| Summary: | 碩士 === 國立臺灣大學 === 動物學研究研究所 === 95 === Autophagy is a catabolic membrane trafficking process conserved in all eukaryotic cells. During autophagic transport, cargos are incorporated into double-membrane vesicles and transported to the lysosomes/vacuole for degradation. In general, autophagy is induced in response to starvation stress for maintaining the cytosolic amino acid pool. In the budding yeast Saccharomyces cerevisiae, one type of selective autophagy, called the cytoplasm-to-vacuole targeting (Cvt) pathway, constitutively delivers at least two resident vacuolar hydrolases aminopeptidase Ι (Ape1) and α-mannosidase (Ams1).
Precursor of Ape1 (prApe1) is transported by either pathways depending on the nutrient condition. In previous studies, prApe1 is found assembling into a higher order complex and associating with its transport receptor Atg19. Through the interaction between Atg19 and Atg11, the complex is recruited to the pre-autophagosomal structure (PAS). Once the complex arrives at the PAS, phosphatidylethanolamine (PE)-conjugated Atg8 binds to Atg19 to ensure incorporation of the complex into the forming Cvt vesicle. However, parts of prApe1 are still successfully transported into the vacuole via autophagy in starved atg11Δ cells. Here we report that prApe1 could not be targeted to PAS and consequently failed to be delivered into the vacuole in atg8Δ atg11Δ double knockout cells. Thus we propose that Atg19 mediates dual prApe1 sorting arms though independent, instead of sequential, interaction with Atg11 and Atg8. In addition, during/after the sorting process, Atg11 is involved in recruitment of Atg9 to the PAS for vesicle formation through direct physical interaction. Furthermore, in the absence of prApe1, some of autophagy proteins including Atg11 are co-localized at multiple sites next to the vacuole, suggesting that these spots are still functional for vesicle formation and Atg11 may not be specific for prApe1 transport but involved in general selective event during autophagy.
Finally, we find that Atg9 is a phosphoprotein, which is hyper-phosphorylated under nitrogen starvation condition. A substitution mutation at the potential phosphorylation site Ser19 results in retarded Cvt pathway and bulk autophagy, along with the decrease of autophagosome generated under starvation, suggesting a close relation between Atg9 Ser19 and autophagy control.
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