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The Atg16L complex specifies the site of LC3 lipidation for membrane biogenesis in autophagy.

ABSTRACT: Two ubiquitin-like molecules, Atg12 and LC3/Atg8, are involved in autophagosome biogenesis. Atg12 is conjugated to Atg5 and forms an approximately 800-kDa protein complex with Atg16L (referred to as Atg16L complex). LC3/Atg8 is conjugated to phosphatidylethanolamine and is associated with autophagosome formation, perhaps by enabling membrane elongation. Although the Atg16L complex is required for efficient LC3 lipidation, its role is unknown. Here, we show that overexpression of Atg12 or Atg16L inhibits autophagosome formation. Mechanistically, the site of LC3 lipidation is determined by the membrane localization of the Atg16L complex as well as the interaction of Atg12 with Atg3, the E2 enzyme for the LC3 lipidation process. Forced localization of Atg16L to the plasma membrane enabled ectopic LC3 lipidation at that site. We propose that the Atg16L complex is a new type of E3-like enzyme that functions as a scaffold for LC3 lipidation by dynamically localizing to the putative source membranes for autophagosome formation.

SUBMITTER: Fujita N

PROVIDER: S-EPMC2366860 | biostudies-other | 2008 May

REPOSITORIES: biostudies-other

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The Atg16L complex specifies the site of LC3 lipidation for membrane biogenesis in autophagy.

Fujita Naonobu N Itoh Takashi T Omori Hiroko H Fukuda Mitsunori M Noda Takeshi T Yoshimori Tamotsu T

Molecular biology of the cell 20080305 5

Two ubiquitin-like molecules, Atg12 and LC3/Atg8, are involved in autophagosome biogenesis. Atg12 is conjugated to Atg5 and forms an approximately 800-kDa protein complex with Atg16L (referred to as Atg16L complex). LC3/Atg8 is conjugated to phosphatidylethanolamine and is associated with autophagosome formation, perhaps by enabling membrane elongation. Although the Atg16L complex is required for efficient LC3 lipidation, its role is unknown. Here, we show that overexpression of Atg12 or Atg16L ...[more]

PMID: 18321988

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Project description:Previous studies indicated that hepatitis B virus (HBV) stimulates autophagy to favor its production. To understand how HBV co-opts autophagy as a proviral machinery, we studied the roles of key autophagy proteins in HBV-replicating liver cell cultures. RNA interference-mediated silencing of Atg5, Atg12, and Atg16L1, which promote autophagophore expansion and LC3 membrane conjugation, interfered with viral core/nucleocapsid (NC) formation/stability and strongly diminished virus yields. Concomitantly, the core/NC membrane association and their sorting to envelope-positive compartments were perturbed. A close inspection of the HBV/autophagy cross talk revealed that the virus depended on Atg12 covalently conjugated to Atg5. In support of this finding, HBV required the E2-like enzymes Atg10 and Atg3, which catalyze or facilitate Atg5-12 conjugation, respectively. Atg10 and Atg3 knockdowns decreased HBV production, while Atg3 overexpression increased virus yields. Mapping analyses demonstrated that the HBV core protein encountered the Atg5-12/16L1 complex via interaction with the intrinsically disordered region of the Atg12 moiety that is dispensable for autophagy function. The role of Atg12 in HBV replication was confirmed by its incorporation into virions. Although the Atg5-12/16L1 complex and Atg3 are essential for LC3 lipidation and, thus, for autophagosome maturation and closure, HBV propagation did not require LC3. Silencing of LC3B, the most abundant LC3 isoform, did not inhibit but rather augmented virus production. Similar augmenting effects were obtained upon overexpression of a dominant negative mutant of Atg4B that blocked the lipid conjugation of the LC3 isoforms and their GABARAP paralogues. Together, our data indicate that HBV subverts early, nondegradative autophagy components as assembly scaffolds, thereby concurrently avoiding autophagosomal destruction.IMPORTANCE Infections with the hepatitis B virus (HBV), an enveloped pararetrovirus, cause about 1 million deaths per year, as current therapies rarely achieve a cure. Understanding the HBV life cycle and concomitant host cell interactions is instrumental to develop new antiviral concepts. Here, we proceeded to dissect the roles of the autophagy machinery in virus propagation. By using RNA interference and overexpression studies in HBV-replicating cell lines, we identified the autophagic Atg5-12/16L1 elongation complex along with Atg10 and Atg3 to be an essential scaffold for HBV nucleocapsid assembly/stability. Deficits in Atg5-12/16L1 and Atg10/Atg3, which normally drive autophagophore membrane expansion, strongly impaired progeny virus yields. HBV gained access to Atg5-12/16L1 via interaction of its core protein with the Atg12 moiety of the complex. In contrast, subsequent autophagosome maturation and closure events were unnecessary for HBV replication, as evidenced by inhibition of Atg8/LC3 conjugation. Interfering with the HBV/Atg12 cross talk may be a tool for virus control.

Dataset Information

The Atg16L complex specifies the site of LC3 lipidation for membrane biogenesis in autophagy.

Publications

The Atg16L complex specifies the site of LC3 lipidation for membrane biogenesis in autophagy.

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