Organelle biogenesis is an essential process for all eukaryotic cells. Compartmentalization in cells occurs by the precise segregation of proteins and membranes during and shortly after the biosynthesis of these macromolecules. The lysosome/vacuole of eukaryotic cells arises from proteins and membranes that are synthesized in the endoplasmic reticulum, transported to the Golgi apparatus, segregated at the Golgi sorting station, and transported to their final destination. The overall aim of this proposal is to identify the components involved with yeast vacuolar assembly and elucidate their role in this complex process with the use of a cell-free transport assay. Using genetic, immunological, and biochemical approaches we will identify and characterize the receptor responsible for the sorting of soluble hydrolases to the yeast vacuole. Once identified, we will done the gene that encodes the receptor, sequence the gene, and prepare gene disruptions to assess the functional role of the receptor. We also plan to utilize our newly developed in vitro Golgi to vacuole transport assay to determine which of the VPS genes encode proteins involved with this membrane transport step. VPS genes found to encode factors required for transport will be cloned, sequenced, and gene disruptions constructed. Antibodies to VPS proteins will be used to assess their intracellular localization and to probe their role in membrane transport in the cell-free assay. Vpslp has been identified as a Golgi-localized GTP-binding protein involved in yeast vacuolar assembly. We will conduct a detailed mutational analysis of VPSI, and investigate the functional significance of its homology to the mammalian force-generating protein, dynamin. Vpslp will be purified and its GTPase and other biochemical activities assessed. Proteins that interact with Vpslp will be identified and characterized. These investigations are designed to elucidate the role played by Vpslp in yeast vacuolar assembly.
A fourth aim i s directed at identifying small GTP-binding proteins involved in yeast vacuolar biogenesis. We have identified a small GTP-binding protein present in purified yeast vacuolar membranes. Based on the identification of a large class of these proteins that are involved in membrane transport events, we plan to determine the functional role of this vacuolar protein by genetic and biochemical analyses. The vacuolar GTP-binding. protein will be purified, protein sequence obtained, and the gene cloned by standard reverse genetic approaches. Mutations will be constructed and their effects assessed in yeast cells by standard yeast genetic methodology. Antibodies to this protein will be used to test for its function in our cell-free Golgi to vacuole transport assay.
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