Studies of the Saccharomyces cerevisiae [alpha]-factor mating pheromone and its recepter (the Ste2 gene product)

The binding of α-factor to the α-factor receptor is the first step in a cascade of cellular events that initiates the mating response. In order to understand the molecular interactions involved in hormone-receptor binding, it is necessary to know the conformation of the messenger peptide in the active site of its receptor and the identity of the binding domain within its receptor. Structure-function studies have been performed on α-factor. Previous studies indicated that a Type II β-turn spanning residues 7 through 10 of the pheromone is important for the biological activity. Based on those results, a series of synthetic analogs were prepared in the laboratory of Dr. Fred Naider (College of Staten Island, Staten Island, New York). The first set of analogs synthesized to study the β-bend region were the cyclic lactam analogs in which the ring size (residues 7-10) was systematically varied. Subsequently, cyclic analogs were synthesized in which residues 7 and 10 were replaced with cysteines to form a disulfide bond which restricted the conformational mobility of the peptide. These analogs were assayed for biological activity and for the ability to bind to the receptor. Although none of the analogs were as active as native α-factor, results suggest a Type II β-turn conformation spanning residues 7-10 with proper orientation of the amine and carboxyl termini around the turn region is required for optimal interaction of α-factor with its receptor and is necessary for biological activity (See Part II and III of dissertation). Determination of the α-factor binding domain within its receptor (Ste2p) was attempted using photoaffinity labeling. α-factor analogs containing p-benzoyl-phenylalanine(BPA) were synthesized, tested for their biological activity and for the ability to bind to Ste2p (Part IV of dissertation). Photoaffinity labeling experiments were conducted using both membrane proteins and partially purified Ste2p. The results of the photoaffinity labeling experiments indicated a 52 kDa protein which was verified to be Ste2p using antibodies (Part V of dissertation). These observations suggest that a photoactivatable α-factor analog is able to covalently label the α-factor receptor and may be suitable for isolating and characterizing the receptor binding site(s).