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© The Rockefeller University Press, 0021-9525/1997//251 $5.00
The Journal of Cell Biology, Volume 136, Number 2, , 1997 251-269

Article

Biogenesis of the Saccharomyces cerevisiae Mating Pheromone a-Factor



Peng Chen, Stephanie K. Sapperstein, Jonathan D. Choi, and Susan Michaelis

Department of Cell Biology and Anatomy, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

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 SDSPAGE. 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.

Abbreviations used in this paper: ABC, ATP-binding cassette; E, extracellular; I, intracellular; M, mature species of a-factor; M(E), extracellular mature a-factor; M(I), intracellular mature a-factor; P0, unmodified a-factor precursor; P1, fully COOH-terminally modified precursor species of a factor; P2, fully COOH-terminally modified, partially NH2-terminally cleaved precursor species of a-factor; PTH, phenylthiohydrantoin; SAM, S-adenosyl-l-methionine; 5-FOA, 5-fluoroorotic acid.

Please address all correspondence to S. Michaelis, Department of Cell Biology and Anatomy, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205. Tel.: (410) 955-8286. Fax: (410) 955-4129. E-mail: susan_michaelis{at}quail.bs.jhu.edu


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