ROLE OF YPT5 INTERACTING COMPONENTS IN ENDOCYTOSIS

我们已经使用了一种合成的致死筛选来鉴定新的基因， 产物与Ytp 51相互作用或作用于相同的途径。YPT 51 P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。我们的筛选结果是 在三个新的内吞作用突变体的分离中，称为ysl（Ypt 5 p 合成致死），两个先前鉴定的内吞作用突变体，RVS 167 和Sac 6，以及已知的空泡蛋白分选突变体（VPS 41）。Rvs167 SAC 6编码脊椎动物两性霉素的酵母同源物，而SAC 6编码 fimmunoglobulin和肌动蛋白结合蛋白。我们项目的目标是 表征Ysl蛋白并确定它们与 Ypt5p。我们将使用各种分析来映射 Ysl蛋白。此外，我们将定位YSL基因产物， Ypt 5 p和Ysl蛋白之间物理相互作用的探针。一 遗传和生物化学方法的组合也将用于 鉴定可能与Ysl蛋白物理相互作用的蛋白质。到 为了帮助我们分析这些蛋白质，我们计划开发一种体外 重建从早期到晚期的转运的转运试验 核内体我们还确定了一个新的假定的t-SNARE的内吞 通路利用遗传和生物化学方法，我们计划表征 这个新发现的陷阱，并确定其他组件， 这些研究应该进一步加深我们对 酵母中的内吞作用。

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.