F. Wickner W. J. Biol. Chem. 1996; 271: 31580-31584Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), mild trypsin treatment activates the intrinsic ATPase activity of SecA by cleaving it at a boundary between the N-terminal ATPase and the C-terminal regulatory domains. We observed about 6-fold elevation of the wild-type ATPase activity upon trypsin treatment (data not shown). When the SecAD580V mutant protein was similarly treated, the activity, which was already ∼8-fold higher than the wild-type enzyme, was further stimulated ∼6-fold (data not shown). This result suggested that the negative regulation by the C-terminal domain was still operating for the full-length mutant enzyme. It in turn suggested that the altered N-terminal domain itself had the increased ATPase activity. To substantiate this point, we constructed clones encoding the N-terminal 68-kDa fragment either with the wild-type sequence (SecA-N68) or with the D580V alteration (SecAD580V-N68), as well as the C-terminal 34-kDa fragment (C34). These fragments were purified using published procedures (12Karamanou S. Vrontou E. Sianidis G. Baud C. Roos T. Kuhn A. Politou A.S. Economou A. Mol. Microbiol. 1999; 34: 1133-1145Crossref PubMed Scopus (118) Google Scholar). SecA-N68 had ATPase activity that was ∼10-fold higher than the intact protein (Fig.3 A). Whereas the full-length SecAD580V preparation used in Fig. 3 was already ∼8-fold higher in the ATPase activity than in the wild type, SecAD580V-N68 showed a further 5.6-fold elevation over the SecAD580V full-length molecule (Fig. 3 A). Thus, SecAD580V-N68 was 4.5-fold higher than SecA-N68 and 47-fold higher than the intact wild-type SecA in its activity to hydrolyze ATP. We then examined whether the inhibitory action of the C34 fragment was still observed against SecAD580V-N68. As shown in Fig. 3 B, the addition of increasing concentrations of C34 resulted in increasing extents of inhibition of the N68 ATPase activity (solid circles) as reported previously (12Karamanou S. Vrontou E. Sianidis G. Baud C. Roos T. Kuhn A. Politou A.S. Economou A. Mol. Microbiol. 1999; 34: 1133-1145Crossref PubMed Scopus (118) Google Scholar). When ATPase activity of SecAD580V-N68 was examined similarly, it was also inhibited by C34 (Fig. 3 B, open circles). The dose-response curves of C34 against the wild-type N68 and SecAD580V-N68 were nearly identical (Fig. 3 B). Physical interactions between C34 and N68 were examined by mixing either wild-type N68 or SecAD580V-N68 with C34 having a C-terminally attached hexahistidine tag (Fig. 3 C). Upon nickel-nitrilotriacetic acid column chromatography, not only N68 but also SecAD580V-N68 was co-eluted with C34-His6 with imidazole. From these results, we conclude that the inter-domain interaction remained unimpaired in the SecAD580V mutant form of SecA. Thus, in the normal SecA protein, the C-terminal domain-dependent regulation is superimposed on the regulation within the N68 ATPase domain. According to the insertion/deinsertion model (4Economou A. Wickner W. Cell. 1994; 78: 835-843Abstract Full Text PDF PubMed Scopus (483) Google Scholar), ATP binding induces the membrane insertion of the SecA-preprotein complex, whereas hydrolysis of ATP occurs only after the above process. Consistent with this model, a nonhydrolyzable ATP analog can drive insertion of about 20 residues of preprotein into the membrane (5Schiebel E. Driessen A.J. Hartl F.U. Wickner W. Cell. 1991; 64: 927-939Abstract Full Text PDF PubMed Scopus (374) Google Scholar). Thus, initiation of translocation is a prerequisite for the SecA-catalyzed ATP hydrolysis. Indeed, intrinsic ATPase of SecA, in the absence of preprotein and membrane, is kept very low. Although the SecA ATPase activity is stimulated significantly by the presence of membranes or anionic phospholipids (membrane ATPase), it is enhanced dramatically by the presence of both preprotein and SecYEG membrane vesicles (translocation ATPase). The present study has focused on the problem of how the intrinsic ATPase activity was kept extremely low in the normal SecA protein. Such information will then be directly relevant to the problem of how this ATPase is activated in the presence of preproteins and the SecYEG integral membrane channel components. We have shown in this paper that SecA ATPase is down-regulated by dual regulatory mechanisms that work independently. The SecAD580V alteration of Asp580 strikingly enhances the translocation-uncoupled ATP hydrolysis activity of SecA. The enhanced activity can be ascribed to the catalysis carried out by the NBS I ATPase site, because the Asp-209 to Asn mutation of the Walker motif in NBS I abolishes it. Because the D580V mutational effect was observed with the isolated N68 fragment, the NBS II region appears to have a direct role in down-regulating the NBS I activity. According to Ramamurthy and Oliver (8Ramamurthy V. Oliver D. J. Biol. Chem. 1997; 272: 23239-23246Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), the NBS II region is included in or close to the regions that are accessible from the periplasmic side of the membrane under certain conditions. It is possible that, when SecA is in the resting state, the NBS II region interacts with the NBS I catalytic region to suppress the intrinsic ATP hydrolysis. Binding of ATP and preprotein as well as interaction with the SecYEG channel components will then trigger the conformational changes of SecA leading to its “membrane-inserted” state (7Eichler J. Wickner W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5574-5581Crossref PubMed Scopus (73) Google Scholar, 8Ramamurthy V. Oliver D. J. Biol. Chem. 1997; 272: 23239-23246Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). This conformational change will simultaneously allow the dissociation of NBS II from NBS I and the resulting release of the inhibition of ATP hydrolytic activity. In the SecAD580V mutant protein, the alteration in the central region in NBS II may disturb the NBS II-NBS I interaction that is required for down-regulating the NBS I ATPase. Thus, in the super-active class of SecA mutants, the ATPase activation step is bypassed, which may make the mutant SecA work better than wild-type SecA in combination with a partially defective channel component that only poorly activates SecA. The regulatory mechanism that operates within the N68 ATPase domain is not the sole mechanism that regulates the intrinsic ATPase of SecA. Our results indicate that the IRA-mediated regulation works independently. The 34-kDa C-terminal regulatory domain largely overlaps the 30-kDa membrane insertion domain of SecA (6Price A. Economou A. Duong F. Wickner W. J. Biol. Chem. 1996; 271: 31580-31584Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Thus, a mechanism similar to that discussed above for the regulatory function of NBS II can be considered for the down-regulation exerted by the C-terminal domain as well; only under the active translocation conditions, the IRA region dissociates from the ATPase domain because of membrane insertion of the 30-kDa domain. SecA ATPase is fully activated under the conditions in which both the C-terminal region and the central NBS II region are engaged in the translocation-driving reactions. Our results demonstrate that in the absence of both the NBS II-mediated and the IRA-mediated regulatory mechanisms, intrinsic SecA ATPase activity is ∼50-fold as high as the wild-type resting activity. The dual regulatory mechanisms may have evolved to avoid such futile consumption of ATP and to couple ATP hydrolysis effectively with polypeptide movement across the membrane. We thank Yoshinori Akiyama for helpful discussion and Yusuke Shimizu for technical support.

Our studies of SecM (secretion monitor) in E. coli have revealed that some amino acid sequences can interact with ribosomal interior components, particularly with gate components of the exit tunnel, thereby interfering with their own translation elongation. Such translation arrest can be regulated by interaction of the N-terminal portion of the nascent polypeptide with other cellular components outside the ribosome. These properties of nascent proteins can in turn provide regulatory mechanisms by which the expression of genetic information at different levels is regulated.

Atg8 and its mammalian homolog LC3, ubiquitin-like proteins (Ubls) required for autophagosome formation, are remarkably unique in that their conjugation target is the lipid phosphatidylethanolamine (PE). Although PE was identified as the sole lipid conjugated with Atg8/LC3 in vivo, phosphatidylserine (PS) can be also a good substrate for its conjugation reaction in vitro. This posed a simple, intriguing question: What confers substrate specificity to lipidation of Atg8/LC3 in vivo? Our recent in vitro studies propose that intracellular milieus such as cytosolic pH and acidic phospholipids in membranes significantly contribute to selective production of the Atg8-PE conjugate.

In macroautophagy (hereafter autophagy), cytoplasmic molecules and organelles are randomly or selectively sequestered within double-membrane vesicles called autophagosomes and delivered to lysosomes or vacuoles for degradation. In selective autophagy, the specificity of degradation targets is determined by autophagy receptors. In the budding yeast <i>Saccharomyces cerevisiae</i>, autophagy receptors interact with specific targets and Atg11, resulting in the recruitment of a protein complex that initiates autophagosome formation. Previous studies have revealed that autophagy receptors are regulated by posttranslational modifications. In selective autophagy of peroxisomes (pexophagy), the receptor Atg36 localizes to peroxisomes by binding to the peroxisomal membrane protein Pex3. We previously reported that Atg36 is phosphorylated by Hrr25 (casein kinase 1δ), increasing the Atg36–Atg11 interaction and thereby stimulating pexophagy initiation. However, the regulatory mechanisms underlying Atg36 phosphorylation are unknown. Here, we show that Atg36 phosphorylation is abolished in cells lacking Pex3 or expressing a Pex3 mutant defective in the interaction with Atg36, suggesting that the interaction with Pex3 is essential for the Hrr25-mediated phosphorylation of Atg36. Using recombinant proteins, we further demonstrated that Pex3 directly promotes Atg36 phosphorylation by Hrr25. A co-immunoprecipitation analysis revealed that the interaction of Atg36 with Hrr25 depends on Pex3. These results suggest that Pex3 increases the Atg36–Hrr25 interaction and thereby stimulates Atg36 phosphorylation on the peroxisomal membrane. In addition, we found that Pex3 binding protects Atg36 from proteasomal degradation. Thus, Pex3 confines Atg36 activity to the peroxisome by enhancing its phosphorylation and stability on this organelle.

Autophagy is an intracellular degradation system that is present in most eukaryotes. In the process of autophagy, double membrane vesicles called autophagosomes sequester a wide variety of cellular constituents and deliver them to lytic organelles: lysosomes in mammals and vacuoles in yeast and plants. Although autophagy used to be considered a non-selective process in its target sequestration into autophagosomes, recent studies have revealed that autophagosomes can also selectively sequester certain proteins and organelles that have become unnecessary or harmful for the cell. We recently discovered that the endoplasmic reticulum (ER) is degraded by autophagy in a selective manner in the budding yeast Saccharomyces cerevisiae, and identified "receptor proteins" that play pivotal roles in this "ER-phagy" pathway. Moreover, several ER-phagy receptors in mammalian cells have also been reported. This report provides an overview of our current knowledge on ER-phagy and discuss their mechanisms, physiological roles, and possible links to human diseases.

The driving force for protein translocation across the bacterial plasma membrane is provided by SecA ATPase, which undergoes striking conformational changes characterized by the membrane insertion and deinsertion cycle. This action of SecA requires the membrane-embedded SecYEG complex. Previously, we have identified a cold-sensitive secY mutation (secY205), affecting the most carboxy-terminal cytosolic domain, that did not allow an ATP-dependent insertion of a SecA-preprotein complex. Thus, this mutant provides an excellent system for genetic analysis of the SecY-SecA interaction.We carried out a systematic isolation of secA mutations that suppressed secY205 cold-sensitivity. A total of 40 independent suppressor mutations were classified into: (i) allele-specific suppressors, acting only against secY205, and (ii) 'super active' suppressors, acting against almost any sec defects. The former class of mutations, presumably with specific effects on the SecY-SecA interaction, clustered in two regions close to the Walker motif A sequences of the two ATP-binding domains. The latter mutations, enhancing general SecA activities, were mostly in or around the minor ATP-binding domain.The Walker motif A regions of SecA are important for the SecA-SecY interaction that leads to the SecA conformational changes required for insertion into the SecYEG channel. The minor ATP-binding domain is important for the down-regulation of SecA activities.

As with the case of the mechanism of autophagosome formation, studies in yeast have taken a leading role in elucidating the molecular basis of target recognition during selective autophagy. Degradation targets are recognized by receptor proteins, which also bind to Atg8 homologs on growing phagophore membranes, leading to the loading of the targets into autophagosomes. However, it remains to be elucidated how these processes are regulated. In yeast, receptors also interact with the scaffold/adaptor protein Atg11, which subsequently recruits core Atg proteins onto receptor-target complexes to initiate autophagosome formation. Recently, we found that Hrr25, a homolog of CSNK1D/casein kinase 1δ, regulates 3 of 4 selective autophagy-related pathways in the budding yeast Saccharomyces cerevisiae by a uniform mechanism: phosphoregulation of the receptor-scaffold interaction.

In selective autophagy, autophagosomes sequester specific targets to be degraded in lysosomes/vacuoles. A new study now provides critical insights into the mechanism by which the autophagosomal membrane closely sticks to the target to avoid incorporating material that should not be degraded.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Recent studies have unveiled the vital role of autophagy in organelle degradation. Our new study in yeast revealed that autophagy targets the endoplasmic reticulum, and even the nucleus, under starvation conditions. Two landmark proteins that direct these organelles to autophagic degradation have been identified, allowing us to dissect the molecular mechanisms and physiological roles of these new pathways.

The ribosome, a ribonucleoprotein machine for protein synthesis, can also serve as an abundant nutrient source under starvation conditions. In a recent issue of <i>Science</i>, Wyant et al. (2018) discovered a specialized "spoon" to "scoop up" more ribosomes for degradation by autophagy.

The secY205 mutant is cold-sensitive for protein export, with an in vitro defect in supporting ATP- and preprotein-dependent insertion of SecA into the membrane. We characterized SecA81 with a Gly516 to Asp substitution near the minor ATP-binding region, which suppresses the secY205 defect at low temperature and exhibits an allele-specific synthetic defect with the same SecY alteration at 42 degrees C. The overproduced SecA81 aggregated in vivo at temperatures above 37 degrees C. Purified SecA81 exhibited markedly enhanced intrinsic and membrane ATPase activities at 30 degrees C, while it was totally inactive at 42 degrees C. The trypsin digestion patterns indicated that SecA81 has some disorder in the central region of SecA, which encompasses residues 421-575. This conformational abnormality may result in unregulated ATPase at low temperature as well as the thermosensitivity of the mutant protein. In the presence of both proOmpA and the wild-type membrane vesicles, however, the thermosensitivity was alleviated, and SecA81 was able to catalyze significant levels of proOmpA-stimulated ATP hydrolysis as well as proOmpA translocation at 42 degrees C. While SecA81 was able to overcome the SecY205 defect at low temperature, the SecY205 membrane vesicles could not significantly support the translocation ATPase or the proOmpA translocation activity of SecA81 at 42 degrees C. The inactivated SecA81 molecules seemed to jam the translocase since it interfered with translocase functions at 42 degrees C. Based on these results, we propose that under preprotein-translocating conditions, the SecYEG channel can stabilize and activate SecA, and that this aspect is defective for the SecA81-SecY205 combination. The data also suggest that the conformation of the central region of SecA is important for the regulation of ATP hydrolysis and for the productive interaction of SecA with SecY.

Reticulophagy (or ER-phagy) is a type of selective autophagy that targets the endoplasmic reticulum (ER). In the process of reticulophagy, part of the ER is fragmented and packed within autophagosomes. However, the underlying mechanism that induces this local remodeling of ER subdomains was poorly understood. Our recent study showed that in the budding yeast Saccharomyces cerevisiae the reticulophagy receptor Atg40 plays an important role in ER remodeling beyond its role as a tether between the ER and the phagophore [1]. Atg40 has an ability to generate positive membrane curvature through the reticulon-like domain and locally forms a super assemblage though its binding to Atg8 at ER-phagophore contacts. These Atg40 assemblages cause folding of the ER subdomains to allow them to be efficiently packed into autophagosomes. Furthermore, our structural analysis identified an evolutionarily conserved short helix that assists strong Atg8-binding of reticulophagy receptors.

Autophagy is involved in diverse cellular functions through degradation of various intracellular constituents, and hence must be tightly controlled. A recent study by Hu et al. (2015) in Nature Cell Biology adds a new layer of autophagy regulation, involving Tor kinase-driven degradation of mRNAs encoding autophagy-related proteins.

Recent studies have revealed that even the nucleus can be degraded by selective macroautophagy (hereafter macronucleophagy). In Saccharomyces cerevisiae, the nuclear envelope (NE) protein Atg39 acts as a macronucleophagy receptor that mediates sequestration of nucleus-derived double-membrane vesicles (NDVs) into phagophores. The outer and inner membranes of these NDVs are derived from the outer and inner nuclear membranes (ONM and INM), respectively, and the lumen contains nucleoplasmic material. Little was known about the mechanisms underlying macronucleophagy, including how the two nuclear membranes are coordinately deformed to generate NDVs and what nuclear components are preferentially loaded into or rather eliminated from NDVs. We found that Atg39 links the ONM and INM through the ONM-embedded transmembrane domain and INM-associated amphipathic helices (APHs). These APHs are important for Atg39 anchoring to the NE and autophagosome formation-coupled Atg39 clustering in the NE. In addition, the overaccumulation of Atg39 in the NE caused NE protrusion toward the cytoplasm depending on the APHs. These results allowed us to propose the mechanism by which Atg39 conducts NDV formation in coordination with autophagosome formation during macronucleophagy.

The polytopic plasma membrane protein Rim21 senses both the elevation of ambient pH and alterations in plasma membrane lipid asymmetry in the Rim101 pathway in budding yeast. Rim21 is known to undergo N-glycosylation, but the site and function of the glycosylation modification is not known. Using a systematic mutation analysis, we found that Rim21 is N-glycosylated at an unconventional motif located in the N-terminal extracellular region. The Rim21 mutant protein that failed to receive N-glycosylation showed prolonged protein lifetime compared to that of WT Rim21 protein. Although both the WT and mutant Rim21 localized to the plasma membrane, they exhibited different biochemical fractionation profiles. The mutant Rim21, but not WT Rim21, was mainly fractionated into the heavy membrane fraction. Further, compared to WT Rim21, mutant Rim21 was more easily solubilized with digitonin but was conversely more resistant to solubilization with Triton X-100. Despite these different biochemical properties from WT Rim21, mutant Rim21 protein could still activate the Rim101 pathway in response to external alkalization. Collectively, N-glycosylation of Rim21 is not indispensable for its activity as a sensor protein, but modulates the residence of Rim21 protein to some microdomains within the plasma membrane with distinct lipid conditions, thereby affecting its turnover.Key words: plasma membrane, lipid asymmetry, N-linked glycosylation, microdomain, Saccharomyces cerevisiae.

During macroautophagy, cytoplasmic constituents are engulfed by autophagosomes. Lysosomes fuse with closed autophagosomes but not with unclosed intermediate structures. This is achieved in part by the late recruitment of the autophagosomal SNARE syntaxin 17 (STX17) to mature autophagosomes. However, how STX17 recognizes autophagosome maturation is not known. Here, we show that this temporally regulated recruitment of STX17 depends on the positively charged C-terminal region of STX17. Consistent with this finding, mature autophagosomes are more negatively charged compared with unclosed intermediate structures. This electrostatic maturation of autophagosomes is likely driven by the accumulation of phosphatidylinositol 4-phosphate (PI4P) in the autophagosomal membrane. Accordingly, dephosphorylation of autophagosomal PI4P prevents the association of STX17 to autophagosomes. Furthermore, molecular dynamics simulations support PI4P-dependent membrane insertion of the transmembrane helices of STX17. Based on these findings, we propose a model in which STX17 recruitment to mature autophagosomes is temporally regulated by a PI4P-driven change in the surface charge of autophagosomes.

Autophagy is a highly conserved intracellular degradation mechanism. The most distinctive feature of autophagy is the formation of double-membrane structures called autophagosomes, which compartmentalize portions of the cytoplasm. The outer membrane of the autophagosome fuses with the vacuolar/lysosomal membrane, leading to the degradation of the contents of the autophagosome. Approximately 30 years have passed since the identification of autophagy-related (ATG) genes and Atg proteins essential for autophagosome formation, and the primary functions of these Atg proteins have been elucidated. These achievements have significantly advanced our understanding of the mechanism of autophagosome formation. This article summarizes our current knowledge on how the autophagosome precursor is generated, and how the membrane expands and seals to complete the autophagosome.

Data for blots and graphs

Microscopy dataset for the investigation of the membrane morphological changes induced by the Atg8 conjugation machinery and the interactions between proteins within this machinery on the membrane. The raw data are in the Olympus format and can be accessed using Olympus software or other suitable software. Please refer to the paper for additional details on the data.

NMR dataset for the investigation of the interaction between proteins in Atg8 conjugation machinery. The raw data can be read and processed by Bruker Topspin or appropriate softwares. The experimental conditions are described in text files named title.<br>

Abstract In selective autophagy of the nucleus (hereafter nucleophagy), nucleus-derived double membrane vesicles (NDVs) are formed, sequestered within autophagosomes, and delivered to lysosomes or vacuoles for degradation. In Saccharomyces cerevisiae , the nuclear envelope (NE) protein Atg39 acts as a nucleophagy receptor, which interacts with Atg8 to target NDVs to forming autophagosomal membranes. In this study, we revealed that Atg39 is anchored to the outer nuclear membrane (ONM) via its transmembrane domain and also associated with the inner nuclear membrane (INM) via membrane-binding amphipathic helices (APHs) in its perinuclear space region, thereby linking these membranes. We also revealed that overaccumulation of Atg39 causes the NE to protrude towards the cytoplasm, and the tips of the protrusions are pinched off to generate NDVs. The APHs of Atg39 are crucial for Atg39 assembly in the NE and subsequent NE protrusion. These findings suggest that the nucleophagy receptor Atg39 plays pivotal roles in NE deformation during the generation of NDVs to be degraded by nucleophagy.

Autophagy is a bulk degradation system highly conserved in eukaryotic cells and involved in a wide variety of biological activities. When autophagy is induced, autophagosomes, double membrane-bound organelles that are specialized for sequestration and transport of cytosolic constituents to lytic compartments for degradation, are formed. This process involves an unconventional membrane dynamics, which is governed by a subset of specific proteins called Atg. Here, we describe the charecteristics of autophagosomal membranes and its formation process, review our current knowledge on the functions and behavior of the Atg proteins, and discuss issues remained to be unraveled.

Autophagy delivers not only cytoplasmic proteins and RNA but also various organelles, such as the endoplasmic reticulum, mitochondria, peroxisomes, and lipid droplets to lysosomes/vacuoles for degradation. Thus, a considerable amount of membrane lipids is also transported into lysosomes/vacuole via autophagy. However, little is known about degradation and recycling of lipids, and the mechanism and physiological significance of these processes, compared with those of proteins and RNA. In this review, we summarize our current knowledge on relationships between lipid metabolism and autophagy and discuss the involvement of autophagy in lipid homeostasis.

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract In autophagy, Atg proteins organize the pre-autophagosomal structure (PAS) to initiate autophagosome formation. Previous studies in yeast revealed that the autophagy-related E3 complex Atg12-Atg5-Atg16 is recruited to the PAS via Atg16 interaction with Atg21, which binds phosphatidylinositol 3-phosphate (PI3P) produced at the PAS, to stimulate conjugation of the ubiquitin-like protein Atg8 to phosphatidylethanolamine. Here, we discover a novel mechanism for the PAS targeting of Atg12-Atg5-Atg16, which is mediated by the interaction of Atg12 with the Atg1 kinase complex that serves as a scaffold for PAS organization. While autophagy is partially defective without one of these mechanisms, cells lacking both completely lose the PAS localization of Atg12-Atg5-Atg16 and show no autophagic activity. As with the PI3P-dependent mechanism, Atg12-Atg5-Atg16 recruited via the Atg12-dependent mechanism stimulates Atg8 lipidation, but also has the specific function of facilitating PAS scaffold assembly. Thus, this study significantly advances our understanding of the nucleation step in autophagosome formation. https://doi.org/10.7554/eLife.43088.001 Introduction Macroautophagy (hereafter autophagy) is a major route for transport of intracellular material into lysosomes or vacuoles in almost all eukaryotes (Ohsumi, 2014; Yang and Klionsky, 2010). In autophagy, a membrane cisterna called the isolation membrane (or phagophore) is generated, expands, becomes spherical, and closes to form a double membrane vesicle called the autophagosome. During the course of this process, various cytoplasmic components, including proteins, RNA, and organelles, are selectively or non-selectively sequestered into the autophagosome. The autophagosome fuses with the lysosome/vacuole to allow degradation of the contents. An increasing number of studies have suggested that autophagy is involved in the regulation of a wide range of cellular functions, and linked to a variety of human diseases (Bento et al., 2016; Dikic and Elazar, 2018; Mizushima, 2018). Isolation of autophagy-defective mutants of the budding yeast Saccharomyces cerevisiae and subsequent analysis of these mutants led to the identification of autophagy-related (ATG/Atg) genes/proteins. Among the over 40 Atg proteins that have been identified to date, 19 are directly involved in the biogenesis of the autophagosome induced under starvation (Nakatogawa et al., 2009; Ohsumi, 2014; Yang and Klionsky, 2010). These ‘core’ Atg proteins constitute six functional units: (i) the Atg1 kinase complex; (ii) Atg9 vesicles; (iii) phosphatidylinositol (PI) 3-kinase (PI3K) complex I; (iv) the Atg2-Atg18 complex; (v) the Atg12 conjugation system; and (vi) the Atg8 conjugation system. In response to starvation, these proteins interact with each other, localize to the site of autophagosome formation in an ordered manner, and organize the pre-autophagosomal structure (PAS) (Nakatogawa et al., 2009; Suzuki et al., 2001; Suzuki and Ohsumi, 2010), in which a precursor of the autophagosomal membrane is generated. The molecular basis of PAS organization, including how Atg proteins are recruited to the PAS, is a key question that needs to be addressed to understand the ‘nucleation’ step in autophagosome formation. The ubiquitin-like protein Atg12 is covalently attached to a lysine residue in Atg5 via ubiquitin-like conjugation reactions, resulting in the Atg12-Atg5 conjugate (Mizushima et al., 1998a; Mizushima et al., 1998b). Atg12-Atg5 non-covalently interacts with Atg16 (Atg16L in mammals) to form the Atg12-Atg5-Atg16/Atg16L complex (hereafter the Atg16/Atg16L complex) (Kuma et al., 2002; Mizushima et al., 2003; Mizushima et al., 1999). The Atg16/Atg16L complex is localized to the PAS (or the site of autophagosome formation) and acts as an E3 enzyme to stimulate the conjugation reaction of ubiquitin-like Atg8/LC3-family proteins to the lipid phosphatidylethanolamine (PE) in autophagosome intermediates (i.e., a still-unknown membrane component of the PAS and the isolation membrane) (Suzuki et al., 2007; Hanada et al., 2007; Ichimura et al., 2000; Fujita et al., 2008; Nakatogawa, 2013). Atg8-PE conjugates promote the expansion of the isolation membrane (Nakatogawa et al., 2007; Xie et al., 2008), and also bind to autophagy receptors that recognize specific degradation targets for their selective sequestration into the autophagosome (Gatica et al., 2018). In both yeast and mammals, the recruitment of the Atg16/Atg16L complex to the site of autophagosome formation depends on PI3-phosphate (PI3P) produced by PI3K complex I (Itakura and Mizushima, 2010; Suzuki et al., 2007). A recent study in mammalian cells revealed that the PROPPIN family protein WIPI2b binds both Atg16L1 and PI3P to target the Atg16L1 complex to autophagosome formation sites (Dooley et al., 2014). In S. cerevisiae, Atg21, one of the three WIPI homologs, was shown to mediate this process in a similar manner (Juris et al., 2015). However, knockout of ATG21 did not completely abrogate the PAS localization of the Atg16 complex or the autophagic activity of cells (Meiling-Wesse et al., 2004; Nair et al., 2010; Strømhaug et al., 2004), suggesting that there is an unknown mechanism which directs the Atg16 complex to the PAS, in addition to the PI3K complex I-PI3P-Atg21 axis. In this study, we identified the Atg1 kinase complex, which forms a scaffold for PAS organization, as a novel interaction partner of the Atg16 complex, and found that this intercomplex interaction collaborates with the PI3P-dependent mechanism to recruit the Atg16 complex to the PAS. In addition to the stimulation of Atg8 lipidation, the Atg16 complex recruited via this newly discovered mechanism has a specific, non-E3 function: the promotion of PAS scaffold assembly. Results An Atg12-dependent, PI3P-independent mechanism targets the Atg16 complex to the PAS To clarify the mechanism that directs the Atg16 complex to the PAS, we carefully analyzed the PAS localization of this complex in cells lacking different Atg proteins (Figure 1). In this analysis, the Atg16 complex was visualized by Atg5-GFP, and the PAS was labeled with the scaffold protein Atg17 fused with mCherry (Suzuki et al., 2007). In the currently accepted model, Atg5 and Atg16 cooperate to target the complex to the PAS, whereas Atg12 is dispensable for this process (Suzuki et al., 2007). It is also believed that PI3P produced by PI3K complex I, which contains Atg14 as a specific subunit, is essential for the localization of the Atg16 complex to the PAS. This PI3P-dependency could, at least in part, be explained by the role of the PI3P-binding protein Atg21 that interacts with Atg16 to recruit the Atg16 complex to the PAS (Nair et al., 2010; Juris et al., 2015). In agreement with this model, the PAS localization of Atg5 was lost by deletion of ATG16 (Figure 1). It was also confirmed that Atg5 localized to the PAS less efficiently in the absence of Atg21. Deletion of ATG14 decreased the PAS localization of Atg5; however, Atg5 still significantly localized to the PAS even without Atg14, to an extent similar to that observed in cells lacking Atg21. In addition, we noticed that the frequency of Atg5 localization to the PAS was decreased in the absence of Atg12, although it abnormally accumulated at the PAS. We found that PAS localization of Atg5 was totally abolished in cells lacking both Atg14 and Atg12 (Figure 1). Disruption of ATG12 also abrogated the residual PAS localization of Atg5 in ATG21 knockout cells. These results suggest that in addition to the previously described PI3P-dependent pathway, there exists an uncharacterized, PI3P-independent mechanism that targets the Atg16 complex to the PAS, which likely involves Atg12. Figure 1 Download asset Open asset Atg12- and PI3P-dependent mechanisms cooperatively act to recruit the Atg16 complex to the PAS. Cells expressing Atg5-GFP and Atg17-mCherry were treated with rapamycin for 90 min, and analyzed by fluorescence microscopy. DIC, Differential interference contrast microscopy. Bars, 5 μm. The ratios of Atg17-mCherry puncta positive for Atg5-GFP to total Atg17-mCherry puncta were calculated, and the mean values are shown with standard deviations (n = 3). **p<0.01; ***p<0.001 (unpaired two-tailed Student’s t-test). https://doi.org/10.7554/eLife.43088.002 The Atg16 complex interacts with the Atg1 complex under autophagy-inducing conditions We proceeded to investigate an Atg12-dependent mechanism for PAS-targeting of the Atg16 complex. Yeast cells expressing FLAG-tagged Atg5 (Atg5-FLAG) were treated with rapamycin, which inhibits Tor kinase complex 1 and thereby induces various starvation responses including autophagy even in the presence of nutrients (Noda and Ohsumi, 1998), followed by immunoprecipitation using anti-FLAG antibody. Mass spectrometry of the immunoprecipitates identified a number of proteins as possible interaction partners of the Atg16 complex, and included most components of the Atg1 complex (Figure 2—figure supplement 1A and B). The Atg1 complex is composed of the protein kinase Atg1 and the regulatory and scaffold proteins Atg13, Atg17, Atg29, and Atg31, and triggers autophagosome formation in response to nutrient starvation (Fujioka et al., 2014; Kamada et al., 2000). Atg1, Atg17, and Atg29 could also be detected in Atg5-FLAG immunoprecipitates by immunoblotting (Figure 2A and Figure 2—figure supplement 1C). When ATG12 or ATG16 was deleted, coimmunoprecipitation of Atg17 with Atg5-FLAG was largely decreased (Figure 2A and Figure 2—figure supplement 1B). We also showed that Atg17 was not precipitated with Atg5-FLAG in the absence of Atg10, which is essential for Atg12 conjugation to Atg5 (Mizushima et al., 1998a) (Figure 2A). Immunoprecipitation of FLAG-tagged Atg16 also precipitated Atg17; however, this was lost by knockout of ATG5 or ATG12 (Figure 2B). Thus, the formation of the Atg16 complex is required for its interaction with the Atg1 complex. We also examined this interaction in cells lacking any of the components of the Atg1 kinase complex. In this analysis, coprecipitation of Atg1 with Atg5-FLAG was also examined to evaluate the effect of ATG17 knockout on the association between the two complexes. The results clearly showed that all the components of the Atg1 complex are important for its interaction with the Atg16 complex (Figure 2C). In addition, the F430A mutation in Atg13, which impairs the formation of the Atg1 complex (Yamamoto et al., 2016), reduced Atg17 precipitation with the Atg16 complex (Figure 2D). These results suggest that the formation of the Atg1 complex is a prerequisite for its association with the Atg16 complex. In contrast, ATG14 deletion did not affect Atg17 precipitation with Atg5-FLAG (Figure 2E), consistent with the idea that this novel intercomplex interaction is involved in the PI3P-independent PAS targeting of the Atg16 complex. Figure 2 with 2 supplements see all Download asset Open asset The Atg16 complex interacts with the Atg1 complex. (A–C, E) Yeast cells expressing Atg5-FLAG (A, C–E) or Atg16-FLAG (B) from each chromosomal locus were treated with rapamycin for 2 hr, and subjected to immunoprecipitation using anti-FLAG antibody. The immunoprecipitates were analyzed by immunoblotting using antibodies against FLAG (A, B), Atg12 (C, E), Atg17 (A–C, E), and Atg1 (C). (D) atg13Δ cells expressing wild-type Atg13, the F375A mutant, or the F430A mutant from centromeric plasmids were treated with rapamycin for 2 hr, subjected to immunoprecipitation using anti-FLAG antibody, and the immunoprecipitates were analyzed by immunoblotting using antibodies against Atg12, Atg13 and Atg17. (F) Yeast cells were treated with or without rapamycin for 2 hr, and coimmunoprecipitation of Atg17 with Atg5-FLAG was examined as described in Figure 2C. (G) Coimmunoprecipitation of Atg17 with Atg5-FLAG was analyzed in cells expressing wild-type Atg1 or the D211A mutant from the original chromosomal locus as described in Figure 2C. https://doi.org/10.7554/eLife.43088.003 We found that the Atg16 complex interacts with the Atg1 complex depending on cell treatment with rapamycin (Figure 2F). Consistent with this result, the interaction was not considerably decreased by the absence of Atg11, which binds to the Atg1 complex but is dispensable for starvation-induced autophagy (Kamada et al., 2000; Kim et al., 2001). Upon nutrient starvation, Atg1, Atg13, and the Atg17-Atg29-Atg31 complex form the Atg1 complex, and multiple copies of the complex further associate with each other, leading to activation of Atg1 kinase via intermolecular autophosphorylation (Yamamoto et al., 2016; Yeh et al., 2011; Yeh et al., 2010). This assemblage of the Atg1 complexes serves as a scaffold to recruit downstream Atg proteins for PAS organization. The interaction between the Atg16 complex and the Atg1 complex was lost in the F375A mutant of Atg13 (Figure 2D), which can form the Atg1 complex, but is defective in its higher order assembly (Yamamoto et al., 2016). By contrast, the D211A mutation in Atg1, which abolishes its kinase activity (Matsuura et al., 1997), did not affect the interaction between the Atg16 and Atg1 complexes (Figure 2G). These results suggest that the Atg16 complex associates with the Atg1 complex following its supramolecular assembly in a manner independent of Atg1 kinase activity. We also examined which subunits mediate the interaction between the Atg16 and Atg1 complexes. Yeast two-hybrid assay suggested that Atg12 could bind Atg17 and Atg31 (Figure 2—figure supplement 2A). In this assay, the Atg12-Atg17 interaction was still observed in atg13Δ atg31Δ cells (Figure 2—figure supplement 2B), in which Atg17 should not interact with the remaining subunits Atg1 and Atg29. By contrast, the Atg12-Atg31 interaction was abolished by ATG17 knockout, suggesting that Atg12 interacted with Atg31 via Atg17 (Figure 2—figure supplement 2C). In addition, immunoprecipitation of Atg12 C-terminally fused with GFP, which is not associated with Atg5 and Atg16, coprecipitated Atg17 in cells lacking the other four subunits of the Atg1 complex, when both of Atg12-GFP and Atg17 were overexpressed. These results suggest that the interaction between Atg17 and Atg12 mediates the association of the two complexes. The Atg16 complex interacts with the Atg1 complex to localize to the PAS Next, we examined the significance of the interaction between the Atg16 and Atg1 complexes in autophagosome formation. Atg12 is a ubiquitin-like protein with an approximately 100 amino acid-long extension at the N terminus (Suzuki et al., 2005). A previous study reported that while the ubiquitin-like domain of Atg12 was essential for autophagy, deletion of the N-terminal region caused a partial defect (Hanada and Ohsumi, 2005). The N-terminal region of Atg12 was not required for Atg12 conjugation to Atg5, the E3 activity of the conjugate, or the interaction of the conjugate with Atg16 (Hanada and Ohsumi, 2005) (Figure 3—figure supplement 1). Thus, the role for the Atg12 N-terminal region remained unknown. We found that the Atg1 complex was hardly coimmunoprecipitated with the Atg16 complex that contained Atg12 lacking the N-terminal 56 residues (Atg12ΔN56) (Figure 3A), suggesting that the Atg12 N-terminal region is involved in the interaction of the Atg16 complex with the Atg1 complex. The results obtained by fluorescence microscopy (Figure 1) suggested that this interaction cooperates with the PI3P-dependent pathway in the recruitment of the Atg16 complex to the PAS. Therefore, we examined the PAS localization of the complex containing Atg12ΔN56 in the absence of Atg21. While expression of wild-type Atg12 rescued a defect in the PAS localization of Atg5-GFP in atg21Δ atg12Δ cells, expression of the Atg12ΔN56 mutant did not (Figure 3B). We also performed an alkaline phosphatase (ALP) assay to assess autophagic activity in the mutant cells. In this assay, a mutant form of the vacuolar phosphatase Pho8 (Pho8Δ60) is expressed in an unprocessed, inactive form in the cytoplasm. This mutant phosphatase is delivered into the vacuole through autophagy. Once inside the vacuole, it is processed into an active form, and its activity can be quantified biochemically (Noda et al., 1995). Consistent with previous results (Hanada and Ohsumi, 2005; Meiling-Wesse et al., 2004; Strømhaug et al., 2004), atg12ΔN56 cells (atg12Δ cells carrying the atg12ΔN56 plasmid) and atg21Δ cells (atg21Δ atg12Δ cells carrying the ATG12WT plasmid) were only partially defective in autophagy (Figure 3C). When these mutations were combined (atg21Δ atg12Δ cells carrying the atg12ΔN56 plasmid), the cells showed almost no autophagic activity. These results suggest that the interaction of the Atg16 complex with the Atg1 complex indeed acts to target the Atg16 complex to the PAS, and that defects caused by the absence of this interaction can be partly compensated by the PI3P-dependent mechanism. If both of these mechanisms are simultaneously compromized, cells totally lose their ability to form the autophagosome. Figure 3 with 1 supplement see all Download asset Open asset The interaction of the Atg16 complex with the Atg1 complex is involved in the PAS targeting of the Atg16 complex. (A) atg12Δ cells expressing wild-type Atg12 or Atg12ΔN56 from centromeric plasmids were treated with rapamycin for 2 hr, and examined for coimmunoprecipitation of Atg17 with Atg5-FLAG as described in Figure 2C. The upper and middle panels were immunoblots obtained using antibodies against Atg5 and FLAG, respectively. Asterisk, non-specific bands. (B) Yeast cells were treated with rapamycin for 2 hr, and the PAS localization of Atg5-GFP was assessed by fluorescence microscopy as described in Figure 1. **p<0.01; ***p<0.001 (unpaired two-tailed Student’s t-test). (C) atg12Δ and atg12Δ atg21Δ cells expressing wild-type Atg12 or Atg12ΔN56 from centromeric plasmids were grown in nutrient-rich medium (open bars) and then starved in SD-N medium for 4 hr (closed bars), and their autophagic activities were evaluated by ALP assay. The mean values are shown with standard deviations (n = 3). *p<0.05; **p<0.01 (unpaired two-tailed Student’s t-test). https://doi.org/10.7554/eLife.43088.006 The Atg16 complex recruited via the Atg12-dependent pathway plays two different roles in PAS organization Previous studies showed that PI3P-dependent PAS recruitment of the Atg16 complex is important for the production of Atg8-PE (Meiling-Wesse et al., 2004; Strømhaug et al., 2004). We asked whether the Atg12-dependent mechanism also contributes to this process. Atg8-PE production is stimulated upon nitrogen starvation (Figure 4A, atg12Δ/pATG12WT). As reported previously (Meiling-Wesse et al., 2004; Strømhaug et al., 2004), ATG21 knockout significantly reduced the level of Atg8-PE, which still gradually increased during nitrogen starvation (atg21Δ atg12Δ/pATG12WT). We found that deletion of the N-terminal region of Atg12 also partially decreased Atg8-PE formation (atg12Δ/patg12ΔN56). In addition, starvation-induced Atg8-PE formation was totally abolished in cells lacking both Atg21 and the N-terminal region of Atg12 (atg21Δ atg12Δ/patg12ΔN56). Of note, the residual amount of Atg8-PE in these mutant cells should represent the conjugates that were produced in the vacuolar membrane depending on the Atg16 complex, which is dispersed throughout the cytoplasm, independent of autophagy (Nakatogawa et al., 2012a). These results demonstrated that the Atg16 complex recruited by the Atg12-dependent mechanism acts as an E3 enzyme and promotes Atg8 lipidation, as was observed with the PI3P-dependent mechanism. Figure 4 Download asset Open asset The Atg16 complex recruited via the association with the Atg1 complex facilitates Atg8 lipidation and PAS scaffold assembly. (A) Yeast cells were incubated in nitrogen starvation medium and examined for the production of Atg8-PE by urea-SDS-PAGE and immunoblotting using anti-Atg8 antibodies (see Materials and methods). The ratio of Atg8-PE to total Atg8 was calculated, and the mean values are shown with standard deviations (n = 3). *p<0.05; **p<0.01 (unpaired two-tailed Student’s t-test). Pgk1 serves as a loading control. (B and C) Yeast cells expressing Atg17-GFP were treated with rapamycin for 90 min (B) or 2 hr (C), and observed under a fluorescence microscope. The proportion of cells containing Atg17-GFP puncta to total cells was calculated, and the mean values are shown with standard deviations (n = 3). **p<0.01 (unpaired two-tailed Student’s t-test). https://doi.org/10.7554/eLife.43088.008 We noticed that Atg17-GFP puncta, which represent PAS scaffold assembly (Yamamoto et al., 2016), were decreased by knockout of ATG12, which disrupts the Atg12-dependent mechanism, whereas the puncta were increased by knockout of ATG14 or ATG21, which impairs the PI3P-dependent mechanism (Figure 1). We confirmed this finding using atg11Δ cells, in which the starvation-induced assembly of the PAS scaffold can be assessed separately from a similar process that occurs under nutrient-replete conditions for the cytoplasm-to-vacuole targeting (Cvt) pathway (Cheong et al., 2008; Kawamata et al., 2008). The results demonstrated that the formation of Atg17-GFP puncta upon rapamycin treatment was defective in the absence of Atg12, Atg5, or Atg16 (Figure 4B). More importantly, Atg17-GFP puncta were also decreased by deletion of the N-terminal region of Atg12 (Figure 4C). These results suggest that the Atg16 complex recruited via the Atg12-dependent mechanism (i.e., the interaction with the Atg1 complex) facilitates starvation-induced PAS scaffold assembly. Discussion Previous studies established a model for PAS targeting of the Atg16 complex: Atg16 interacts with Atg21, which binds PI3P produced by PI3K complex I at the PAS (Juris et al., 2015; Nair et al., 2010; Meiling-Wesse et al., 2004; Strømhaug et al., 2004) (Figure 5, PI3P-dependent targeting). This model nicely explained the PI3P-dependency of the process. However, disruption of ATG21 or ATG14 did not totally abolish PAS localization of the Atg16 complex, suggesting the existence of another pathway that targets this complex to the PAS in a PI3P-independent manner. In this study, we discovered that the Atg16 complex also interacts with the Atg1 complex via the N-terminal region of Atg12 to localize to the PAS (Figure 5, Atg12-dependent targeting). Thus, the Atg16 complex is recruited to the PAS through two different pathways. Although disrupting either of the pathways caused partial defects, in the absence of both of these pathways, the Atg16 complex hardly localized to the PAS, and autophagy was completely blocked. These results suggested that these pathways function in a partially redundant manner. Consistent with this idea, we showed that these pathways cooperatively act to stimulate Atg8-PE formation in response to starvation (Figure 4A). However, the Atg1 complex serves as a scaffold to initiate PAS organization, whereas Atg21 is recruited at a later step, following the production of PI3P by PI3K complex I (Figure 5), suggesting that there is a functional difference between these pathways. Indeed, we found that the Atg12-dependent pathway, but not the PI3P-dependent pathway, is involved in PAS scaffold assembly. Thus, the Atg16 complex recruited by the Atg12-dependent pathway has a specific, non-E3 role in the initiation of PAS organization. Given the complementary relationship with the PI3P-dependent pathway, it is likely that the Atg16 complex recruited through the association of the Atg1 complex also contributes to Atg8 lipidation at later stages (Figure 5, dashed arrow). Figure 5 Download asset Open asset Model for the PAS recruitment of the Atg16 complex. The Atg16 complex is recruited to the PAS through two different pathways (Atg12-dependent targeting and PI3P-dependent targeting). Upon autophagy induction (starvation or TORC1 inactivation), the Atg1 complex is assembled, and multiple copies of Atg1 complexes further form a higher order assembly. During the process, the Atg16 complex associates with Atg1 complexes via the N-terminal region of Atg12, promoting PAS scaffold assembly. The Atg16 complex recruited at this stage also facilitates lipidation of Atg8 at a later stage in PAS organization (dashed arrow). As reported previously, following the recruitment of PI3K complex I and the production of PI3P by this complex, the Atg16 complex localizes to the PAS via the interaction with the PI3P-binding protein Atg21 to stimulate Atg8 lipidation. https://doi.org/10.7554/eLife.43088.009 The interaction between the Atg16 complex and the Atg1 complex required both of the complexes to be intact. In addition, higher order assembly of Atg1 complexes was a prerequisite for the interaction (Figure 2D). We speculate that the Atg16 complex, in which Atg12-Atg5-Atg16 is dimerized by the homodimerization of Atg16 (Fujioka et al., 2010), simultaneously binds to two copies of the Atg1 complexes via the N-terminal regions of Atg12 (Figure 5). This mode of interaction can crosslink the Atg1 complexes, resulting in the facilitation of supramolecular assembly of Atg1 complexes to form the PAS scaffold. In this study, we showed that the association between the Atg16 and Atg1 complexes requires the N-terminal region of Atg12. Although there is no sequence similarity between the N-terminal regions of yeast and mammalian Atg12, a recent study revealed that the Atg16L complex also associates with the ULK1 complex (corresponding to the Atg1 complex) in mammalian cells (Nishimura et al., 2013). This intercomplex association was mediated by the interaction between Atg16L and FIP200, which is a mammalian counterpart of yeast Atg17, and important for the localization of the Atg16L complex to the isolation membrane. Thus, although the underlying mechanisms are different, the recruitment of the Atg16/Atg16L complex to autophagosome intermediates through association with the Atg1/ULK1 complex is a common process during autophagosome formation in yeast and mammals. It is still unclear how this process cooperates with the PI3P (WIPI2)-dependent mechanism in mammalian cells. The Atg16L complex may also facilitate supramolecular assembly of ULK complexes in the initiation of autophagosome formation. Materials and methods Yeast strains and media Request a detailed protocol S. cerevisiae strains used in this study are listed in Table 1. Gene knockout and tagging were performed as described previously (Janke et al., 2004). Yeast cells were grown at 30°C in YPD medium (1% yeast extract, 2% peptone, and 2% glucose) for immunoprecipitation analysis or in SD+CA+ATU medium [0.17% yeast nitrogen base without amino acids and ammonium sulfate (YNB w/o aa and as), 0.5% ammonium sulfate, 0.5% casamino acids, and 2% glucose supplemented with 0.002% adenine sulfate, 0.002% tryptophan, and 0.002% uracil] for fluorescence microscopy. Cells carrying pRS316-derived plasmids expressing Atg12, Atg13, and their mutants were cultured in SD+CA+ATU without uracil. To induce autophagy, cells were treated with 0.2 μg/mL rapamycin or incubated in SD-N medium (0.17% YNB w/o aa and as and 2% glucose). Table 1 Yeast strains used in this study. https://doi.org/10.7554/eLife.43088.010 NameGenotypeFiguresReferenceW303-1aMATa ade2-1 ura3-1 his3-11,15 trp1-1 leu2-3,112 can1-100-(Thomas and Rothstein, 1989)ScKH146W303-1A, ade2::ADE2 ATG5-EGFP-kanMX6 ATG17-2×mCherry-hphNT11AThis studyScKH153ScKH146 atg16Δ::natNT21AThis studyScKH182ScKH146 atg21Δ::zeoNT31AThis studyScKH151ScKH146 atg14Δ::natNT21AThis studyScKH149ScKH146 atg12Δ::natNT21AThis studyScKH162ScKH146 atg14Δ::natNT2 atg12Δ::zeoNT31AThis studyScTK623ScKH146 atg21Δ::natNT2 atg12Δ::zeoNT31AThis studyBJ2168MATa leu2 trp1 ura3-52 prb1-1122 pep4-3 prc1-407 gal22A-C, 2E, 2F, 2-S1A, 2-S1C(Jones, 1991)MAN169BJ2168 ATG5-TEV-3×FLAG-kanMX42A, 2C, 2E, 2F, 2-S1A, 2-S1CThis studyScKH10MAN169 atg16Δ::natNT22A, 2E, 2-S1AThis studyScKH32MAN169 atg12Δ::natNT22A, 3AThis studyScKH96MAN169 atg10Δ::natNT22AThis studyScKH90BJ2168 ATG16-TEV-3×FLAG-kanMX42BThis studyScKH92ScKH90 atg5Δ::natNT22BThis studyScKH93ScKH90 atg12Δ::natNT22BThis studyScKH141MAN169 atg1Δ::natNT22CThis studyScKH99MAN169 atg13Δ::natNT22C, 2DThis studyScKH216MAN169 atg17Δ::natNT22CThis studyScKH101MAN169 atg29Δ::natNT22CThis studyScKH143MAN169 atg31Δ::natNT22CThis studyScKH98MAN169 atg14Δ::natNT22EThis studyScKH97MAN169 atg11Δ::natNT22FThis studyScYH3184BJ2168 leu2::LEU22GThis studyScKH66ScHY3184 ATG5-TEV-3×FLAG-kanMX42GThis studyScKH68ScHY3184 atg1D211A-hphNT1 ATG5-TEV-3×FLAG-kanMX42GThis studyAH109MATa trp1-901 leu2-3, 112 ura3-52 his3-200 gal4Δ gal80ΔLYS2::GAL1UAS-GAL1TATA-HIS3 MEL1 GAL2UAS-GAL2TATA-ADE2 URA3::MEL1UAS-MEL1TATA-lacZ2-S2AClontechScTK967AH109 atg13Δ::natNT2 atg31Δ::hphNT12-S2BThis studyScTK968AH109 atg17Δ::natNT22-S2CThis studyScTK877BJ2168 atg11Δ::LEU2 atg29Δ::zeoNT3 atg31Δ::hphNT1 atg1Δ::natNT2 atg13Δ::Klura3 PADH1-ATG17-CgTRP12-S2DThis studyScTK958ScTK877 KlURA3-PADH1-ATG12-EGFP-kanMX42-S2DThis studyScTK649ScTK623 his3-11::pRS3033BThis studyScTK650ScTK623 his3-11::pRS303-ATG123BThis studyScTK651ScTK623 his3-11::pRS303-atg12ΔN563BThis studyBY4741MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0-(Brachmann et al

Eukaryotic cells have developed various organelles, and therein separated subsets of reactions, to perform diverse cellular functions in an organized manner. They have also been equipped with elaborate mechanisms to strictly control quality and quantity of organelles, and deficiencies in these mechanisms can lead to diseases in humans. Autophagy, an intracellular degradation pathway, proceeds through the following steps: (1) a double-membraned vesicle called the autophagosome is formed in the cytoplasm to sequester degradation targets, (2) the autophagosome fuses with the lysosomal or vacuolar membrane, and (3) the sequestered material is degraded by hydrolases in lysosomes/vacuoles.1 While autophagy randomly degrades cytoplasmic constituents under nutrient-limiting conditions, it can also selectively degrade certain molecules or structures via landmark proteins called autophagy receptors.2 These proteins localize to specific targets, and also bind to Atg8-family proteins on expanding autophagosomal membranes, leading to the sequestration of the targets by autophagosomes. Previous studies have shown that receptor-mediated selective autophagy largely contributes to degradation of some organelles, including mitochondria and peroxisomes, as well as protein aggregates and invading pathogens.2 However, how extensively selective autophagy is involved in organelle homeostasis and regulation remained to be investigated. In a recent study, we have revealed that when starved, yeast cells actively degrade their own endoplasmic reticulum (ER) and nucleus via selective autophagy, which involves 2 novel receptors.3

Autophagy is an intracellular degradation system conserved from yeast to mammal that isolates the cellular materials and organelles into a double membrane vesicle, termed an autophagosome, for degradation. Autophagosome formation depends on Atg8 and Atg12 conjugation systems, which produce Atg8-phosphatidylethanolamine (PE) conjugate as a final product that plays a crucial role for promoting autophagy. Another conjugate, Atg12-Atg5, functions as E3 and promotes transfer of Atg8 from Atg3 (E2) to PE. Here, we identified the minimum binding region of Atg3 for Atg12 by in vitro pull-down assay using Arabidopsis thaliana (At) homologs and crystallized the AtAtg12b-AtAtg3 peptide complex. The obtained crystal (P64, a = 128.5, b = 128.5, c = 163.2 Å) diffracted X-rays to 3.2 Å resolution, and its structure was determined by the molecular replacement method. AtAtg12b forms a swapping dimer in the crystal. The side-chain of AtAtg3 Met157 is bound deeply to the hydrophobic pocket of AtAtg12b consisting of Phe30, Val41, and Phe44. The importance of AtAtg3 Met157 for AtAtg12b binding was confirmed by mutational analysis. These data establish the basis for E2-E3 interaction in the plant Atg system.

Autophagy is an intracellular degradation pathway, in which degradation targets are sequestered by a double membrane vesicle called the autophagosome, transported to, and degraded in the lysosome or vacuole. Recent studies revealed that some organelles, such as mitochondria and peroxisomes, are marked by “autophagy receptors”, and selectively engulfed by the autophagosome. Here, I describe our recent discovery of selective autophagy of the endoplasmic reticulum and nucleus in the budding yeast Saccharomyces cerevisiae.