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

蛋白质的选择性识别、分类和运输 胞内细胞器通过囊泡载体传输到另一个细胞器代表着一种 所有真核细胞的基本特征。最近五月的一次汇聚 对酵母和哺乳动物细胞的研究表明， 肌醇磷脂在膜调节中发挥重要作用 除了他们作为第二信使的经典角色之外的贩运 细胞表面的信号转导。我们建议继续使用我们的 酵母中强大的遗传和分子方法可用于 描述引导水泡的复杂分子相互作用- 介导蛋白质从高尔基体到溶酶体样液泡的分选。我们 已经分离出大量酵母突变株(VP)，这些突变株展示了 空泡蛋白分选存在严重缺陷。在基因产品中 在这些VPS突变体中，我们发现了一种蛋白激酶， Vps15p和磷脂酰肌醇3-激酶，Vps34p。每一种激酶 活性对高尔基体到液泡的蛋白质运输是必不可少的 传递途径。Vps15蛋白激酶似乎调节蛋白质 Vps34p的选择性膜募集和激活分选。 在这项拨款中，我们建议描述Vps34 PtdIns- 3-激酶及其脂类产物PtdIns3P在溶酶体蛋白中的作用 分类。这笔赠款的具体目标是使用基因， 分子、形态和生化方法：1)鉴定 Vps15p/Vps34p激酶的上游调节和下游作用 级联，2)表征PtdIns3P在酵母中的周转途径和3) 分析Vps34p在调节高尔基体到内体转运中的作用。 这些研究将对人类健康具有普遍的重要性，因为 溶酶体水解酶的错误定位已被提出用来增强肿瘤 细胞生长和增加某些癌症的转移潜能 有助于水解胞外基质成分 目标组织。此外，PI-3-激酶信号通路已经被 在控制有丝分裂、细胞生长、 分化和细胞凋亡。

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.