PROTEIN SORTING TO THE LYSOSOME-LIKE VACUOLE IN YEAST

酵母中溶酶体样液泡的蛋白质分类

基本信息

批准号：
3281760
负责人：
SCOTT D EMR
金额：
$ 19.03万
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：
1983
资助国家：
美国
起止时间：
1983-12-01 至 1995-11-30
项目状态：
已结题

项目摘要

The selective recognition, sorting and vesicle-mediated transport of proteins through the secretory pathway 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 yeast mutants that exhibit severe and specific defects in protein delivery to the lysosome-like vacuole in yeast. The defective genes in these mutants 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. 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 medical importance of this sorting pathway is exemplified by the serious lysosomal storage diseases (e.g. I-cell disease, pseudo-Hurler polydystrophy as well as other diseases (e.g. osteoporosis and the progression of certain types of cancer) that result from, or are correlated with, mislocalization of lysosomal hydrolases. Eight VPS genes have been cloned and sequenced in the lab. To determine where and how the products of these genes act to direct the sorting and delivery of vacuolar hydrolases, we propose to: 1) use indirect immunofluorescence and cell fractionation techniques to determine the subcellular location of the VPS gene products, 2) isolate temperature- conditional vps alleles to analyze the onset kinetics of the vacuolar protein sorting defects and to identify potential intermediates in the sorting process, 3) use genetic suppression and chemical cross-linking agents to identify and characterize interactions among VPS gene products, 4) employ an in vitro assay that reconstitutes vacuolar protein delivery to characterize the stage specific requirements and functions of Vps proteins, and 5) clone and sequence functional homologs for certain VPS genes from cDNA libraries of Schizosaccharomyces pombe as well as mammalian (human) cell types to begin an analysis of the functional role VPS gene products play in other eukaryotes. It is anticipated that these studies will result in a detailed view of the organization and control of protein sorting and vesicular traffic between the Golgi complex and the vacuole/lysosome in yeast and other eukaryotes.

选择性识别、分选和囊泡介导的转运通过分泌途径的蛋白质代表了一个基本特征所有真核细胞。指导的精确机制和机械这些过程尚不清楚。我们已经分离出大量在蛋白质递送方面表现出严重且特定缺陷的酵母突变体酵母中的溶酶体样液泡。这些缺陷基因突变体可能编码细胞蛋白质分类的成分设备。了解分子和生化基础关于这些突变体（vps）中的液泡蛋白分选缺陷，我们提出表征某些 VPS 基因及其产物所起的作用液泡酶从高尔基复合体的分选和输送液泡。酵母和其他酵母之间的基本相似之处真核细胞在其蛋白质传递途径中，与酵母中可用的强大遗传和分子方法，使酵母成为解决这些问题的理想有机体。医学上的重要性这种分选途径的例子是严重的溶酶体储存疾病（例如 I 细胞病、假性多发性营养不良症以及其他疾病（例如骨质疏松症和某些类型癌症的进展）由溶酶体的错误定位引起或与之相关水解酶。实验室已克隆并测序了 8 个 VPS 基因。确定这些基因的产物在何处以及如何发挥作用来指导分选和液泡水解酶的递送，我们建议：1）使用间接免疫荧光和细胞分级分离技术来确定 VPS基因产物的亚细胞定位，2)分离温度- 条件 vps 等位基因分析液泡的起始动力学蛋白质分选缺陷并识别潜在的中间体分选过程，3）使用基因抑制和化学交联识别和表征 VPS 基因产物之间相互作用的试剂， 4) 采用体外测定法重建液泡蛋白递送至表征 Vps 蛋白的阶段特定要求和功能， 5) 克隆并测序某些 VPS 基因的功能同源物粟酒裂殖酵母以及哺乳动物（人）的 cDNA 文库细胞类型开始分析 VPS 基因产物的功能作用在其他真核生物中发挥作用。预计这些研究将产生详细了解蛋白质分选的组织和控制高尔基复合体和液泡/溶酶体之间的囊泡交通酵母和其他真核生物。