PROTEIN TRANSLOCATION IN YEAST

酵母中的蛋白质易位

• 批准号: 2178781
• 负责人: Peter Walter
• 金额: $ 20.31万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-12-01 至 1995-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2178781
• 关键词: Saccharomyces cerevisiae; alleles; complementary DNA; electroporation; endoplasmic reticulum; gel filtration chromatography; gene expression; gene mutation; genetic manipulation; genetic transcription; guanine nucleotide binding protein; intracellular transport; laboratory mouse; laboratory rabbit; membrane permeability; molecular cloning; nucleic acid probes; nucleic acid sequence; protein sequence; protein transport; site directed mutagenesis; structural genes; suppressor mutations

The primary objective of the proposed research is to understand, mechanistically, how the correct proteins are targeted to and translocated across the endoplasmic reticulum membrane in the simple eukaryote Saccharomyces cerevisiae. We will test our current hypothesis that there are two parallel targeting pathways, one dependent on the signal recognition particle (SRP), the other one SRP-independent. Our aim is to identify all the gene products catalyzing the reactions in both pathways, and to understand their molecular function and importance in vivo. Specifically, i) we will identify and sequence the genes encoding all subunits of yeast SRP and SRP receptor; ii) we will use in vitro protein translocation assays or assays for partial reactions to analyze in detail their mechanism of action. We aim to identify novel components functioning in targeting and translocation and clone their genes; iii) we will use random mutagenesis to isolate conditional (cs, ts) mutations in SRP, SRP receptor and newly identified components. These mutations and site- directed mutations in predicted GTP binding sites, and in regions of predicted RNA-RNA, RNA-protein or protein-protein interactions will be used to analyze in vivo and in vitro the role of the gene products; iv) we will isolate additional genes involved in protein targeting and translocation using improved genetic selections. Such mutations will be analyzed as to their involvement in either the SRP-dependent or the SRP-independent pathway; v) we will use suppressor analysis of mutations in each pathway to identify interacting gene products. The proposed biochemical and genetic analyses aim at a precise molecular understanding of the protein targeting and translocation process. We wish to learn which aspects of the process are unique to yeast and which aspects can be generalized to other eukaryotic cells and bacteria. Ultimately, we hope that through the power of a combined genetic and biochemical approach, we will identify the cellular constituents of the protein translocation machinery that are essential for translocation, as well as those that are modulatory.

拟议研究的主要目的是了解，