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

酵母 VPS15/VPS34 激酶复合物在酵母分泌蛋白分选中的作用

• 批准号: 6448183
• 负责人: SCOTT D EMR
• 金额: $ 15.83万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-04-20 至 2002-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6448183
• 关键词: Golgi apparatus; Saccharomyces cerevisiae; biological signal transduction; enzyme activity; enzyme complex; fungal genetics; gene expression; intracellular transport; lysosomes; membrane activity; mutant; phosphatidylinositol 3 kinase; phosphorylation; protein kinase; protein localization; protein structure function; protein transport; site directed mutagenesis; vesicle /vacuole

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.

蛋白质的选择性识别、分选和运输 细胞内细胞器通过囊泡载体转移到另一个细胞器代表 所有真核细胞的基本特征。最近可能出现的收敛 对酵母和哺乳动物细胞的研究表明 磷酸肌醇在细胞膜的调节中发挥着重要作用 除了作为第二信使的经典角色外，人口贩运 细胞表面的信号转导。我们建议继续使用 酵母中可用的强大遗传和分子方法 表征指导囊泡的复杂分子相互作用 介导从高尔基体到溶酶体样液泡的蛋白质分选。我们 分离出大量酵母突变体（vps），这些突变体表现出 液泡蛋白质分选的严重缺陷。基因产物中 在这些 vps 突变体中，我们鉴定出了一种蛋白激酶， Vps15p 和磷脂酰肌醇 3-激酶 Vps34p。每个激酶 活性对于高尔基体到液泡的蛋白质运输至关重要 传递途径。 Vps15 蛋白激酶似乎可以调节蛋白质 通过选择性膜募集和激活 Vps34p 进行分选。 在这笔赠款中，我们建议描述 Vps34 PtdIns 的作用： 3-激酶及其脂质产物 PtdIns3P 在溶酶体蛋白中发挥作用 排序。赠款的具体目标是利用遗传、 分子、形态学和生化方法：1) 识别 Vps15p/Vps34p 激酶的上游调节因子和下游效应 级联，2) 表征酵母中 PtdIns3P 的周转途径，3) 分析Vps34p在调节高尔基体向内体转运中的作用。 这些研究对人类健康具有普遍重要意义，因为 溶酶体水解酶的错误定位已被提出可增强肿瘤 细胞生长并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 显示在有丝分裂、细胞生长的控制中发挥重要作用， 分化和凋亡。