SORTING AND TRANSPORT OF YEAST MEMBRANE PROTEINS

这项研究的总体目标是开发一种机械 对TGN和液泡膜蛋白分选的认识 在酿酒酵母中组装。酵母菌已经证明 是一个很好的模型系统，既可以用来识别蛋白质 调控真核细胞的膜运输并研究 这些蛋白质发挥作用的分子机制。遗传 分析表明，酵母反式高尔基体网络(TGN) 膜蛋白是通过检索保留下来的，并产生了大量的 酵母保留和回收相关GRD基因的收集 TGN膜蛋白。使用删除和站点定向 酵母TGN膜上的静态滞留信号--诱变 蛋白质二肽基氨基肽酶A(DPAPA)将被鉴定并 特色化的。非常大的GRD蛋白质集合已经被 最新鉴定的将用于研究分子基础 TGN中的静态保留和TGN膜蛋白的提取 从泡前隔室出来的。化验方法将发展为 确定单个GRD蛋白在静态保留中的功能 或检索步骤。 遗传分析还揭示了大量酵母基因的集合 不仅编码酵母液泡质子转运的亚基 ATPase(V-ATPase)，还有三个编码蛋白质的基因，分别是 不是酶的亚基，而是组装 V-ATPase。使用遗传和生化方法，V- ATPase组装因子将被表征以决定是否 它们在内质网中形成一个稳定的复合体，这个复合体是否 取决于底物(v-ATPase)的持续合成 亚单位)用于组装反应。这三个人的个人角色 V-ATPase组装因子将通过交联法、共... 免疫沉淀和密度梯度离心法 确定它们在V-ATPase组装中的机械性参与。至 发展对组装途径的详细的分子理解 V-ATPase复合体及其相互作用的动力学和程度 组装因子与V-ATPase整合膜亚基 将在组装反应的早期进行监测。附加功能 对于装配因素将进行调查，如护送V- ATPase进入内质网衍生的小泡并伴随着该复合体到 顺位高尔基人。

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.