Our long-term objectives are to define the pathway by which two haploid yeast cells fuse to become one diploid cell. Related to fertilization, conjugation is a fundamental process common to all sexually reproducing organisms. Conjugation also has close parallels to cell fusion events during development. We propose to continue the analysis of genes required for the three major steps in the pathway; cell fusion, nuclear migration and nuclear envelope fusion. Many of the genes required for cell and nuclear fusion have homologues in all eukaryotic organisms and their study should provide important clues to human cell biology, fertility and disease. In yeast and higher organisms, secretory vesicles accumulate at the site of cell fusion. We hypothesize that cell fusion is based upon the localized and regulated exocytosis of a subset of secretory vesicles. We will test this model by examining biochemically a putative cargo molecule in wild type and mutant cells. We will use both genetic and biochemical methods to elucidate how two key cell fusion proteins, Fus2p and the conserved Fus3p MAP kinase, facilitate and regulate cell fusion. Finally, we will examine the role of a newly identified GTPase. Nuclear migration is a fundamental microtubule-dependent process in fertilization, mitosis and early development. Kar9p, a novel cortical protein required for nuclear migration, acts to orient the microtubules in mating and mitosis. We will test the hypothesis that Kar9p captures and stabilizes cytoplasmic microtubules by examining microtubule dynamics in vivo. Using biochemical and genetic methods we will identify the proteins that Kar9p interacts with, determine whether Kar9p is sufficient to orient microtubules, and determine that basis for Kar9p s regulated localization in the cell cycle. Homotypic nuclear envelope fusion occurs at the SPB as the last step in conjugation. Kar5p, an integral nuclear envelope protein at the SPB, is the only known mating-induced fusion protein. To test the hypothesis that Kar5p recruits fusion proteins to the SPB, we will identify Kar5p-associated proteins using both biochemical and genetic approaches. We will map the domains of Kar5p, investigate the role of a subset of translocation proteins and determine whether two proteins closely related to Cdc48p are the mating specific NSF-related fusion proteins. Finally, we propose to complete a large screen for bilateral mating defective mutants screen to identify the complete set of genes required for cell and nuclear fusion.
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