Formation process of autophagosome is traced with Apg8/Aut7p in yeast

We characterized Apg8/Aut7p essential for autophagy in yeast. Apg8p was tra nscriptionally upregulated in response to starvation and mostly existed as a protein bound to membrane under both growing and starvation conditions. I mmunofluorescence microscopy revealed that the intracellular localization o f Apg8p changed drastically after shift to starvation. Apg8p resided on uni dentified tiny dot structures dispersed in the cytoplasm at growing phase. During starvation, it was localized on large punctate structures, some of w hich were confirmed to be autophagosomes and autophagic bodies by immuno-EM . Besides these structures, we found that Apg8p was enriched on isolation m embranes and in electron less-dense regions, which should contain Apg8p-loc alized membrane- or lipid-containing structures. These structures would rep resent intermediate structures during autophagosome formation. Here, we als o 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 Ap g8/Aut7p, delivery of autophagosome to the vacuole along microtubule. Moreo ver, it is revealed that autophagosome formation is severely impaired in th e apg8 null mutant. Apg8p would play an important role in the autophagosome formation.