Membrane Delivery to the Yeast Autophagosome from the Golgi–Endosomal System

Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 0QH, UK.
Molecular biology of the cell (Impact Factor: 4.47). 11/2010; 21(22):3998-4008. DOI: 10.1091/mbc.E10-05-0457
Source: PubMed


While many of the proteins required for autophagy have been identified, the source of the membrane of the autophagosome is still unresolved with the endoplasmic reticulum (ER), endosomes, and mitochondria all having been evoked. The integral membrane protein Atg9 is delivered to the autophagosome during starvation and in the related cytoplasm-to-vacuole (Cvt) pathway that occurs constitutively in yeast. We have examined the requirements for delivery of Atg9-containing membrane to the yeast autophagosome. Atg9 does not appear to originate from mitochondria, and Atg9 cannot reach the forming autophagosome directly from the ER or early Golgi. Components of traffic between Golgi and endosomes are known to be required for the Cvt pathway but do not appear required for autophagy in starved cells. However, we find that pairwise combinations of mutations in Golgi-endosomal traffic components apparently only required for the Cvt pathway can cause profound defects in Atg9 delivery and autophagy in starved cells. Thus it appears that membrane that contains Atg9 is delivered to the autophagosome from the Golgi-endosomal system rather than from the ER or mitochondria. This is underestimated by examination of single mutants, providing a possible explanation for discrepancies between yeast and mammalian studies on Atg9 localization and autophagosome formation.

    • "A more recent study has shown that Atg9 is concentrated in a yeast compartment comprising clusters of vesicles and tubules that are often adjacent to the mitochondria (Mari et al. 2010). However, another study which examined the molecular requirements for delivery of Atg9-containing membrane to the yeast autophagosome indicated that Atg9 is unlikely to be delivered to PAS from the mitochondria, or an early secretory pathway compartment such as the ER, but more likely from the Golgi-endosomal membranes (Ohashi and Munro 2010). Rubinsztein and colleagues have shown that the plasma membrane could be a potential source of at least some types of autophagosomes characterized by Atg16L1-positive 'early autophagosome precursors' formed by clathrin-dependent endocytic mechanism (Ravikumar et al. 2010). "
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    ABSTRACT: Abstract Recent advances have revealed much about the signaling events and molecular components associated with autophagy. Although it is clear that there are multiple points of intersection and connection between autophagy and known vesicular membrane transport processes between membrane compartments, autophagy is modulated by a distinct set of molecular components, and the autophagosome has a unique membrane composition. A key question that has yet to be resolved with regards to autophagosome formation is its membrane source. Various evidences have indicated that membranes from the endoplasmic reticulum (ER), mitochondria, Golgi, endosomes and the plasma membrane could all potentially act as a source of autophagosomal membrane in non-specialized macroautophagy. Recent investigations have generated advances in terms of the mitochondria's involvement in starvation-induced autophagy, and refined localization of autophagosome formation to ER-mitochondria contact sites. On the other hand, data accumulates on the delivery of membrane sources to the pre-autophagosome structure by Atg9-containing mobile carriers, which likely originated from the Golgi-endosome system. The answer to the question on the origin of membrane materials for autophagosome biogenesis awaits further reconciliation of these different findings.
    Molecular Membrane Biology 11/2013; 30(8). DOI:10.3109/09687688.2013.850178 · 1.69 Impact Factor
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    • "A SNARE complex contains four parallel SNARE helix bundles, supplied by a Qa SNARE and either separate Qb and Qc SNAREs or a SNAP protein that contains both Qb and Qc SNARE domains, all located on the membrane of the first vesicle, and an R SNARE anchored to the second vesicle (Hong, 2005). Yeast SNARE proteins Vam3, Vam7, Vti1, and Ykt6 have all been suggested to play a role in fusion of autophagosomes with the vacuole, the equivalent of metazoan lysosomes (Dilcher et al., 2001; Ishihara et al., 2001; Ohashi and Munro, 2010). Vam3 and Vam7 have no clear homologues in metazoa. "
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    ABSTRACT: During autophagy, phagophores capture portions of cytoplasm and form double-membrane autophagosomes to deliver cargo for lysosomal degradation. How autophagosomes gain competence to fuse with late endosomes and lysosomes is not known. In this paper, we show that Syntaxin17 is recruited to the outer membrane of autophagosomes to mediate fusion through its interactions with ubisnap (SNAP-29) and VAMP7 in Drosophila melanogaster. Loss of these genes results in accumulation of autophagosomes and a block of autolysosomal degradation during basal, starvation-induced, and developmental autophagy. Viable Syntaxin17 mutant adults show large-scale accumulation of autophagosomes in neurons, severe locomotion defects, and premature death. These mutant phenotypes cannot be rescued by neuron-specific inhibition of caspases, suggesting that caspase activation and cell death do not play a major role in brain dysfunction. Our findings reveal the molecular mechanism underlying autophagosomal fusion events and show that lysosomal degradation and recycling of sequestered autophagosome content is crucial to maintain proper functioning of the nervous system.
    The Journal of Cell Biology 05/2013; 201(4):531-9. DOI:10.1083/jcb.201211160 · 9.83 Impact Factor
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    • "Fusion between the late endosome and lysosome uses syntaxin 7 (Stx7) (Qa), Vti1b (Qb), and Stx8 (Qc) on the late endosome and VAMP7 (R) on the lysosome (Pryor et al., 2004). Regarding the autophagy pathway, it has been suggested that Vam3 (Qa) (Darsow et al., 1997; Ohashi and Munro, 2010), Vam7 (Qc) (Ohashi and Munro, 2010; Sato et al., 1998), Ykt6 (R) (Dilcher et al., 2001; Klionsky, 2005), and Vti1 (Qb) (Fischer von Mollard and Stevens, 1999; Ishihara et al., 2001) are involved in autophagosome-vacuole fusion in yeast, and VAMP7 (R) (Fader et al., 2009), VAMP8 (R), and Vti1b (Qb) (Furuta et al., 2010) in mammals. However, these studies have not identified specific SNARE protein(s) on the autophagosomal membrane. "
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    ABSTRACT: The lysosome is a degradative organelle, and its fusion with other organelles is strictly regulated. In contrast to fusion with the late endosome, the mechanisms underlying autophagosome-lysosome fusion remain unknown. Here, we identify syntaxin 17 (Stx17) as the autophagosomal SNARE required for fusion with the endosome/lysosome. Stx17 localizes to the outer membrane of completed autophagosomes but not to the isolation membrane (unclosed intermediate structures); for this reason, the lysosome does not fuse with the isolation membrane. Stx17 interacts with SNAP-29 and the endosomal/lysosomal SNARE VAMP8. Depletion of Stx17 causes accumulation of autophagosomes without degradation. Stx17 has a unique C-terminal hairpin structure mediated by two tandem transmembrane domains containing glycine zipper-like motifs, which is essential for its association with the autophagosomal membrane. These findings reveal a mechanism by which the SNARE protein is available to the completed autophagosome.
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