GOLGI PROTEIN LOCALIZATION IN SACCHAROMYCES CEREVISIASE

居民的定位需要外壳蛋白网状蛋白 蛋白质到高尔基体，用于正确地将蛋白质分选到溶酶体，以及 受体介导的内吞作用。目前，只有少数几种蛋白质 高尔基体蛋白质定位所需的基因已经被确定，并且 这些蛋白质与网状蛋白作用定位驻留的机制 高尔基体蛋白的研究还不完全清楚。因此，这项提议 重点是分离和表征额外的成分 酵母菌体内的高尔基体定位机制 酿酒。将在基因中鉴定与笼状蛋白相互作用的蛋白质 筛选导致细胞合成生长缺陷的突变 表达编码的功能受损的胞质蛋白重链 Chc1-ts等位基因。新的突变体(标记为sct，用于合成致死的chcl. 来自初步筛查的TS)已使用三种生物学方法分类 鉴定那些特定影响高尔基蛋白的突变的方法 本地化。这类SCT中的蛋白质定位缺陷 突变体的特征是通过分析两个基因的错误定位 分别来自跨高尔基体和内侧高尔基体的驻留蛋白，以及 一种来自内质网。两个具有强烈影响的突变体 高尔基蛋白的定位将得到更广泛的分析。首先， 将对每个基因的野生型拷贝进行克隆、测序和检查 与已知蛋白质同源。第二，免疫荧光和密度 将使用梯度离心法和分馏法来定位每个 蛋白质进入亚细胞隔间。最后，原生的 免疫沉淀和交联实验将被用来检测 Sct蛋白与所需的其他蛋白之间的直接相互作用 用于高尔基体蛋白质定位。

The coat protein clathrin is required for localization of resident proteins to the Golgi, for proper sorting of proteins to the lysosome, and for receptor-mediated endocytosis. At present, only a few proteins required for Golgi protein localization have been identified, and the mechanism by which these proteins act with clathrin to localize resident Golgi proteins is incompletely understood. Therefore, this proposal focuses on the isolation and characterization of additional components of the Golgi localization machinery in the organism, Saccharomyces cerevisiae. Clathrin-interacting proteins will be identified in a genetic screen for mutations that cause a synthetic growth defect in cells expressing a functionally-compromised clathrin heavy chain encoded by the chcl-ts allele. New mutants (denoted sct for synthetic lethal with chcl- ts) from a preliminary screen have been classified using three biological assays to identify those mutations that specifically affect Golgi protein localization. The protein localization defect in this class of sct mutants will be characterized by assaying for the mislocalization of two resident proteins each from the trans and medial Golgi compartments, and one from the endoplasmic reticulum. Two mutants with strong effects on Golgi protein localization will be analyzed more extensively. First, the wild type copy of each gene will be cloned, sequenced, and examined for homology to known proteins. Second, immunofluorescence and density gradient centrifugation and fractionation, will be used to localize each protein to a subcellular compartment. Finally, native immunoprecipitations and cross-linking experiments will be used to detect direct interactions between the sct proteins and other proteins required for Golgi protein localization.