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Mechanism and functions of membrane binding by the Atg5-Atg12/Atg16 complex during autophagosome formation.

ABSTRACT: Autophagy is a conserved process for the bulk degradation of cytoplasmic material. Triggering of autophagy results in the formation of double membrane-bound vesicles termed autophagosomes. The conserved Atg5-Atg12/Atg16 complex is essential for autophagosome formation. Here, we show that the yeast Atg5-Atg12/Atg16 complex directly binds membranes. Membrane binding is mediated by Atg5, inhibited by Atg12 and activated by Atg16. In a fully reconstituted system using giant unilamellar vesicles and recombinant proteins, we reveal that all components of the complex are required for efficient promotion of Atg8 conjugation to phosphatidylethanolamine and are able to assign precise functions to all of its components during this process. In addition, we report that in vitro the Atg5-Atg12/Atg16 complex is able to tether membranes independently of Atg8. Furthermore, we show that membrane binding by Atg5 is downstream of its recruitment to the pre-autophagosomal structure but is essential for autophagy and cytoplasm-to-vacuole transport at a stage preceding Atg8 conjugation and vesicle closure. Our findings provide important insights into the mechanism of action of the Atg5-Atg12/Atg16 complex during autophagosome formation.

SUBMITTER: Romanov J

PROVIDER: S-EPMC3501226 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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Mechanism and functions of membrane binding by the Atg5-Atg12/Atg16 complex during autophagosome formation.

Romanov Julia J Walczak Marta M Ibiricu Iosune I Schüchner Stefan S Ogris Egon E Kraft Claudine C Martens Sascha S

The EMBO journal 20121012 22

Autophagy is a conserved process for the bulk degradation of cytoplasmic material. Triggering of autophagy results in the formation of double membrane-bound vesicles termed autophagosomes. The conserved Atg5-Atg12/Atg16 complex is essential for autophagosome formation. Here, we show that the yeast Atg5-Atg12/Atg16 complex directly binds membranes. Membrane binding is mediated by Atg5, inhibited by Atg12 and activated by Atg16. In a fully reconstituted system using giant unilamellar vesicles and ...[more]

PMID: 23064152

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Project description:The interactions between viruses and cellular autophagy have been widely reported. On the one hand, autophagy is an important innate immune response against viral infection. On the other hand, some viruses exploit the autophagy pathway for their survival and proliferation in host cells. Vaccinia virus is a member of the family of Poxviridae which includes the smallpox virus. The biogenesis of vaccinia envelopes, including the core envelope of the immature virus (IV), is not fully understood. In this study we investigated the possible interaction between vaccinia virus and the autophagy membrane biogenesis machinery. Massive LC3 lipidation was observed in mouse fibroblast cells upon vaccinia virus infection. Surprisingly, the vaccinia virus induced LC3 lipidation was shown to be independent of ATG5 and ATG7, as the atg5 and atg7 null mouse embryonic fibroblasts (MEFs) exhibited the same high levels of LC3 lipidation as compared with the wild-type MEFs. Mass spectrometry and immunoblotting analyses revealed that the viral infection led to the direct conjugation of ATG3, which is the E2-like enzyme required for LC3-phosphoethanonamine conjugation, to ATG12, which is a component of the E3-like ATG12–ATG5-ATG16 complex for LC3 lipidation. Consistently, ATG3 was shown to be required for the vaccinia virus induced LC3 lipidation. Strikingly, despite the high levels of LC3 lipidation, subsequent electron microscopy showed that vaccinia virus-infected cells were devoid of autophagosomes, either in normal growth medium or upon serum and amino acid deprivation. In addition, no autophagy flux was observed in virus-infected cells. We further demonstrated that neither ATG3 nor LC3 lipidation is crucial for viral membrane biogenesis or viral proliferation and infection. Together, these results indicated that vaccinia virus does not exploit the cellular autophagic membrane biogenesis machinery for their viral membrane production. Moreover, this study demonstrated that vaccinia virus instead actively disrupts the cellular autophagy through a novel molecular mechanism that is associated with aberrant LC3 lipidation and a direct conjugation between ATG12 and ATG3.

Dataset Information

Mechanism and functions of membrane binding by the Atg5-Atg12/Atg16 complex during autophagosome formation.

Publications

Mechanism and functions of membrane binding by the Atg5-Atg12/Atg16 complex during autophagosome formation.

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