Screening for compounds targeted to novel rapamycin-independent autophagic mechanism

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In this grant, we established monitoring system of rapamycin-independent autophagic protein degradation utilizing GFP-tagged Ypk1 localization change under fluorescent microscope. In this assay, inhibitory activity of compounds could be monitored by the loss of vacuolar localization of Ypk1 when vacuolar peptidase deficient strain(pep4Δ) is used.This assay should be utilized along with primary screening system of normal protein degradation, namely Western blotting analysis after SDS-PAGE to monitor the degree of Ypk1 proteolysis. We also screened a set of yeast protein kinase mutant strains to understand the autophagic degradation of Ypk1. This screening was based on the hypothesis that intracellular signaling event is involved in the Ypk1 degradation. We have identified a pair of protein kinase that is responsible of Ypk1 phosphorylation both in vivo and in vitro. Data obtained here will be fundamentally required for future screening to target novel autophagic pathway.