YPTL GTPASE AND VESICULAR TRANSPORT

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DESCRIPTION: Elucidation of the mechanism by which proteins are sorted into the correct compartments is fundamental to our understanding of cell growth. Protein transport between intracellular compartments is mediated by membranous vesicles, and the rab/Ypt family of GTPases plays a major role in regulating the targeting of these vesicles in all eukaryotic cells. The long term goal of the project is to understand how vesicular transport operates and the specific role(s) that rab/Ypt GTPases have in its regulation. Mechanisms that underlie vesicle fusion are highly conserved between yeast and man. This proposal specifically addreses the function of Ypt GTPases in secretion, using Ypt1 of baker's yeast as a model. The proposed research will focus on proteins that interact with Ypt GTPases and the mechanisms by which they function as effectors or regulators. Existing as well as newly identified genes, will be examined for their role in protein transport analyzing potential secretory defects in mutants, and studying intracellular localization of their products. Overexpression studies will be used to distinguish putative regulators that modulate nucleotide cycling of Ypt1, from those that funciton as Ypt1 effectors, which typically do not affect this cycling. These genes will be used to address the following questions: 1) What are the roles of effector proteins of Ypt GTPases? Rab proteins, like other ras-related GTPases, are believed to function through the regulation of downstream effectors in a GTP-dependent manner, to transmit a signal from the GTP-bound form to the membrane docking and/or fusion apparatus. To determine the exact mechanism by which Ypt1 effectors function, the behavior of mutants in these genes will be examined with respect to visicle budding, targeting, uncoating or fusion, and the nature of their interaction with Ypt1. 2) What is the role of accessory factors that regulate nucleotide cycling of Ypt GTPases? Cycling between the GTP- and GDP-bound forms, facilitated by accessory factors, is considered crucial for rab function. However, little is known regarding how these factors find their rab targets of how they facilitate nucleotide exchange and hydrolysis. Putative regulators will be tested for these activities. To resolve issues concerning their spoecificity and mechanism of action, their interactions with Ypt and other proteins that funciton in secretion will be examined in vivo and in vitro. Finally, interaction among exocytic Ypt proteins themselves will be explored.