BIOCHEMICAL ANALYSIS OF THE YEAST A-FACTOR RECEPTOR

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Interaction between peptide pheromones and cell surface receptors controls cell division and other physiological events during mating in the yeast Saccharomyces cerevisiae. Haploid cells of the a mating type secrete a peptide pheromone, a factor, to which cells of the yield mating type respond. The response includes changes in transcription of a set of genes and arrest of the cell division cycle in G1. It is believed that a factor binds to a specific receptor on the surface of yield cells and thereby creates an internal signal(s) that triggers the physiological response. Our long-term goal is to understand how binding of pheromone to receptor generates an internal signal and what the specific intracellular targets of that signal are. Thus, we expect to understand the pathway whereby binding of ligand at the cell surface can control complex physiological processes such as cell division. This is a fundamental biological problem. Peptide hormones that control cell division are ubiquitous in eukaryotic organisms. Yeast offer the unique opportunity to combine biochemical and genetic approaches to this problem. We have identified the gene, STE3, that very likely encodes the a-factor receptor. In the proposed work we will determine by direct binding assays whether this gene indeed encodes the receptor. Using a combination of gene fusion methods and antibody probes to different portions of the STE3 product, we will determine the topology of the receptor in the membrane -- what parts of the receptor are extracellular and whether it traverses the membrane seven times as the primary amino acid sequence suggests. We will purify the receptor to determine its subunit composition and to permit detailed analysis of pheromone binding. For instance, using physical techniques, we will determine whether the structure of the receptor changes when pheromone is bound. We will also determine whether the location of the receptor changes when ligand is bound. In particular, do the receptor molecules cluster at one position on the cell surface, and are the receptor-ligand complexes internalized? Finally, we will investigate the nature of the intracellular chemical signal generated by binding of pheromone to receptor.