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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.
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