Atg4 recycles inappropriately lipidated Atg8 to promote autophagosome biogenesis

Abstract

Atg8 is a ubiquitin-like protein required for autophagy in the budding yeast Saccharomyces cerevisiae. A ubiquitin-like system mediates the conjugation of the C terminus of Atg8 to the lipid phosphatidylethanolamine (PE), and this conjugate (Atg8-PE) plays a crucial role in autophagosome formation at the phagophore assembly site/pre-autophagosomal structure (PAS). The cysteine protease Atg4 processes the C terminus of newly synthesized Atg8 and also delipidates Atg8 to release the protein from membranes. While the former is a prerequisite for lipidation of Atg8, the significance of the latter in autophagy has remained unclear. Here, we show that autophagosome formation is significantly retarded in cells deficient for Atg4-mediated delipidation of Atg8. We find that Atg8-PE accumulates on various organelle membranes including the vacuole, the endosome and the ER in these cells, which depletes unlipidated Atg8 and thereby attenuates its localization to the PAS. Our results suggest that the Atg8-PE that accumulates on organelle membranes is erroneously produced by lipidation system components independently of the normal autophagic process. It is also suggested that delipidation of Atg8 by Atg4 on different organelle membranes promotes autophagosome formation. Considered together with other results, we propose that Atg4 acts to compensate for the intrinsic defect in the lipidation system; it recycles Atg8-PE generated on inappropriate membranes to maintain a reservoir of unlipidated Atg8 that is required for autophagosome formation at the PAS.

Differential Function of the Two Atg4 Homologues in the Aggrephagy Pathway in Caenorhabditis elegans

“…The LGG-1(G116 end) transgene also rescues, although not completely, defective degradation of protein aggregates in atg-4.1; atg-4.2 double mutants. In ⌬atg4⌬atg8 yeast cells, expressing Atg8G116 also partially suppresses the defect in autophagy (35). Thus, the deconjugation activity is not completely required for autophagic flux.…”

“…A recent study showed that yeast Atg4 also recycles Atg8-PE pro- duced on inappropriate membranes to maintain a cytoplasmic pool of unlipidated Atg8 that can undergo lipidation and participate in autophagosome formation at the pre-autophagosomal structure (PAS) (35). The mutant form of LGG-1, in which the conjugated glycine residue is directly exposed, rescues defective degradation of protein aggregates in atg-4.1 mutants.…”

Differential processing of Arabidopsis ubiquitin-like Atg8 autophagy proteins by Atg4 cysteine proteases

“…The exposed Gly residue is then implicated in adduct with PE. Atg4 is also required for a deconjugation step in which Atg8-PE is removed from the outer membrane of autophagosome for Atg8 recycling and autophagosome completion (7,8).…”

Autophagic Processes in Yeast: Mechanism, Machinery and Regulation

“…The hydrolysis of this lipid is critical in releasing the Atg machinery from the completed autophagosome, and it appears to be requisite for the fusion of these vesicles with the vacuole (Cebollero et al 2012b). Accordingly, autophagosomes with Atg8 on their surface fuse inefficiently with the vacuole Nakatogawa et al 2012). This fusion step involves components that are common to other transport processes that terminate at the vacuole.…”