ROLE OF THE YEAST VPS15 PROTEIN KINASE IN INTRACELLULAR PROTEIN SORTING

酵母 VPS15 蛋白激酶在细胞内蛋白分选中的作用

基本信息

批准号：
6237382
负责人：
SCOTT D EMR
金额：
$ 16.35万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
1997
资助国家：
美国
起止时间：
1997-06-20 至 1998-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6237382
关键词：
Saccharomyces cerevisiae biological signal transduction fungal genetics hydrolase intracellular transport lysosomes mutant phosphatidylinositols phosphoprotein phosphatase phosphorylation protein kinase protein structure function protein transport site directed mutagenesis vesicle /vacuole

项目摘要

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. The precise mechanisms and machinery that direct these processes are not yet known. We have isolated a large collection of mutant that exhibit severe defects in protein delivery to the yeast lysosome-like vacuole. The defective genes in these mutant are likely to encode components of the cells' protein sorting apparatus. Toward an understanding of the molecular and biochemical basis of the vacuolar protein sorting defects in these mutants (vps), we propose to characterize the roles certain of the VPS genes and their products play in the sorting and delivery of vacuolar enzymes from the Golgi complex to the vacuole in yeast. The fundamental similarities between yeast and other eukaryotic cells in their pathways for protein delivery, together with the powerful genetic and molecular approaches available in yeast, make yeast an ideal organism for addressing these problems. The yeast mutants we have isolated exhibit a similar phenotype to many transformed mammalian cell lines that are known to aberrantly secret lysosomal proteases. The mislocalization of these proteases has been proposed to potentiate the metastasis of tumor cells. The proposed studies focus on two of the VPS genes, VPS15 and VPS34. Mutations in each gene result in a common set of phenotypes suggesting that each may facilitate the same step in the vacuolar protein delivery pathway. The sequence of the VPS15 gene predicts a protein with the following features: a concensus site for N-terminal myristoylation, a region of homology with the regulatory subunit of protein phosphatase 2A (PP2A). The VPS34 gene product shares significant sequence similarity with the catalytic subunit of mammalian phosphatidylinositol-3-kinase. VPS34 P13-kinase activity may be regulated by the VPS15 gene product. We have found that overexpression of the Vps34 protein can suppress the defects caused by mutation within the kinase domain of the VPS15 gene product, but will not suppress a null allele of VPS15. Using both genetic and biochemical approaches, we propose to characterize: 1) the functional role of the Vps15 kinase domain, as well as protein phosphorylation, in vacuolar protein sorting, 2) the significance of the sequence homology in Vps15p to the PP2A regulatory subunit, 3) the subcellular location and possible site of action for both the Vps15 and Vps34 proteins, and 4) the potential interaction of Vps15 and Vps34 proteins with each other and possibly other cellular components (e.g., PP2A).

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。精确的机制和指导这些过程的机械尚不清楚。我们有分离出大量表现出严重缺陷的突变体将蛋白质递送至酵母溶酶体样液泡。有缺陷的基因这些突变体可能编码细胞蛋白质的成分分选装置。为了理解分子和这些细胞中液泡蛋白分选缺陷的生化基础突变体（vps），我们建议描述 VPS 的某些角色基因及其产物在液泡的分类和传递中发挥作用从高尔基复合体到酵母液泡的酶。基本的酵母和其他真核细胞在途径上的相似之处用于蛋白质输送，以及强大的遗传和分子酵母中可用的方法，使酵母成为理想的生物体解决这些问题。我们分离出的酵母突变体表现出与许多转化的哺乳动物细胞系相似的表型已知异常秘密的溶酶体蛋白酶。错误定位这些蛋白酶被认为可以增强肿瘤的转移细胞。拟议的研究重点关注两个 VPS 基因：VPS15 和 VPS34。每个基因的突变都会导致一组共同的表型，这表明每个都可以促进液泡蛋白质递送中的相同步骤途径。 VPS15 基因的序列预测了一种蛋白质以下功能：N 端肉豆蔻酰化的共识位点、与蛋白磷酸酶2A调节亚基同源的区域（PP2A）。 VPS34基因产物具有显着的序列相似性与哺乳动物磷脂酰肌醇-3-激酶的催化亚基。 VPS34 P13 激酶活性可能受 VPS15 基因产物调节。我们发现Vps34蛋白的过度表达可以抑制 VPS15 基因激酶结构域内突变引起的缺陷产品，但不会抑制 VPS15 的无效等位基因。两者同时使用遗传和生化方法，我们建议表征：1） Vps15 激酶结构域以及蛋白质的功能作用磷酸化，在液泡蛋白质分选中，2) 的意义 Vps15p 与 PP2A 调节亚基的序列同源性，3) Vps15 和 Vps15 的亚细胞位置和可能的作用位点 Vps34 蛋白，以及 4) Vps15 和 Vps34 的潜在相互作用蛋白质彼此之间以及可能的其他细胞成分（例如， PP2A）。