Polarized cell growth is important for the development of both unicellular and multicellular organisms. The process of polarized cell growth requires selection of a site and the targeted assembly of cytoskeletal and secretory components at that site. To help elucidate the molecular mechanisms and steps by which these processes occur, we propose to analyze the function, targetting and assembly of several proteins important for asymmetric growth and division in Saccharomyces cerevisiae. The Spa2 protein (Spa2p) localizes to cortical sites of polarized growth and functions in both bud site selection and mating projection formation. Spa2p has a potential coiled-coil domain, and may be a cytoskeletal protein. Extraction and biochemical fractionation experiments will be performed to determine if Spa2p exhibits properties expected of a cytoskeletal protein, and if it is associated with membrane compartments. The ability of Spa2p to oligomerize and form higher order structures will be studied. To understand how proteins are transported to and assembled at sites of cell growth, we will determine the domains of Spa2p that target it to the cell periphery, attempt to identify proteins that interact with these sequences, and examine the roles of specific motor proteins (e.g. Myo2p, Smy1p) in the Spa2p targetting process. Finally, Spa2p accumulates at both the incipient bud site and at cytokinesis it redistributes to the neck; the mechanism of its localization to the bud site and its redistribution at cytokinesis, including the possible role of phosphorylation, will be examined. In addition to analyzing Spa2p, we plan to analyze in detail the molecular mechanisms of pheromone-induced morphogenesis in yeast. Yeast cells grow toward pheromone sources; we have found that the carboxy terminus of the Ste2p mating pheromone receptor is important for proper orientation toward the pheromone source. To understand how this domain functions, we will determine the sequences that are important for projection orientation and identify and characterize proteins which interact with these sequences. Such proteins may be signaling molecules, regulators of the actin cytoskeleton or other growth control components. We will also investigate the role of known cytoskeletal and polarity components in projection formation and orientation. New mutants defective in projection formation and/or projection orientation will be sought using genetic screens, and by generating and analyzing a collection of yeast strains containing mutations in pheromone-induced genes. Such mutants and their corresponding genes will be characterized extensively. Finally, to understand how projection formation may be regulated, the role of the Pcl/Pho85 complex in pheromone-induced morphogenesis will be investigated. We expect that these studies will enhance our understanding of asymmetric growth in yeast. Because many aspects of this process (particularly mating projection formation) resemble cell differentiation events in mammals, these studies should provide insights into general mechanisms of polarized growth in eucaryotes.
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