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.
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