BIOGENESIS OF THE SACCHAROMYCES-CEREVISIAE MATING PHEROMONE A-FACTOR

The Saccharomyces cerevisiae mating pheromone a-factor is a prenylated and carboxyl methylated extracellular peptide signaling molecule. Bio genesis of the a-factor precursor proceeds via a distinctive multistep pathway that involves COOH-terminal modification, NH2-terminal proteo lysis, and a nonclassical export mechanism. In this study, we examine the formation and fate of a-factor biosynthetic intermediates to more precisely define the events that occur during a-factor biogenesis. We have identified four distinct a-factor biosynthetic intermediates (P0, P1, P2, and M) by metabolic labeling, immunoprecipitation, and SDS-PA GE. We determined the biochemical composition of each by defining thei r NH2-terminal amino acid and COOH-terminal modification status. Unexp ectedly, we discovered that not one, but two NH2-terminal cleavage ste ps occur during the biogenesis of a-factor. In addition, we have shown that COOH-terminal prenylation is required for the NH2-terminal proce ssing of a-factor and that all the prenylated a-factor intermediates ( P1, P2, and M) are membrane bound, suggesting that many steps of a-fac tor biogenesis occur in association with membranes. We also observed t hat although the biogenesis of a-factor is a rapid process, it is inhe rently inefficient, perhaps reflecting the potential for regulation. P revious studies have identified gene products that participate in the COOH-terminal modification (Ram1p, Ram2p, Ste14p), NH2-terminal proces sing (Ste24p, Axl1p), and export (Ste6p) of a-factor. The intermediate s defined in the present study are discussed in the context of these b iogenesis components to formulate an overall model for the pathway of a-factor biogenesis.