Heterotrimeric G Protein-Mediated Cellular Polarization in Yeast

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Cellular polarization is essential to morphogenesis, immune response, neuronal development, chemotropism, and motility. The budding yeast, Saccharomyces cerevisiae, is a well studied model eukaryote that exhibits numerous polarization phenomena, including signal-induced changes in cell shape and nuclear position. In the life cycle of S. cerevisiae, two haploid cells of opposite mating type, MATa and MATa, can conjugate to form a MATa/a diploid cell. Each mating type constitutively secretes a peptide mating pheromone that transforms vegetatively growing cells of opposite type into gametes. To prepare haploids for cellular and nuclear fusion, pheromone triggers the induction of mating specific genes, arrest in the G1 phase of the cell cycle, polarized growth toward the mating partner, and nuclear migration to the tip of the mating projection. The molecular mechanisms underlying transmission of the mating signal are well understood. Communication of the signal from the plasma membrane to the cytoplasm is mediated by a receptor-coupled heterotrimeric G protein. When occupied by ligand, the pheromone receptors activate the pheromone-responsive Ga protein (encoded by GPA1) via guanine nucleotide exchange and the concomitant dissociation of Ga?GTP from the Gbg dimer (encoded by STE4 and STE18). The signal is then transmitted by Gbg to a mitogen?activated protein (MAP) kinase cascade. The MAP kinase module consists of Ste11 (the MEKK), Ste7 (the MEK), and Fus3 (the MAPK). In addition to stimulating the mating signal, Gbg serves as a positional cue. It directs the dual function protein, Far1, to the growth site. Far1 serves as a scaffold. It brings together elements that stimulate polarization of the actin cytoskeleton. Under physiological conditions, the Gbg-Far1 complex is presumed to assemble in the region of the cell surface that experiences the highest concentration of pheromone, and to mark this area for growth. Thus, the cell orients its growth toward the source of pheromone. This is called chemotropism. Recent results implicate the pheromone-responsive Ga protein, Gpa1, in control of signal-induced polarization and nuclear movement. In cells responding to pheromone, Gpa1 interacts directly with the mating-specific MAP kinase, Fus3, and with Kar3, a kinesin-like protein that is essential for nuclear movement during mating. Disruption of the Gpa1-Fus3 interaction confers defects in chemotropism and nuclear migration. The goals of this investigation are to determine which chemotropic factors depend on the recruitment of Fus3 by Gpa1, and to determine how Gpa1 affects nuclear movement during mating. In the work funded by NSF, the following hypothesis will be tested: Gpa1 co-localizes Fus3 and Kar3 so that Fus3 can activate Kar3. Because there is no precedent for regulation of kinesins by Ga proteins or MAP kinases, and because it is not known how kinesins are activated, this work promises to be of great value to those studying microtubule-associated motor proteins. The broader impact of this investigation is that it will enhance our understanding of chemotropism and nuclear movement, two fundamental aspects of cellular function. Students at all levels will be involved in this research.