ROLE OF THE YEAST VPS15/VPS34 KINASE COMPLEX IN YEAST SECRETORY PROTEIN SORTING

蛋白质的选择性识别、分选和转运， 通过囊泡载体将细胞内细胞器连接到另一个细胞器代表了 所有真核细胞的基本特征。最近五月的一次集会 在酵母和哺乳动物细胞中的研究已经证明， 磷脂酰肌醇在调节细胞膜 除了作为第二信使的传统作用外， 细胞表面的信号转导。我们建议继续使用 酵母中强大的遗传和分子方法， 描述了复杂的分子相互作用， 介导的从高尔基体到溶酶体样空泡的蛋白质分选。我们 已经分离出大量酵母突变体（VPS）， 液泡蛋白分选的严重缺陷。在基因产物中 在这些VPS突变体中，我们已经鉴定了一种蛋白激酶， Vps 15 p和磷脂酰肌醇3-激酶Vps 34 p。每个激酶 活性是蛋白质在高尔基体到液泡的运输所必需的 传递途径Vps 15蛋白激酶似乎调节蛋白质 通过选择性膜募集和Vps 34 p的活化进行分选。 在本资助中，我们建议描述Vps 34 PtdIn的角色- 3-激酶及其脂质产物PtdIns 3 P在溶酶体蛋白中发挥作用 分类赠款的具体目标是利用遗传， 分子、形态学和生物化学方法：1）鉴定 Vps 15 p/Vps 34 p激酶的上游调节因子和下游效应 级联，2）表征酵母中PtdIns 3 P的周转途径和3） 分析Vps 34 p在调节高尔基体向核内体转运中的作用。 这些研究将对人类健康具有普遍重要性， 已经提出溶酶体水解酶的错误定位可以增强肿瘤的生长， 细胞生长和增加某些癌症的转移潜力， 有助于细胞外基质成分的水解， 靶组织。此外，PI 3-激酶信号通路已被发现。 显示在控制有丝分裂，细胞生长， 分化和凋亡。

The selective recognition, sorting and transport of proteins from one intracellular organelle to another via vesicular carriers represents an essential feature of all eukaryotic cells. A convergency of may recent studies in both yeast and mammalian cells has demonstrated that phosphoinositides play critical roles in the regulation of membrane trafficking in addition to their classical roles as second messengers in signal transduction at the cell surface. We propose to continue our use of the powerful genetic and molecular approaches available in yeast to characterize the complex molecular interactions that direct vesicle- mediated protein sorting from the Golgi to the lysosome-like vacuole. We have isolated a large collection of yeast mutants (vps) that exhibit severe defects in vacuolar protein sorting. Among the gene products affected in these vps mutants, we have identified a protein kinase, Vps15p, and a phosphatidylinositol 3-kinase, Vps34p. Each of the kinase activities is essential for protein transport in the Golgi-to-vacuole delivery pathway. The Vps15 protein kinase appears to regulate protein sorting by the selective membrane recruitment and activation of Vps34p. In this grant, we propose to characterize the role that the Vps34 PtdIns- 3-kinase and its lipid product, PtdIns3P, play in lysosomal protein sorting. The specific objectives of the grant are to use genetic, molecular, morphological and biochemical approaches to: 1) Identify upstream regulators and downstream effects of the Vps15p/Vps34p kinase cascade, 2) Characterize the turnover pathway for PtdIns3P in yeast and 3) Analyze the role of Vps34p in regulating Golgi to endosome transport. These studies will be of general importance to human health because mislocalization of lysosomal hydrolases has been proposed to enhance tumor cell growth and increase the metastatic potential of certain cancers by contributing to the hydrolysis of extracellular matrix components of target tissues. In addition, PI 3-kinase signaling pathways have been shown to play an important role in the control of mitosis, cell growth, differentiation and apoptosis.