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. Biogenesis of the a-factor precursor proceeds via a distinctive multistep pathway that involves COOH-terminal modification. NH2-terminal proteolysis, 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-PAGE. We determined the biochemical composition of each by defining their NH2-terminal amino acid and COOH-terminal modification status. Unexpectedly, we discovered that not one, but two NH2-terminal cleavage steps occur during the biogenesis of a-factor. In addition, we have shown that COOH-terminal prenylation is required for the NH2-terminal processing of a-factor and that all the prenylated a-factor intermediates (P1, P2, and M) are membrane bound, suggesting that many steps of a-factor biogenesis occur in association with membranes. We also observed that although the biogenesis of a-factor is a rapid process, it is inherently inefficient, perhaps reflecting the potential for regulation. Previous studies have identified gene products that participate in the COOH-terminal modification (Ram1p, Ram2p, Ste14p), NH2-terminal processing (Ste24p, Axl1p), and export (Ste6p) of a-factor. The intermediates defined in the present study are discussed in the context of these biogenesis components to formulate an overall model for the pathway of a-factor biogenesis.
Chen
P
P
Department of Cell Biology and Anatomy, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Sapperstein
S K
SK
Choi
J D
JD
Michaelis
S
S
D000595:Amino Acid Sequence; D001692:Biological Transport; D005656:Fungal Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008074:Lipoproteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D008745:Methylation; D008969:Molecular Sequence Data; D010675:Pheromones / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D011498:Protein Precursors / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D017368:Protein Prenylation; D011499:Protein Processing, Post-Translational; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013045:Species Specificity
D005656:Fungal Proteins; D008074:Lipoproteins; C095063:MFA2 protein, S cerevisiae; D010675:Pheromones; D011498:Protein Precursors; D029701:Saccharomyces cerevisiae Proteins