SORTING AND TRANSPORT OF YEAST MEMBRANE PROTEINS

这项研究的总体目标是开发一种机制， 了解TGN和液泡膜蛋白分选， 在酵母Saccharomyces creatinae中组装。 酵母已被证明 是一个很好的模型系统， 调节真核细胞中的膜运输，并用于研究 这些蛋白质发挥作用的分子机制。 遗传 分析表明，酵母trans-Golgi网络（TGN） 膜蛋白被保留的检索，并产生了一个大的 参与酵母保留和回收的GRD基因的集合 TGN膜蛋白。 使用删除和定点 诱变，酵母TGN膜上的静态保留信号 蛋白二肽氨基肽酶A（DPAPA）将被鉴定， 表征了 大量的Grd蛋白质已经被 最近发现的将用于研究分子基础， TGN中的静态保留和TGN膜蛋白的回收 从前液泡区室中分离出来。 将开发试验， 确定在静态保留中单个Grd蛋白的功能 或检索步骤。 遗传分析也揭示了大量的酵母基因 不仅编码酵母液泡质子转运的亚基 ATP酶（V-ATP酶），但也有三个基因编码的蛋白质， 不是酶的亚基，而是组装酶所需的亚基。 V-ATPase。 使用遗传和生物化学方法，V- 将表征ATP酶组装因子以确定是否 它们在内质网中形成稳定的复合物， 依赖于底物的持续合成（v-ATP酶 亚基）进行组装反应。 三人各自的角色 V-ATP酶的组装因子将通过交联、共-交联和共-交联来研究。 免疫沉淀和密度梯度离心方法， 确定它们参与V-ATP酶组装的机制。 到 发展一个详细的分子理解的组装途径， V-ATP酶复合物，动力学和之间的关联程度 组装因子和V-ATP酶整合膜亚基 将在组装反应的早期进行监测。 附加功能 对于组装因素将进行调查，如护送V- ATP酶进入ER衍生的囊泡，并陪伴复合物进入 顺式高尔基体

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.