G Protein Mediated Downregulation of a MAP Kinase in Yeast

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Collecting information about the environment, integrating it and responding in accordance with changing conditions are fundamental abilities required of all cells. In eukaryotes, one of the most common strategies for detecting and transmitting information across the plasma membrane depends on heptihelical receptors coupled to heterotrimeric G proteins. Haploid cells of the budding yeast Saccharomyces cerevisiae use a receptor coupled G protein both to sense and adapt to mating pheromone secreted by cells of opposite mating type. The G By subunit of the G protein couples to a MAP kinase cascade, the activation of which blocksproliferation and induces the differentiation of vegetative cells into gametes. Yeast cells that are unable to mate eventually resume growth, even when continuously exposed to pheromone. Thus, the mating response pathway of yeast, like analogous signaling systems of higher eukaryotes, can adapt to a continuous signal. We have established that the pheromone responsive G a protein, Gpa1, stimulates adaptation to pheromone, and that most likely it does so by two independent mechanisms. First, it is thought that Gpa1 stimulates an unknown protein to bind and inactivate G aa. Second, there is evidence that Gpa1 inhibits the pheromone responsive MAP kinase, Fus3. The latter mechanism may serve both to inactivate Fus3, and to remove itfrom the nucleus. It is believed that the movement of MAP kinases into and out of the nucleus is a primary method of signal transduction regulation, but the ways in which nucleocytoplasmic transport is controlled are poorly understood. Recently, a model was posited in which Gpa1 stimulates long-term downregulation of the mating signal by inducing vacuolar localization of Fus3 and the phosphatase that regulates it, Msg5 is proposed. In this investigation, the two most important tenets of this model will be tested. Genetic and biochemical approaches will be used to determine whether Gpa1 stimulates the export of Fus3 from the nucleus during recovery from pheromone treatment, and whether Fus3 is translocated in complex with Msg5. A second goal of the project is to test the possibility that Gpa1 inhibits Fus3 kinase activity, and if so, to determine whether this effect is mediated directly, by allosteric interaction, or indirectly, via Msg5. In addition to testing specific hypotheses, a proven protocol will be used to isolate novel alleles of FUS3 that confer defects in Gpa1-mediated adaptation. The mutant forms of Fus3 will be characterized according to their kinase activity, localization, and ability to associate with Gpa1. The results of these experiments are expected to elucidate the mechanisms by which Gpa1 downregulates Fus3. Because adaptive mechanisms are a fundamental means of regulating signaling pathways, and because G proteins and MAP kinase cascades play important signaling roles in all eukaryotic cells, important precedents are likely to result from this work.