Authorship：Lead author   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

DOI： 10.1038/nsmb.2822

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Other Link： http://www.nature.com/articles/nsmb.2822

Language：English   Publisher：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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Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

DOI： 10.1038/ncomms4207

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Other Link： http://www.nature.com/articles/ncomms4207

Publishing type：Research paper (scientific journal)   Publisher：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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Other Link： http://journals.biologists.com/jcs/article-pdf/doi/10.1242/jcs.122960/2044897/jcs122960.pdf

Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1074/jbc.m112.411454

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Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

DOI： 10.1038/nsmb.2451

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Other Link： http://www.nature.com/articles/nsmb.2451

Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1074/jbc.m112.394205

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1074/jbc.m112.397570

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1074/jbc.c112.387514

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.str.2012.04.018

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Authorship：Lead author   Publishing type：Research paper (scientific journal)   Publisher：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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Authorship：Lead author   Publishing type：Research paper (scientific journal)   Publisher：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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Authorship：Lead author   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1074/jbc.m109.041756

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Publishing type：Research paper (scientific journal)   Publisher：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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Publishing type：Research paper (scientific journal)   Publisher：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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Authorship：Lead author   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1016/j.febslet.2004.12.018

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1074/jbc.m410272200

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1074/jbc.m404591200

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Publishing type：Research paper (scientific journal)   Publisher：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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Publishing type：Research paper (scientific journal)   Publisher：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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Authorship：Lead author   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/s0092-8674(02)01053-x

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