Noboru Mizushima

The University of Tokyo, Tōkyō, Japan

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Publications (178)1830.89 Total impact

  • Peidu Jiang, Noboru Mizushima
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    ABSTRACT: Autophagy is an intracellular degradation system that delivers cytoplasmic materials to the lysosome or vacuole. This system plays a crucial role in various physiological and pathological processes in living organisms ranging from yeast to mammals. Thus, an accurate and reliable measure of autophagic activity is necessary. However, autophagy involves dynamic and complicated processes that make it difficult to analyze. The term "autophagic flux" is used to denote overall autophagic degradation (i.e., delivery of autophagic cargo to the lysosome) rather than autophagosome formation. Immunoblot analysis of LC3 and p62/SQSTM1, among other proteins, has been widely used to monitor autophagic flux. Here, we describe basic protocols to measure the levels of endogenous LC3 and p62 by immunoblotting in cultured mammalian cells. Copyright © 2014. Published by Elsevier Inc.
    Methods (San Diego, Calif.). 12/2014;
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    ABSTRACT: Mitochondria are dynamic organelles, and their fusion and fission regulate cellular signaling, development, and mitochondrial homeostasis including mtDNA distribution. Cardiac myocytes have a specialized cytoplasmic structure where large mitochondria are aligned into tightly packed myofibril bundles; however, recent studies have revealed that mitochondrial dynamics also play important roles in the formation and maintenance of cardiomyocytes. Here we precisely analyzed the role of mitochondrial fission in vivo. The mitochondrial fission GTPase, Drp1, is highly expressed in developing neonatal heart, and muscle-specific Drp1-KO mice showed neonatal lethality due to dilated cardiomyopathy. The Drp1 ablation in heart and primary cultured cardiomyocytes resulted in severe mtDNA nucleoid clustering, and led to mosaic deficiency of mitochondrial respiration. The functional and structural alteration of mitochondria also led to immature myofibril assembly and defective cardiomyocyte hypertrophy. Thus, the dynamics of mtDNA nucleoids regulated by mitochondrial fission is required for neonatal cardiomyocyte development by promoting homogeneous distribution of active mitochondria throughout the cardiomyocytes.
    Molecular and cellular biology. 10/2014;
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    ABSTRACT: Mitochondria are dynamic organelles that change their morphology by active fusion and fission in response to cellular signaling and differentiation [1 and 2]. The in vivo role of mitochondrial fission in mammals has been examined by using tissue-specific knockout (KO) mice of the mitochondria fission-regulating GTPase Drp1 [3 and 4], as well as analyzing a human patient harboring a point mutation in Drp1 [5], showing that Drp1 is essential for embryonic and neonatal development and neuronal function. During oocyte maturation and aging, structures of various membrane organelles including mitochondria and the endoplasmic reticulum (ER) are changed dynamically [6 and 7], and their organelle aggregation is related to germ cell formation and epigenetic regulation [8, 9 and 10]. However, the underlying molecular mechanisms of organelle dynamics during the development and aging of oocytes have not been well understood. Here, we analyzed oocyte-specific mitochondrial fission factor Drp1-deficient mice and found that mitochondrial fission is essential for follicular maturation and ovulation in an age-dependent manner. Mitochondria were highly aggregated with other organelles, such as the ER and secretory vesicles, in KO oocyte, which resulted in impaired Ca2+ signaling, intercellular communication via secretion, and meiotic resumption. We further found that oocytes from aged mice displayed reduced Drp1-dependent mitochondrial fission and defective organelle morphogenesis, similar to Drp1 KO oocytes. On the basis of these findings, it appears that mitochondrial fission maintains the competency of oocytes via multiorganelle rearrangement.
    Current biology : CB. 09/2014;
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    ABSTRACT: Cells exposed to extreme physicochemical or mechanical stimuli die in an uncontrollable manner, as a result of their immediate structural breakdown. Such an unavoidable variant of cellular demise is generally referred to as /`accidental cell death/' (ACD). In most settings, however, cell death is initiated by a genetically encoded apparatus, correlating with the fact that its course can be altered by pharmacologic or genetic interventions. /`Regulated cell death/' (RCD) can occur as part of physiologic programs or can be activated once adaptive responses to perturbations of the extracellular or intracellular microenvironment fail. The biochemical phenomena that accompany RCD may be harnessed to classify it into a few subtypes, which often (but not always) exhibit stereotyped morphologic features. Nonetheless, efficiently inhibiting the processes that are commonly thought to cause RCD, such as the activation of executioner caspases in the course of apoptosis, does not exert true cytoprotective effects i
    Cell death and differentiation 09/2014; · 8.24 Impact Factor
  • Noboru Mizushima, Mayurbhai Himatbhai Sahani
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    ABSTRACT: The ATG genes are highly conserved in eukaryotes including yeasts, plants, and mammals. However, these genes appear to be only partially present in most protists. Recent studies demonstrated that, in the apicomplexan parasites Plasmodium (malaria parasites) and Toxoplasma, ATG8 localizes to the apicoplast, a unique nonphotosynthetic plastid with 4 limiting membranes. In contrast to this established localization, it remains unclear whether these parasites can induce canonical macroautophagy and if ATG8 localizes to autophagosomes. Furthermore, the molecular function of ATG8 in its novel workplace, the apicoplast, is totally unknown. Here, we review recent studies on ATG8 in Plasmodium and Toxoplasma, summarize both consensus and controversial findings, and discuss its potential role in these parasites.
    Autophagy 08/2014; 10(9). · 12.04 Impact Factor
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    ABSTRACT: Autophagy is mediated by a unique organelle, the autophagosome. Autophagosome formation involves a number of autophagy-related (ATG) proteins and complicated membrane dynamics. Although the hierarchical relationships of ATG proteins have been investigated, how individual ATG proteins or their complexes contribute to the organization of the autophagic membrane remains largely unknown. Here, systematic ultrastructural analysis of mouse embryonic fibroblasts and HeLa cells deficient in various ATG proteins revealed that the emergence of the isolation membrane (phagophore) requires FIP200/RB1CC1, ATG9A, and PtdIns 3-kinase activity. By contrast, small premature isolation membrane- and autophagosome-like structures were generated in cells lacking VMP1 and ATG2A/B, respectively. The isolation membranes could elongate in cells lacking ATG5, but these did not mature into autophagosomes. We also found that ferritin clusters accumulated at the autophagosome formation site together with p62/SQSTM1 in autophagy-deficient cells. These results reveal the specific functions of these representative ATG proteins in autophagic membrane organization and ATG-independent recruitment of ferritin to the autophagosome formation site.
    Journal of cell science. 07/2014;
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    ABSTRACT: Autophagy is one of the major degradation pathways for cytoplasmic components. The autophagic isolation membrane is a unique membrane whose content of unsaturated fatty acids is very high. However, the molecular mechanisms underlying formation of this membrane, including the roles of unsaturated fatty acids, remain to be elucidated. From a chemical library consisting of structurally diverse compounds, we screened for novel inhibitors of starvation-induced autophagy by measuring LC3 puncta formation in mouse embryonic fibroblasts stably expressing GFP-LC3 (GFP-LC3 MEFs). One of the inhibitors we identified, 2, 5-pyridinedicarboxamide, N2, N5-bis[5-[(dimethylamino)carbonyl]-4-methyl-2-thiazolyl], has a molecular structure similar to that of a known stearoyl-CoA desaturase (SCD) 1 inhibitor. To determine whether SCD1 inhibition influences autophagy, we examined the effects of the SCD1 inhibitor 28c. This compound strongly inhibited starvation-induced autophagy, as determined by LC3 puncta formation, immunoblot analyses of LC3, electron-microscopic observations, and p62/SQSTM1 accumulation. Overexpression of SCD1 or supplementation with oleic acid which is a catalytic product of SCD1 abolished the inhibition of autophagy by 28c. Furthermore, 28c suppressed starvation-induced autophagy without affecting mTOR activity, and also inhibited rapamycin-induced autophagy. In addition to inhibiting formation of LC3 puncta, 28c also inhibited formation of ULK1, WIPI1, Atg16L, and p62/SQSTM1 puncta. These results suggest that SCD1 activity is required for the earliest step of autophagosome formation.
    The Journal of biological chemistry. 07/2014;
  • Atsushi Yamamoto, Noboru Mizushima, Satoshi Tsukamoto
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    ABSTRACT: Autophagy is a dynamically regulated intracellular degradation system that is important for cellular processes such as amino acid production during starvation and intracellular quality control. Previously, we reported that autophagy is suppressed in oocytes, but is rapidly up-regulated after fertilization. During this period, autophagy is thought to be important for the generation of amino acids from the bulk degradation of maternal proteins that have accumulated during oogenesis. However, the mechanism of autophagy induction after fertilization is presently unknown. In most cell types, autophagy is negatively controlled by mammalian target of rapamycin complex 1 (mTORC1), which is typically regulated by amino acids and insulin or related growth factors. In this study, we determined the role of mTORC1 in fertilization-induced autophagy. On the basis of the phosphorylation status of mTORC1 substrates, we found that mTORC1 activity was relatively high in metaphase II (MII) oocytes, but was rapidly decreased within 3 h of fertilization. However, chemical inhibition of mTORC1 by Torin1 or PP242 in MII oocytes or fertilized embryos did not induce autophagy. In addition, activation of mTORC1 by cycloheximide did not inhibit fertilization-induced autophagy in fertilized embryos. By contrast, the phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 effectively suppressed autophagy in these embryos. These data suggest that, even though autophagy induction and post-fertilization mTORC1 activity are inversely correlated with each other, as observed in other cell types, mTORC1 suppression is neither essential nor sufficient for fertilization-induced autophagy, highlighting a unique feature of the regulation mechanism of autophagy-mediated intracellular turnover in early embryos.
    Biology of Reproduction 05/2014; · 4.03 Impact Factor
  • Takako Watanabe-Asano, Akiko Kuma, Noboru Mizushima
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    ABSTRACT: Protein synthesis inhibitors such as cycloheximide (CHX) are known to suppress protein degradation including autophagy. The fact that CHX inhibits autophagy has been generally interpreted to indicate that newly synthesized protein is indispensable for autophagy. However, CHX is also known to increase the intracellular level of amino acids and activate mTORC1 activity, a master negative regulator of autophagy. Accordingly, CHX can affect autophagic activity through inhibition of de novo protein synthesis and/or modulation of mTORC1 signaling. In this study, we investigated the effects of CHX on autophagy using specific autophagy markers. We found that CHX inhibited starvation-induced autophagy but not Torin1-induced autophagy. CHX also suppressed starvation-induced puncta formation of GFP-ULK1, an early-step marker of the autophagic process which is regulated by mTORC1. CHX activated mTORC1 even under autophagy-inducible starvation conditions. Finally, the inhibitory effect of CHX on starvation-induced autophagy was cancelled by the mTOR inhibitor Torin1. These results suggest that CHX inhibits starvation-induced autophagy through mTORC1 activation and also that autophagy does not require new protein synthesis at least in the acute phase of starvation.
    Biochemical and Biophysical Research Communications 03/2014; · 2.28 Impact Factor
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    ABSTRACT: Membrane fusion is generally controlled by Rabs, SNAREs, and tethering complexes. Syntaxin 17 (STX17) was recently identified as the autophagosomal SNARE, which is required for autophagosome-lysosome fusion in mammals and Drosophila. In this study, to better understand the mechanism of autophagosome-lysosome fusion, we searched for STX17-interacting proteins. Immunoprecipitation and mass spectrometry analysis identified VPS33A and VPS16, which are components of the HOPS tethering complex. We further confirmed that all HOPS components were coprecipitated with STX17. Knockdown of VPS33A, VPS16, or VPS39 blocked autophagic flux and caused accumulation of STX17- and LC3-positive autophagosomes. The endocytic pathway was also affected by knockdown of VPS33A, as previously reported, but not by knockdown of STX17. By contrast, UVRAG, a known HOPS-interacting protein, did not interact with the STX17-HOPS complex and may not be directly involved in autophagosome-lysosome fusion. Collectively, these results suggest that, in addition to the well-established function in the endocytic pathway, HOPS promotes autophagosome-lysosome fusion through interaction with STX17.
    Molecular biology of the cell 02/2014; · 5.98 Impact Factor
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    ABSTRACT: SQSTM1/p62 (sequestosome 1) is a multifunctional signaling molecule, involved in a variety of cellular pathways. SQSTM1 is one of the best-known autophagic substrates, and is therefore widely used as an indicator of autophagic degradation. Here we report that the expression level of SQSTM1 can be restored during prolonged starvation. Upon starvation, SQSTM1 is initially degraded by autophagy. However, SQSTM1 is restored back to basal levels during prolonged starvation in mouse embryonic fibroblasts and HepG2 cells, but not in HeLa and HEK293 cells. Restoration of SQSTM1 depends on its transcriptional upregulation, which is triggered by amino acid starvation. Furthermore, amino acids derived from the autophagy-lysosome pathway are used for de novo synthesis of SQSTM1 under starvation conditions. The restoration of SQSTM1 is independent of reactivation of MTORC1 (mechanistic target of rapamycin complex 1). These results suggest that the expression level of SQSTM1 in starved cells is determined by at least 3 factors: autophagic degradation, transcriptional upregulation, and availability of lysosomal-derived amino acids. The results of this study also indicate that the expression level of SQSTM1 does not always inversely correlate with autophagic activity.
    Autophagy 01/2014; 10(3). · 12.04 Impact Factor
  • Han-Ming Shen, Noboru Mizushima
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    ABSTRACT: In the past decade, autophagy studies have largely focused on the early stage of autophagy: the molecular mechanisms leading to autophagosome formation. Recently, however, we have observed significant progress in understanding the role of lysosomes, the specific cellular organelle that degrades cellular components delivered via autophagy. The discoveries include connections between autophagy and lysosomal biogenesis, activation, reformation, and turnover, as well as the identification of an autophagosomal SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein in control of autophagosome-lysosome fusion. We illustrate these findings in the context of the underlying molecular mechanisms and the relevance to human health and disease.
    Trends in Biochemical Sciences 12/2013; · 13.08 Impact Factor
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    ABSTRACT: Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response.
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    Peidu Jiang, Noboru Mizushima
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    ABSTRACT: Autophagy is a major intracellular degradative process that delivers cytoplasmic materials to the lysosome for degradation. Since the discovery of autophagy-related (Atg) genes in the 1990s, there has been a proliferation of studies on the physiological and pathological roles of autophagy in a variety of autophagy knockout models. However, direct evidence of the connections between ATG gene dysfunction and human diseases has emerged only recently. There are an increasing number of reports showing that mutations in the ATG genes were identified in various human diseases such as neurodegenerative diseases, infectious diseases, and cancers. Here, we review the major advances in identification of mutations or polymorphisms of the ATG genes in human diseases. Current autophagy-modulating compounds in clinical trials are also summarized.Cell Research advance online publication 10 December 2013; doi:10.1038/cr.2013.161.
    Cell Research 12/2013; · 10.53 Impact Factor
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    ABSTRACT: Induction of autophagy requires the ULK1 protein kinase complex and the Vps34 lipid kinase complex. PI3P synthesised by Vps34 accumulates in omegasomes, membrane extensions of the ER within which some autophagosomes form, whereas the ULK1 complex is thought to target autophagosomes independently of PI3P, and its functional relation to omegasomes is unclear. Here we show that the ULK1 complex colocalizes with omegasomes in a PI3P-dependent way. Live imaging of Atg13 (a ULK1 complex component), omegasomes and LC3 establishes and annotates for the first time a complete sequence of steps leading to autophagosome formation as follows: Upon starvation, ULK1 complex forms puncta associated with the ER and sporadically with mitochondria. If PI3P is available, these puncta become omegasomes. Subsequently, the ULK1 complex exits omegasomes and autophagosomes bud off. If PI3P is unavailable, ULK1 puncta are greatly reduced in number and duration. Atg13 (a component of the ULK1 complex) contains a region with affinity for acidic phospholipids, required for translocation to punctate structures and autophagy progression.
    Journal of Cell Science 09/2013; · 5.88 Impact Factor
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    ABSTRACT: Macroautophagy is an essential, homeostatic process involving degradation of cell's own components; it plays a role in catabolizing cellular components such as protein or lipids, and damaged or excess organelles. Here, we show that in Atg5(-/-) cells, sialyl oligosaccharides specifically accumulated in the cytosol. Accumulation of these glycans was observed under non-starved conditions, suggesting that non-induced, basal autophagy is essential for their catabolism. Interestingly, once accumulated in the cytosol, sialyl glycans cannot be efficiently catabolized by resumption of the autophagic process, suggesting that functional autophagy is important for preventing sialyl oligosaccharides from accumulating in the cytosol. Moreover, knockdown of sialin, a lysosomal transporter of sialic acids, resulted in a significant reduction of sialyl oligosaccharides, implying that autophagy affects the substrate specificity of this transporter. This study thus provides a surprising link between basal autophagy and catabolism of N-linked glycans.
    Journal of Biological Chemistry 07/2013; · 4.65 Impact Factor
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    ABSTRACT: Autophagosome formation is governed by sequential functions of autophagy-related (ATG) proteins. Although their genetic hierarchy in terms of localization to the autophagosome formation site has been determined, their temporal relationships remain largely unknown. In this study, we comprehensively analyzed the recruitment of mammalian ATG proteins to the autophagosome formation site by live-cell imaging, and determined their temporal relationships. Although ULK1 and ATG5 are separated in the genetic hierarchy, they synchronously accumulate at pre-existing VMP1-positive punctate structures, followed by recruitment of ATG14, ZFYVE1 and WIPI1. Only a small number of ATG9 vesicles appear to be associated with these structures. Finally, LC3 and SQSTM1/p62 accumulate synchronously, while the other ATG proteins dissociate from the autophagic structures. These results suggest that autophagosome formation takes place on the VMP1-containing domain of the endoplasmic reticulum or a closely related structure, where ULK1 and ATG5 complexes are synchronously recruited.
    Autophagy 07/2013; 9(10). · 12.04 Impact Factor
  • Takako Naito, Akiko Kuma, Noboru Mizushima
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    ABSTRACT: Autophagy is a highly inducible intracellular degradation process. It is generally induced by nutrient starvation and suppressed by food intake. Mammalian (or mechanistic) target of rapamycin complex 1 (mTORC1) is considered to be the major regulator of autophagy, but the precise mechanism of in vivo regulation remains to be fully characterized. Here, we examined the autophagy-suppressive effect of glucose, insulin, and amino acids in the liver and muscle in mice starved for 1 day. Refeeding after starvation with a standard mouse chow rapidly suppressed autophagy in both tissues, and this suppression was inhibited by rapamycin administration almost completely in the liver and partially in muscle, confirming that mTORC1 is indeed an crucial regulator in vivo. As glucose administration showed no major suppressive effect on autophagy, we examined the role of insulin and amino acids using hyperinsulinemic-euglycemic clamp and intravenous amino acid infusion techniques. Insulin administration showed a clear effect on the mTORC1-autophagy pathway in muscle, but had only a very weak effect in the liver. By contrast, amino acids were able to regulate the mTORC1-autophagy pathway in the liver, but less effectively in muscle. These results suggest that autophagy is differentially regulated by insulin and amino acids in a tissue-dependent manner.
    Journal of Biological Chemistry 06/2013; · 4.65 Impact Factor
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    ABSTRACT: The lens of the eye is composed of fiber cells, which differentiate from epithelial cells and undergo programmed organelle degradation during terminal differentiation. Although autophagy, a major intracellular degradation system, is constitutively active in these cells, its physiological role has remained unclear. We have previously shown that Atg5-dependent macroautophagy is not necessary for lens organelle degradation, at least during the embryonic period. Here, we generated lens-specific Atg5 knockout mice and showed that Atg5 is not required for lens organelle degradation at any period of life. However, deletion of Atg5 in the lens results in age-related cataract, which is accompanied by accumulation of polyubiquitinated and oxidized proteins, p62, and insoluble crystallins, suggesting a defect in intracellular quality control. We also produced lens-specific Pik3c3 knockout mice to elucidate the possible involvement of Atg5-independent alternative autophagy, which is proposed to be dependent on Pik3c3 (also known as Vps34), in lens organelle degradation. Deletion of Pik3c3 in the lens does not affect lens organelle degradation, but leads to congenital cataract and a defect in lens development after birth likely due to an impairment of the endocytic pathway. Taken together, these results suggest that clearance of lens organelles is independent of macroautophagy. These findings also clarify the physiological role of Atg5 and Pik3c3 in quality control and development of the lens, respectively.
    Journal of Biological Chemistry 03/2013; · 4.65 Impact Factor
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    Eisuke Itakura, Noboru Mizushima
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    ABSTRACT: The phagophore (also called isolation membrane) elongates and encloses a portion of cytoplasm, resulting in formation of the autophagosome. After completion of autophagosome formation, the outer autophagosomal membrane becomes ready to fuse with the lysosome for degradation of enclosed cytoplasmic materials. However, the molecular mechanism for how the fusion of completed autophagosomes with the lysosome is regulated has not been fully understood. We discovered syntaxin 17 (STX17) as an autophagosomal soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE). STX17 has a hairpin-type structure mediated by two transmembrane domains, each containing glycine zipper motifs. This unique transmembrane structure contributes to its specific localization to completed autophagosomes but not to phagophores. STX17 interacts with SNAP29 and the lysosomal SNARE VAMP8, and all of these proteins are required for autophagosome-lysosome fusion. The late recruitment of STX17 to completed autophagosomes could prevent premature fusion of the lysosome with unclosed phagophores.
    Autophagy 03/2013; 9(6). · 12.04 Impact Factor

Publication Stats

31k Citations
1,830.89 Total Impact Points

Institutions

  • 2013–2014
    • The University of Tokyo
      • Faculty & Graduate School of Medicine
      Tōkyō, Japan
  • 2006–2014
    • Tokyo Medical and Dental University
      • Department of Physiology and Cell Biology
      Edo, Tōkyō, Japan
    • National Institute of Genetics
      • Department of Cell Genetics
      Mishima, Shizuoka-ken, Japan
  • 2012
    • University of Michigan
      • Life Sciences Institute
      Ann Arbor, MI, United States
  • 2007–2011
    • University of Texas Southwestern Medical Center
      • Department of Microbiology
      Dallas, TX, United States
  • 2007–2010
    • Keio University
      • Department of Physiology
      Tokyo, Tokyo-to, Japan
  • 2003–2009
    • Hokkaido University
      • Graduate School of Pharmaceutical Sciences
      Sapporo-shi, Hokkaido, Japan
    • The Graduate University for Advanced Studies
      • Department of Molecular Physiology
      Miura, Kanagawa-ken, Japan
  • 2008
    • University of Washington Seattle
      • Department of Immunology
      Seattle, WA, United States
    • Saitama University
      Saitama, Saitama, Japan
    • Columbia University
      New York City, New York, United States
  • 2005–2007
    • Tokyo Metropolitan Institute of Medical Science
      Edo, Tōkyō, Japan
  • 2005–2006
    • Harvard Medical School
      • Department of Cell Biology
      Boston, MA, United States
  • 1998–2005
    • National Institute for Basic Biology
      Okazaki, Aichi, Japan
  • 2004
    • Osaka University
      • Department of Oral and Molecular Microbiology
      Ibaraki, Osaka-fu, Japan
    • Japan Science and Technology Agency (JST)
      Edo, Tōkyō, Japan
  • 2000
    • University of California, Davis
      Davis, California, United States