Formation Process of Autophagosome Is Traced with Apg8/Aut7p in Yeast

Abstract

We characterized Apg8/Aut7p essential for autophagy in yeast. Apg8p was transcriptionally upregulated in response to starvation and mostly existed as a protein bound to membrane under both growing and starvation conditions. Immunofluorescence microscopy revealed that the intracellular localization of Apg8p changed drastically after shift to starvation. Apg8p resided on unidentified tiny dot structures dispersed in the cytoplasm at growing phase. During starvation, it was localized on large punctate structures, some of which were confirmed to be autophagosomes and autophagic bodies by immuno-EM. Besides these structures, we found that Apg8p was enriched on isolation membranes and in electron less-dense regions, which should contain Apg8p-localized membrane- or lipid-containing structures. These structures would represent intermediate structures during autophagosome formation. Here, we also showed that microtubule does not play an essential role in the autophagy in yeast. The result does not match with the previously proposed role of Apg8/Aut7p, delivery of autophagosome to the vacuole along microtubule. Moreover, it is revealed that autophagosome formation is severely impaired in the apg8 null mutant. Apg8p would play an important role in the autophagosome formation.

Atg27 Is Required for Autophagy-dependent Cycling of Atg9

“…Atg8 is an ubiquitin-like protein that is conjugated to phosphatidylethanolamine (Kirisako et al, 1999; and is one of two Atg proteins that remain associated with the completed autophagosome membrane. Similar to Pex14-GFP, Atg8 is degraded after delivery into the vacuole, whereas the GFP moiety is again relatively stable (Shintani et al, 2002).…”

Autophagy in organelle homeostasis: Peroxisome turnover

“…The membrane-recruited form of Atg8 is lipidated. The lipidation process involves specific posttranslational modifications of the protein via a process that shares features with the ubiquitin conjugation mechanism Kirisako et al,1999). Lipidation requires cleavage of the arginine residue located at the extreme C terminus, which is removed by the cysteine protease Atg4.…”

“…For example, in rat normal kidney epithelial cells the cytochalasins, microfilament depolymerizing agents, interfere with formation of the autophagosomes (Aplin et al,1992), whereas microtubule inhibitors block fusion of the autophagosome with the lysosome in both rat hepatocytes and normal rat kidney epithelial cells (Aplin et al,1992;Seglen et al,1996). However, microtubules in yeast are not essential for autophagy (Kirisako et al,1999;. In contrast, the actin cytoskeleton is required for two types of selective autophagy including pexophagy .…”

A current perspective of autophagosome biogenesis

“…Since the Atg12-Atg5-Atg16(L1) complex accumulates on the isolation membrane and its precursor, Atg8/ LC3-PE conjugation takes place on these membranes. PE-conjugated Atg8/LC3 stably associates with autophagy-related structures, including isolation membranes and completed autophagosomes, and is therefore widely used as a marker for microscopic analysis of autophagy [53,54]. In addition, PE-conjugated Atg8/LC3 displays increased mobility in SDS-PAGE gels compared to its unconjugated form, and can be specifically detected by western blotting.…”