Pheromone response in the yeast Saccharomyces cerevisiae provides a microbial model for studying general features of hormone action and membrane protein traffic. When a-factor pheromone binds to specific receptors on the surface of yeast a cells, it causes the cells to arrest division in the Gi phase of the cycle. The occupied receptors are continually internalized and resynthesized. After prolonged exposure to a-factor, the cells adapt to the pheromone and reenter the mitotic cycle. The receptor belongs to the same structural class as rhodopsin and the B-adrenergic receptor and signal transduction is mediated by a heterotrimeric GTP-binding regulatory protein (G protein). The basic understanding obtained from detailed study of the yeast system should provide important insight into the design of drugs that target this general class of receptor and should provide insight into diseases associated with membrane protein traffic. A combined genetic and biochemical approach will be applied to this problem. In the next budget period, we will focus on intracellular traffic of the receptor and the G protein. We will test the relationship of receptor oligomerization with endocytosis and signa transduction by isolating receptor mutants that are defective in oligomerization and testing for defects in recepto traffic and signaling. The positions of the amino acids altered in these mutants will suggest the domains of the receptor that mediate this process. The events that control the exit of receptors from the endosome will be ordered by examining the properties of double mutants that affect this process. The cellular processes that control the movement of G proteins during a-factor response will be tested by using pulse-chase analysis in mutant cells with specific protein trafficking defects. We will develop an assay that will allow us to test for traffic of endocytosed proteins from the endosome to the cell surface. Such an assay will allow us to initiate a genetic analysis of the receptor recycling pathway.
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