PROTEIN TRANSLOCATION IN YEAST

酵母中的蛋白质易位

• 批准号: 3292746
• 负责人: Peter Walter
• 金额: $ 13.79万
• 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 1991-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3292746
• 关键词: Saccharomyces; alleles; antibody formation; complementary DNA; endoplasmic reticulum; fungal genetics; gel filtration chromatography; gene expression; gene mutation; genetic manipulation; genetic transcription; laboratory mouse; laboratory rabbit; membrane permeability; molecular cloning; molecular genetics; nucleic acid probes; protein transport; structural genes

Our primary objective is understanding the mechanism by which certain classes of proteins are selectively targeted to and translocated across the endoplasmic reticulum membrane in the yeast Saccharomyces cerevisiae. Yeast components of the cellular machinery that catalyzes this process will be biochemically identified, characterized and purified. In subsequent genetic analyses the cloned genes will be perturbed and the effects studied in vivo and in vitro. Specifically: i) A yeast in vitro protein translocation assay will be used to purify soluble, as well as membrane-bound translocation factors. Further in vitro assays will be designed to elucidate their function. The genes encoding such factors will be cloned using molecular probes generated to the purified material. ii) Additionally, we will purify and clone the yeast analogs of those components that are already well defined in the mammalian protein translocation system, namely SRP and SRP receptor, which are expected to be present also in yeast. iii) Genes identified in i) or ii) will be disrupted and the phenotype of the resulting null alleles will be characterized. Once a secretion-impaired phenotype has been identified, it will be used to isolate conditional alleles. iv) Interacting components will be identified by isolation of extragenic suppressors of either ts or cs or of in vitro engineered mutations. v) The 'reverse genetic' approaches outlined above will be complemented by a biochemical characterization of mutants isolated by classical genetic means by screening for alterations in the yeast translocation machinery. Ultimately, the power of a combined biochemical and genetic approach should lead to a complete molecular description of the protein translocation machinery and its essential and modulatory constituents, and allow us to gain an understanding of the mechanism of protein movement across membranes. The proposed research is clearly of a most basic nature and there is no doubt that it will be of profound significance for an understanding of physiology and pathology at the cellular and molecular level.

我们的主要目标是了解某些机制 蛋白质类别被选择性地靶向并易位 酿酒酵母中的内质网膜。 催化此过程的细胞机械的酵母成分将 在生化上鉴定，表征和纯化。在后续 遗传分析的克隆基因将受到干扰，并研究了效果 体内和体外。 具体：i）体外蛋白质的酵母菌 易位测定将用于净化可溶性，以及 膜结合的易位因子。 进一步的体外测定将是 旨在阐明其功能。 编码这些因素的基因将 使用生成纯化材料的分子探针克隆。 ii） 此外，我们将纯化并克隆这些类似物的酵母类似物 哺乳动物蛋白质中已经定义得很好的成分 易位系统，即SRP和SRP受体，预计为 也存在于酵母中。 iii）在i）或ii）中确定的基因将是 被破坏，由此产生的零等位基因的表型将是 特征。 一旦确定了分泌受损的表型， 将用于隔离条件等位基因。 iv）相互作用的组件 通过隔离TS或 CS或体外工程突变。 v）“反向遗传” 上面概述的方法将通过生化补充 通过经典遗传手段隔离的突变体的表征 筛选酵母易位机械的更改。 最终，合并的生化和遗传方法的力量应 导致蛋白质易位的完整分子描述 机械及其基本和调节成分，使我们能够 了解跨越蛋白质运动的机制 膜。 拟议的研究显然具有最基本的性质， 毫无疑问，对于一个人来说，它将具有深远的意义 了解细胞和分子的生理学和病理学 等级。