Autophagosomes form at ER-mitochondria contact sites

1] Department of Genetics, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan [2] Laboratory of Intracellular Membrane Dynamics, Graduate School of Frontier Biosciences, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan [3].
Nature (Impact Factor: 41.46). 03/2013; 495(7441). DOI: 10.1038/nature11910
Source: PubMed


Autophagy is a tightly regulated intracellular bulk degradation/recycling system that has fundamental roles in cellular homeostasis. Autophagy is initiated by isolation membranes, which form and elongate as they engulf portions of the cytoplasm and organelles. Eventually isolation membranes close to form double membrane-bound autophagosomes and fuse with lysosomes to degrade their contents. The physiological role of autophagy has been determined since its discovery, but the origin of autophagosomal membranes has remained unclear. At present, there is much controversy about the organelle from which the membranes originate-the endoplasmic reticulum (ER), mitochondria and plasma membrane. Here we show that autophagosomes form at the ER-mitochondria contact site in mammalian cells. Imaging data reveal that the pre-autophagosome/autophagosome marker ATG14 (also known as ATG14L) relocalizes to the ER-mitochondria contact site after starvation, and the autophagosome-formation marker ATG5 also localizes at the site until formation is complete. Subcellular fractionation showed that ATG14 co-fractionates in the mitochondria-associated ER membrane fraction under starvation conditions. Disruption of the ER-mitochondria contact site prevents the formation of ATG14 puncta. The ER-resident SNARE protein syntaxin 17 (STX17) binds ATG14 and recruits it to the ER-mitochondria contact site. These results provide new insight into organelle biogenesis by demonstrating that the ER-mitochondria contact site is important in autophagosome formation.

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Available from: Atsuo Amano
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    • "Indeed, a growing list of membrane trafficking regulators impinges on autophagosome formation and maturation. These have been reviewed in detail recently (Lamb et al, 2013a,b) and include small GTPases (Itoh et al, 2008; Zoppino et al, 2010; Moreau et al, 2012), RabGAPs (GTPase activating proteins) (Itoh et al, 2011; Longatti et al, 2012; Popovic et al, 2012), SNARE proteins (Itakura et al, 2012; Hamasaki et al, 2013; Puri et al, 2013; Moreau et al, 2014), sorting nexins (Knaevelsrud et al, 2013) and vesicle tethering complexes (Liang et al, 2008). Unpicking the functions of these components will permit a better understanding of the mechanisms of autophagosome formation. "
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    ABSTRACT: Macroautophagy requires membrane trafficking and remodelling to form the autophagosome and deliver its contents to lysosomes for degradation. We have previously identified the TBC domain-containing protein, TBC1D14, as a negative regulator of autophagy that controls delivery of membranes from RAB11-positive recycling endosomes to forming autophagosomes. In this study, we identify the TRAPP complex, a multi-subunit tethering complex and GEF for RAB1, as an interactor of TBC1D14. TBC1D14 binds to the TRAPP complex via an N-terminal 103 amino acid region, and overexpression of this region inhibits both autophagy and secretory traffic. TRAPPC8, the mammalian orthologue of a yeast autophagy-specific TRAPP subunit, forms part of a mammalian TRAPPIII-like complex and both this complex and TBC1D14 are needed for RAB1 activation. TRAPPC8 modulates autophagy and secretory trafficking and is required for TBC1D14 to bind TRAPPIII. Importantly, TBC1D14 and TRAPPIII regulate ATG9 trafficking independently of ULK1. We propose a model whereby TBC1D14 and TRAPPIII regulate a constitutive trafficking step from peripheral recycling endosomes to the early Golgi, maintaining the cycling pool of ATG9 required for initiation of autophagy.
    Preview · Article · Dec 2015 · The EMBO Journal
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    • "3F3A labeling localizes Gp78 to the mitochondria-associated ER or MAM (Wang et al., 2000; Goetz et al., 2007), and we therefore tested whether S538 phosphorylation affected ER proximity to mitochondria . Cos7 cells were cotransfected with pOct-dsRed and FLAG-Gp78, Gp78 S538 mutants, or Gp78 RFmut and then labeled for the MAM marker syntaxin17 (Hamasaki et al., 2013) and, to identify transfected cells, anti-FLAG. Three-dimensional confocal stacks were acquired, and overlap of the syntaxin17-labeled MAM and total mitochondria was quantified. "
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    ABSTRACT: Gp78 is an ERAD-associated E3 ubiquitin ligase that induces degradation of the mitofusin mitochondrial fusion proteins and mitochondrial fission. Gp78 is localized throughout the ER, however the anti-Gp78 3F3A monoclonal antibody (mAb) recognizes Gp78 selectively in mitochondria-associated ER domains. Epitope mapping localized the epitope of 3F3A and a commercial anti-Gp78 mAb to an eight amino acid (533-541 in mouse Gp78 isoform 2) motif, that forms part of a highly conserved 41 amino acid region containing 14-3-3 and WW binding domains and a p38 MAP kinase (p38 MAPK) consensus site on serine 538 (S538). 3F3A binds selectively to non-phosphorylated S538 Gp78. Using 3F3A as a reporter, Gp78 S538 phosphorylation was induced by serum starvation and shown to be mediated by p38 MAPK. Mass spectroscopy analysis of Gp78 phosphopeptides confirmed S538 as a major p38 MAPK phosphorylation site on Gp78. Gp78 S538 phosphorylation limited its ability to induce mitochondrial fusion and degrade MFN1 and MFN2 but did not impact in vitro Gp78 ubiquitin E3 ligase activity. Phosphomimetic Gp78 S538D mutation prevented Gp78 promotion of ER-mitochondria interaction and SB203580 inhibition of p38 MAPK increased ER-mitochondria association. p38 MAPK phosphorylation of Gp78 S538 therefore regulates Gp78-dependent ER-mitochondria association and mitochondria motility. © 2015 by The American Society for Cell Biology.
    Preview · Article · Sep 2015 · Molecular biology of the cell
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    • "Thus, the PS lipids on the extracellular membrane leaflet of the vesicle are in a compartment that is topologically equivalent to the lumenal membrane leaflet of the double-membraned autophagosome-like organelle. The ER is a major membrane source for autophagosomes (Hamasaki et al., 2013) and has lumenal membrane leaflet enriched in PS lipids (Kay et al., 2012). Given that the isolated PS vesicles also contain ER-resident proteins (Figure S2B), it is highly likely that the autophagosome-like organelles , and thereby the released PS vesicles, originated from ER and/or ER-derived replication organelle membranes. "
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    ABSTRACT: A central paradigm within virology is that each viral particle largely behaves as an independent infectious unit. Here, we demonstrate that clusters of enteroviral particles are packaged within phosphatidylserine (PS) lipid-enriched vesicles that are non-lytically released from cells and provide greater infection efficiency than free single viral particles. We show that vesicular PS lipids are co-factors to the relevant enterovirus receptors in mediating subsequent infectivity and transmission, in particular to primary human macrophages. We demonstrate that clustered packaging of viral particles within vesicles enables multiple viral RNA genomes to be collectively transferred into single cells. This study reveals a novel mode of viral transmission, where enteroviral genomes are transmitted from cell-to-cell en bloc in membrane-bound PS vesicles instead of as single independent genomes. This has implications for facilitating genetic cooperativity among viral quasispecies as well as enhancing viral replication. Copyright © 2015 Elsevier Inc. All rights reserved.
    Full-text · Article · Feb 2015 · Cell
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