SORTING AND TRANSPORT OF YEAST MEMBRANE PROTEINS

酵母膜蛋白的分选和运输

• 批准号: 6179510
• 负责人: Tom Hall Stevens
• 金额: $ 19.37万
• 依托单位: UNIVERSITY OF OREGON
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-04-01 至 2001-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6179510
• 关键词: Golgi apparatus; Saccharomyces cerevisiae; adenosinetriphosphatase; aminopeptidase; endoplasmic reticulum; enzyme mechanism; fungal genetics; immunoprecipitation; laboratory mouse; laboratory rabbit; membrane proteins; molecular chaperones; molecular cloning; protein biosynthesis; protein transport; site directed mutagenesis; vesicle /vacuole

The overall goal of this research is to develop a mechanistic understanding of TGN and vacuole membrane protein sorting and assembly in the yeast Saccharomyces crevisiae. Yeast has proved to be an excellent model system, both for identifying the proteins regulating membrane traffic in eukaryotic cells and for investigating the molecular mechanisms by which these proteins function. Genetic analyses have revealed that a yeast trans-Golgi network (TGN) membrane protein is retained by retrieval, and yielded a large collection of GRD genes involved in the retention and retrieval of yeast TGN membrane proteins. Using deletion and site-directed mutagenesis, the static retention signal on the yeast TGN membrane protein dipeptidyl aminopeptidase A (DPAPA) will be identified and characterized. The very large collection of Grd proteins that has been recently identified will be used to investigate the molecular basis for static retention in the TGN and retrieval of TGN membrane proteins from the prevacuolar compartment. Assays will be developed to determine functions for the individual Grd proteins in static retention or the retrieval step. Genetic analysis has also revealed a large collection of yeast genes encoding not only subunits of the yeast vacuolar proton-translocating ATPase (V-ATPase), but also three genes encoding proteins that are not subunits of the enzyme but instead are required for assembly of the V-ATPase. Using both genetic and biochemical approaches, the V- ATPase assembly factors will be characterized to determine whether they form a stable complex in the ER and whether this complex depends on the continued synthesis of substrates (the v-ATPase subunits) for the assembly reaction. The individual roles for the three V-ATPase assembly factors will be investigated by cross-linking, co- immunoprecipitation and density gradient centrifugation approaches to determine their mechanistic involvement in V-ATPase assembly. To develop a detailed molecular understanding of the assembly pathway of the V-ATPase complex, the kinetics and degree of association between the assembly factors and the V-ATPase integral membrane subunits will be monitored early in the assembly reaction. Additional functions for the assembly factors will be investigated such as escorting the V- ATPase into ER derived vesicles and chaperoning the complex to the cis-Golgi.

这项研究的总体目标是开发机械 了解TGN和液泡膜蛋白分类和 在酵母糖果实中的组装。 酵母已经证明 成为一个出色的模型系统，既可以识别蛋白质 调节真核细胞中的膜流量并研究 这些蛋白质功能的分子机制。 遗传 分析表明，酵母跨性高尔基网络（TGN） 膜蛋白通过检索保留，并产生较大的 涉及酵母保留和检索的GRD基因的收集 TGN膜蛋白。 使用删除和站点定向 诱变，酵母TGN膜上的静态保留信号 将鉴定蛋白质二肽基氨基肽酶A（DPAPA），并 特征。 大量的GRD蛋白质已经 最近确定的将用于研究分子基础 TGN膜蛋白的TGN和检索中的静态保留率 从prevacuall室。 测定将开发 确定静态保留中各个GRD蛋白的功能 或检索步骤。 遗传分析还揭示了大量的酵母基因 不仅编码酵母液泡质子转换的亚基 ATPase（V-ATPase），也是编码蛋白质的三个基因 不是酶的亚基，而是需要 V-ATPase。 使用遗传和生化方法，V- 将对ATPase组装因子进行表征，以确定是否 它们在ER中形成一个稳定的复合物，以及该复合物是否 取决于底物的持续合成（V-ATPase 亚基）用于组装反应。 三个角色的个人角色 V-ATPase组装因子将通过交联，共同进行研究 免疫沉淀和密度梯度离心方法 确定它们在V-ATPase组件中的机械介入。 到 对组装途径的详细分子理解 V-ATPase复合物，动力学和关联程度 装配因子和V-ATPase积分膜亚基 将在组装反应的早期进行监测。 其他功能 对于组装因素，将研究诸如护送V- ATPase进入ER衍生的囊泡，并将复合物伴 顺式高尔基。