ROLE OF YPT5 INTERACTING COMPONENTS IN ENDOCYTOSIS

我们已经使用了一种合成的致命筛选来识别新的基因 产物与ytp51相互作用或作用于相同的途径。Ypt51P是一款小型 GTP结合蛋白(Rab 5的三种酵母同源物之一)，介导 早期和晚期内体之间的膜运输。我们的屏幕结果是 分离到三个新的内吞突变体，命名为YSL(Ypt5p 合成致死)，两个先前发现的内吞突变株rvs167 和Sac6，以及一个已知的空泡蛋白分类突变体(Vps41)。RVS167 编码脊椎动物两栖动物的酵母同系物，而SAC6编码 纤毛蛋白和肌动蛋白结合蛋白。我们项目的目标是 确定YSL蛋白的特性并确定它们与 Ypt5p。我们将使用各种分析来映射 YSL蛋白。此外，我们还将YSL基因产品本地化，并 Ypt5p和YSL蛋白之间的物理相互作用的探针。一个 遗传和生化方法的结合也将用于 确定可能与YSL蛋白发生物理作用的蛋白质。至 为了帮助我们分析这些蛋白质，我们计划开发一种体外 重新构建从早期到晚期的运输分析 内生体。W还发现了一种新的可能存在于细胞内的t型陷阱。 路径。利用遗传和生化方法，我们计划将 这一新发现的陷阱并确定了可能 与之互动。这些研究应该会加深我们对 酿酒酵母的内吞作用。

We have used a synthetic lethal screen to identify new genes whose products interact with ytp51 or act on the same pathway. Ypt51p is a small GTP-binding protein (one of three yeast homologue of Rab 5) that mediates membrane traffic between early and late endosomes. Our screen has resulted in the isolation of three new endocytosis mutants, called ysl (Ypt5p synthetic lethal), two previously identified endocytosis mutants, rvs167 and sac6, and a known vacuolar protein sorting mutant (vps41). Rvs167 encodes the yeast homologue of vertebrate amphiphysin, while SAC6 encodes fimbrin, and actin binding protein. The goal of our project is to characterize the Ysl proteins and to determine their relationship to Ypt5p. We will use a variety of assays to map the execution point of the Ysl proteins. In addition, we will localize the YSL gene products and probe for physical interactions between Ypt5p and the Ysl proteins. A combination of genetic and biochemical approaches will also be used to identify proteins that may physically interact with the Ysl protein. To aid in our analysis of these proteins, we plan to develop an in vitro transport assay that reconstitutes transport from the early to the late endosome. W have also identified a new putative t-SNARE on the endocytic pathway. Using genetic and biochemical approaches we plan to characterize this newly identified SNARE and identify other components that may interact with it. These studies should further our understanding of endocytosis in the yeast Saccharomyces cerevisae.