The selective recognition, sorting and transport of proteins from one intracellular organelle to another via vesicular carriers represents an essential feature of all eukaryotic cells. A convergency of may recent studies in both yeast and mammalian cells has demonstrated that phosphoinositides play critical roles in the regulation of membrane trafficking in addition to their classical roles as second messengers in signal transduction at the cell surface. We propose to continue our use of the powerful genetic and molecular approaches available in yeast to characterize the complex molecular interactions that direct vesicle- mediated protein sorting from the Golgi to the lysosome-like vacuole. We have isolated a large collection of yeast mutants (vps) that exhibit severe defects in vacuolar protein sorting. Among the gene products affected in these vps mutants, we have identified a protein kinase, Vps15p, and a phosphatidylinositol 3-kinase, Vps34p. Each of the kinase activities is essential for protein transport in the Golgi-to-vacuole delivery pathway. The Vps15 protein kinase appears to regulate protein sorting by the selective membrane recruitment and activation of Vps34p. In this grant, we propose to characterize the role that the Vps34 PtdIns- 3-kinase and its lipid product, PtdIns3P, play in lysosomal protein sorting. The specific objectives of the grant are to use genetic, molecular, morphological and biochemical approaches to: 1) Identify upstream regulators and downstream effects of the Vps15p/Vps34p kinase cascade, 2) Characterize the turnover pathway for PtdIns3P in yeast and 3) Analyze the role of Vps34p in regulating Golgi to endosome transport. These studies will be of general importance to human health because mislocalization of lysosomal hydrolases has been proposed to enhance tumor cell growth and increase the metastatic potential of certain cancers by contributing to the hydrolysis of extracellular matrix components of target tissues. In addition, PI 3-kinase signaling pathways have been shown to play an important role in the control of mitosis, cell growth, differentiation and apoptosis.
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