担当区分：責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：Informa UK Limited  

DOI： 10.1080/15548627.2022.2123638

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1016/j.molcel.2022.08.008

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担当区分：筆頭著者,　責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：eLife Sciences Publications, Ltd  

Monitoring autophagic flux is necessary for most autophagy studies. The autophagic flux assays currently available for mammalian cells are generally complicated and do not yield highly quantitative results. Yeast autophagic flux is routinely monitored with the green fluorescence protein (GFP)-based processing assay, whereby the amount of GFP proteolytically released from GFP-containing reporters (e.g. GFP-Atg8), detected by immunoblotting, reflects autophagic flux. However, this simple and effective assay is typically inapplicable to mammalian cells because GFP is efficiently degraded in lysosomes while the more proteolytically resistant red fluorescent protein (RFP) accumulates in lysosomes under basal conditions. Here, we report a HaloTag (Halo)-based reporter processing assay to monitor mammalian autophagic flux. We found that Halo is sensitive to lysosomal proteolysis but becomes resistant upon ligand binding. When delivered into lysosomes by autophagy, pulse-labeled Halo-based reporters (e.g. Halo-LC3 and Halo-GFP) are proteolytically processed to generate Halo^ligand when delivered into lysosomes by autophagy. Hence, the amount of free Halo^ligand detected by immunoblotting or in-gel fluorescence imaging reflects autophagic flux. We demonstrate the applications of this assay by monitoring the autophagy pathways, macroautophagy, selective autophagy, and even bulk nonselective autophagy. With the Halo-based processing assay, mammalian autophagic flux and lysosome-mediated degradation can be monitored easily and precisely.

DOI： 10.7554/elife.78923

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その他リンク： https://cdn.elifesciences.org/articles/78923/elife-78923-v2.xml

掲載種別：研究論文（学術雑誌）   出版者・発行元：Wiley  

DOI： 10.1002/1873-3468.14329

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その他リンク： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/1873-3468.14329

掲載種別：研究論文（学術雑誌）   出版者・発行元：Informa UK Limited  

DOI： 10.1080/15548627.2022.2059168

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掲載種別：研究論文（学術雑誌）   出版者・発行元：The Company of Biologists  

ABSTRACT

TMEM41B and VMP1 are endoplasmic reticulum (ER)-localizing multi-spanning membrane proteins required for ER-related cellular processes such as autophagosome formation, lipid droplet homeostasis and lipoprotein secretion in eukaryotes. Both proteins have a VTT domain, which is similar to the DedA domain found in bacterial DedA family proteins. However, the molecular function and structure of the DedA and VTT domains (collectively referred to as DedA domains) and the evolutionary relationships among the DedA domain-containing proteins are largely unknown. Here, we conduct a remote homology search and identify a new clade consisting mainly of bacterial proteins of unknown function that are members of the Pfam family PF06695. Phylogenetic analysis reveals that the TMEM41, VMP1, DedA and PF06695 families form a superfamily with a common origin, which we term the DedA superfamily. Coevolution-based structural prediction suggests that the DedA domain contains two reentrant loops facing each other in the membrane. This topology is biochemically verified by the substituted cysteine accessibility method. The predicted structure is topologically similar to that of the substrate-binding region of Na+-coupled glutamate transporter solute carrier 1 (SLC1) proteins. A potential ion-coupled transport function of the DedA superfamily proteins is discussed.

This article has an associated First Person interview with the joint first authors of the paper.

DOI： 10.1242/jcs.255877

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その他リンク： http://journals.biologists.com/jcs/article-pdf/doi/10.1242/jcs.255877/1804874/jcs255877.pdf

掲載種別：研究論文（学術雑誌）   出版者・発行元：Springer Science and Business Media LLC  

DOI： 10.1038/s41594-020-00520-2

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その他リンク： https://www.nature.com/articles/s41594-020-00520-2

担当区分：筆頭著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：Springer Science and Business Media LLC  

DOI： 10.1038/s41594-019-0204-3

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その他リンク： http://www.nature.com/articles/s41594-019-0204-3

掲載種別：研究論文（学術雑誌）   出版者・発行元：eLife Sciences Publications, Ltd  

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.

DOI： 10.7554/elife.43088

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その他リンク： https://cdn.elifesciences.org/articles/43088/elife-43088-v2.xml

掲載種別：研究論文（学術雑誌）   出版者・発行元：Informa UK Limited  

DOI： 10.1080/15548627.2018.1532262

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Rockefeller University Press  

Macroautophagy is an evolutionarily conserved catabolic mechanism that delivers intracellular constituents to lysosomes using autophagosomes. To achieve degradation, lysosomes must fuse with closed autophagosomes. We previously reported that the soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) protein syntaxin (STX) 17 translocates to autophagosomes to mediate fusion with lysosomes. In this study, we report an additional mechanism. We found that autophagosome–lysosome fusion is retained to some extent even in STX17 knockout (KO) HeLa cells. By screening other human SNAREs, we identified YKT6 as a novel autophagosomal SNARE protein. Depletion of YKT6 inhibited autophagosome–lysosome fusion partially in wild-type and completely in STX17 KO cells, suggesting that YKT6 and STX17 are independently required for fusion. YKT6 formed a SNARE complex with SNAP29 and lysosomal STX7, both of which are required for autophagosomal fusion. Recruitment of YKT6 to autophagosomes depends on its N-terminal longin domain but not on the C-terminal palmitoylation and farnesylation that are essential for its Golgi localization. These findings suggest that two independent SNARE complexes mediate autophagosome–lysosome fusion.

DOI： 10.1083/jcb.201712058

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Wiley  

DOI： 10.1002/1873-3468.12901

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Informa UK Limited  

DOI： 10.1080/15548627.2017.1327940

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掲載種別：研究論文（学術雑誌）   出版者・発行元：EMBO  

DOI： 10.15252/embj.201695189

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その他リンク： https://onlinelibrary.wiley.com/doi/full-xml/10.15252/embj.201695189

担当区分：筆頭著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1016/j.devcel.2016.06.015

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担当区分：筆頭著者,　責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1074/jbc.m115.684233

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担当区分：責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：Proceedings of the National Academy of Sciences  

Significance

Autophagy is a highly conserved degradative process in eukaryotes. In response to starvation, a number of autophagosome-related (Atg) proteins are recruited, and these proteins govern the process of autophagosome formation. Atg9 vesicles are thought to play an essential role in the nucleation step, but it remains unclear how Atg9 vesicles are localized to the site of autophagosome formation. In this study, we found that Atg9 interacts with the HORMA (from Hop1, Rev7, and Mad2) domain of Atg13. Atg13 mutants lacking the Atg9-binding region fail to recruit Atg9 vesicles to the site of autophagosome formation and exhibit severe defects in autophagy. Thus, the HORMA domain of Atg13 facilitates recruitment of Atg9 vesicles during autophagosome formation. Our studies provide a molecular insight into how Atg9 vesicles become part of the autophagosomal membrane.

DOI： 10.1073/pnas.1421092112

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担当区分：筆頭著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：Springer Science and Business Media LLC  

DOI： 10.1038/nsmb.2822

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その他リンク： http://www.nature.com/articles/nsmb.2822

掲載種別：研究論文（学術雑誌）   出版者・発行元：Springer Science and Business Media LLC  

DOI： 10.1038/ncomms4207

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その他リンク： http://www.nature.com/articles/ncomms4207

記述言語：英語   出版者・発行元：TAYLOR & FRANCIS INC  

Phosphatidylinositol 3-phosphate (PtdIns3P) is a phospholipid essential for autophagy, but the detailed distribution of PtdIns3P in the membrane of autophagosomes, autophagic bodies, and other organelles is unclear due to technical difficulties. In the present study, we examined PtdIns3P distribution in autophagic membranes with an electron microscopy method called the quick-freeze freeze-fracture replica labeling method (QF-FRL), which can define the distribution of membrane lipids at the nanometer scale. In this method, membranes are split into 2 leaflets so that membrane asymmetry, i.e., differences between the 2 leaflets, can be defined unambiguously. As a result, PtdIns3P in the yeast autophagosome was found to exist much more abundantly in the lumenal leaflet (i.e., the leaflet facing the space between the outer and inner autophagosomal membranes) than in the cytoplasmic leaflet. In contrast, PtdIns3P in the mammalian autophagosome was confined to the cytoplasmic leaflet, showing an opposite asymmetry from that found in yeast. In yeast deleted for 2 cytoplasmic PtdIns3P phosphatases, Ymr1 and Sjl3, PtdIns3P distributed in an equivalent density in the 2 leaflets of the autophagosome membrane, suggesting that the asymmetry in wild-type yeast is generated as a result of unilateral PtdIns3P hydrolysis. The contrasting PtdIns3P distribution revealed in the present study suggested that formation of autophagic membranes may proceed in different ways in yeast and mammals.

DOI： 10.4161/auto.28489

Web of Science

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掲載種別：研究論文（学術雑誌）   出版者・発行元：The Company of Biologists  

Autophagy is a bulk degradation system mediated by biogenesis of autophagosomes under starvation conditions. In Saccharomyces cerevisiae, a membrane sac called the isolation membrane (IM) is generated from the pre-autophagosomal structure (PAS); ultimately, the IM expands to become a mature autophagosome. Eighteen Atg (autophagy-related) proteins are engaged in autophagosome formation at the PAS. However, the cup-shaped IM was visualized just as a dot by fluorescence microscopy, posing a challenge to further understanding the detailed functions of Atg proteins during IM expansion. Here, we visualized expanding IMs as cup-shaped structures using fluorescence microscopy by enlarging a selective cargo of autophagosomes, and finely mapped the localizations of Atg proteins. The PAS scaffold proteins (Atg13 and Atg17) and phosphatidylinositol 3-kinase complex I were localized to a dot at the junction between the IM and the vacuolar membrane, termed the vacuole-IM contact site (VICS). By contrast, Atg1, Atg8, and the Atg16–Atg12–Atg5 complex labeled both the VICS and the cup-shaped IM. We designate this localization the ‘IM’ pattern. The Atg2–Atg18 complex and Atg9 localized at the edge of the IM as two or three dots, in close proximity to the endoplasmic reticulum (ER) via ER exit sites. Thus, we designate these dots as the ‘IM edge’ pattern. These data suggest that Atg proteins play individual roles at spatially distinct localizations during IM expansion. These findings will facilitate detailed investigations of the function of each Atg protein during autophagosome formation.

DOI： 10.1242/jcs.122960

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その他リンク： http://journals.biologists.com/jcs/article-pdf/doi/10.1242/jcs.122960/2044897/jcs122960.pdf

掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1074/jbc.m112.411454

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Springer Science and Business Media LLC  

DOI： 10.1038/nsmb.2451

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その他リンク： http://www.nature.com/articles/nsmb.2451

掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1074/jbc.m112.394205

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1074/jbc.m112.397570

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1074/jbc.c112.387514

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1016/j.str.2012.04.018

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担当区分：筆頭著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：Rockefeller University Press  

During the process of autophagy, cytoplasmic materials are sequestered by double-membrane structures, the autophagosomes, and then transported to a lytic compartment to be degraded. One of the most fundamental questions about autophagy involves the origin of the autophagosomal membranes. In this study, we focus on the intracellular dynamics of Atg9, a multispanning membrane protein essential for autophagosome formation in yeast. We found that the vast majority of Atg9 existed on cytoplasmic mobile vesicles (designated Atg9 vesicles) that were derived from the Golgi apparatus in a process involving Atg23 and Atg27. We also found that only a few Atg9 vesicles were required for a single round of autophagosome formation. During starvation, several Atg9 vesicles assembled individually into the preautophagosomal structure, and eventually, they are incorporated into the autophagosomal outer membrane. Our findings provide conclusive linkage between the cytoplasmic Atg9 vesicles and autophagosomal membranes and offer new insight into the requirement for Atg9 vesicles at the early step of autophagosome formation.

DOI： 10.1083/jcb.201202061

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担当区分：筆頭著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：Proceedings of the National Academy of Sciences  

Mitochondria import most of their resident proteins from the cytosol, and the import receptor Tom20 of the outer-membrane translocator TOM40 complex plays an essential role in specificity of mitochondrial protein import. Here we analyzed the effects of Tom20 binding on NMR spectra of a long mitochondrial presequence and found that it contains two distinct Tom20-binding elements. In vitro import and cross-linking experiments revealed that, although the N-terminal Tom20-binding element is essential for targeting to mitochondria, the C-terminal element increases efficiency of protein import in the step prior to translocation across the inner membrane. Therefore Tom20 has a dual role in protein import into mitochondria: recognition of the targeting signal in the presequence and tethering the presequence to the TOM40 complex to increase import efficiency.

DOI： 10.1073/pnas.1014918108

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担当区分：筆頭著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1074/jbc.m109.041756

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Rockefeller University Press  

Mitochondrial protein traffic requires coordinated operation of protein translocator complexes in the mitochondrial membrane. The TIM23 complex translocates and inserts proteins into the mitochondrial inner membrane. Here we analyze the intermembrane space (IMS) domains of Tim23 and Tim50, which are essential subunits of the TIM23 complex, in these functions. We find that interactions of Tim23 and Tim50 in the IMS facilitate transfer of precursor proteins from the TOM40 complex, a general protein translocator in the outer membrane, to the TIM23 complex. Tim23–Tim50 interactions also facilitate a late step of protein translocation across the inner membrane by promoting motor functions of mitochondrial Hsp70 in the matrix. Therefore, the Tim23–Tim50 pair coordinates the actions of the TOM40 and TIM23 complexes together with motor proteins for mitochondrial protein import.

DOI： 10.1083/jcb.200808068

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Rockefeller University Press  

Newly synthesized mitochondrial proteins are imported into mitochondria with the aid of protein translocator complexes in the outer and inner mitochondrial membranes. We report the identification of yeast Tam41, a new member of mitochondrial protein translocator systems. Tam41 is a peripheral inner mitochondrial membrane protein facing the matrix. Disruption of the TAM41 gene led to temperature-sensitive growth of yeast cells and resulted in defects in protein import via the TIM23 translocator complex at elevated temperature both in vivo and in vitro. Although Tam41 is not a constituent of the TIM23 complex, depletion of Tam41 led to a decreased molecular size of the TIM23 complex and partial aggregation of Pam18 and -16. Import of Pam16 into mitochondria without Tam41 was retarded, and the imported Pam16 formed aggregates in vitro. These results suggest that Tam41 facilitates mitochondrial protein import by maintaining the functional integrity of the TIM23 protein translocator complex from the matrix side of the inner membrane.

DOI： 10.1083/jcb.200603087

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担当区分：筆頭著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：Wiley  

DOI： 10.1016/j.febslet.2004.12.018

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1074/jbc.m410272200

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1074/jbc.m404591200

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Rockefeller University Press  

Mitochondrial outer and inner membranes contain translocators that achieve protein translocation across and/or insertion into the membranes. Recent evidence has shown that mitochondrial β-barrel protein assembly in the outer membrane requires specific translocator proteins in addition to the components of the general translocator complex in the outer membrane, the TOM40 complex. Here we report two novel mitochondrial outer membrane proteins in yeast, Tom13 and Tom38/Sam35, that mediate assembly of mitochondrial β-barrel proteins, Tom40, and/or porin in the outer membrane. Depletion of Tom13 or Tom38/Sam35 affects assembly pathways of the β-barrel proteins differently, suggesting that they mediate different steps of the complex assembly processes of β-barrel proteins in the outer membrane.

DOI： 10.1083/jcb.200405138

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掲載種別：研究論文（学術雑誌）   出版者・発行元：The Company of Biologists  

Nearly all mitochondrial proteins are synthesized in the cytosol and subsequently imported into mitochondria with the aid of translocators: the TOM complex in the outer membrane, and the TIM23 and TIM22 complexes in the inner membrane. The TOM complex and the TIM complexes cooperate to achieve efficient transport of proteins to the matrix or into the inner membrane and several components, including Tom22, Tim23, Tim50 and small Tim proteins, mediate functional coupling of the two translocator systems. The TOM complex can be disconnected from the TIM systems and their energy sources (ATP andΔΨ), however, using alternative mechanisms to achieve vectorial protein translocation across the outer membrane

DOI： 10.1242/jcs.00667

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担当区分：筆頭著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1016/s0092-8674(02)01053-x

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