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 fundamental and 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 are conserved in all eukaryotic organisms; their study will provide important clues to human cell biology, fertility and disease. In yeast and higher organisms, secretory vesicles localize to incipient sites of cell fusion, as ceils polarize toward each other. We hypothesize that cell fusion ensues after the localized and regulated exocytosis of a subset of secretory vesicles. We will use genetic, cell biological and biochemical methods to elucidate the roles of two key cell fusion proteins, Fus3p, a conserved MAP-kinase, and Fus2p, a putative Rho-GEF. We will test the hypothesis that Fus3p's phosphorylation of a conserved formin, Bni1p, is critical for polarization. We will test the hypothesis that transport of Fus2p and an amphiphysin, Rvs161p, to the cortex, and the subsequent activation of a Rho-protein, are key regulatory events for polarized exocytosis and cell fusion. Nuclear migration is a fundamental microtubule-dependent process in fertilization and mitosis. Kar4p is a transcription factor that promotes nuclear migration during mating by inducing the expression of Kar3p, a kinesin motor protein. Microarray experiments suggest that Kar4p plays a broader role in the pheromone response, including negatively regulating mitotically expressed genes. Understanding the role of Kar4p will provide insight into how cells modify cell processes in response to developmental cues. Identification of Kar4p regulated genes will identify new candidate cell and nuclear fusion proteins. Homotypic nuclear membrane fusion is a paradigm for ER remodeling and organelle fusion. Kar5p and Prm3p are nuclear envelope membrane proteins that are required for nuclear fusion, induced during mating, and localized to the spindle pole body (SPB), where fusion occurs. We propose to study their roles in recruiting other nuclear fusion proteins to the SPB and characterize the protein complex required for membrane fusion. We will use multiple microscopic methods to characterize the events and topology of nuclear envelope fusion.
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