Screening for compounds targeted to novel rapamycin-independent autophagic mechanism

本研究利用GFP标记的Ypk 1在荧光显微镜下的定位变化，建立了非雷帕霉素依赖性自噬蛋白降解的监测系统。在本试验中，当使用空泡肽酶缺陷型菌株（pep 4 Δ）时，化合物的抑制活性可以通过Ypk 1空泡定位的丧失来监测，该试验应沿着正常蛋白降解的初级筛选系统，即SDS-PAGE后的Western印迹分析，以监测Ypk 1蛋白降解的程度。我们还筛选了一组酵母蛋白激酶突变株，以了解Ypk 1的自噬降解。该筛选基于细胞内信号传导事件参与Ypk 1降解的假设。我们已经确定了一对蛋白激酶，这是负责YPK 1磷酸化在体内和体外。本文获得的数据将为未来筛选新的自噬通路提供基础。

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.