PROTEIN TRANSLOCATION IN YEAST

酵母中的蛋白质易位

• 批准号: 3292742
• 负责人: Peter Walter
• 金额: $ 17.61万
• 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/3292742
• 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 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， 从机械上讲，正确的蛋白质如何靶向和易位 跨越简单真核生物中的内质网膜 酿酒酵母。 我们将检验我们目前的假设 是两个平行的靶向途径，一个取决于信号 识别粒子（SRP），另一个独立于SRP。 我们的目标是 识别所有催化两种途径反应的基因产物， 并了解它们在体内的分子功能和重要性。 具体而言，i）我们将识别并对所有编码的基因进行测序 酵母SRP和SRP受体的亚基； ii）我们将使用体外蛋白 易位分析或部分反应的测定法，以详细分析 他们的作用机理。 我们旨在确定功能的新型组件 在靶向和易位并克隆其基因； iii）我们将使用 SRP中有条件（CS，TS）突变的随机诱变，SRP 受体和新确定的成分。 这些突变和部位 - 预测的GTP结合位点中的定向突变以及 将使用预测的RNA-RNA，RNA-蛋白质或蛋白质 - 蛋白质相互作用 在体内和体外分析基因产物的作用； iv）我们会的 分离蛋白靶向和易位的其他基因 使用改进的遗传选择。 将分析此类突变 他们参与了与SRP依赖性或SRP无关的参与 途径； v）我们将在每种途径中使用抑制剂分析 识别相互作用的基因产物。 提出的生化和遗传分析旨在精确的分子 了解蛋白质靶向和易位过程。 我们希望 了解该过程的哪些方面是酵母独有的以及哪些方面 可以推广到其他真核细胞和细菌。 最终，我们 希望通过结合遗传和生化方法的力量 我们将确定蛋白质易位的细胞成分 对于易位至关重要的机械以及 调节。