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

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.