title: A novel a-factor-related peptide of Saccharomyces cerevisiae that exits the cell by a Ste6p-independent mechanism.
abstract: Many secreted signaling molecules are synthesized as precursors that undergo multiple maturation steps to generate their mature forms. The Saccharomyces cerevisiae mating pheromone a-factor is a C-terminally isoprenylated and carboxylmethylated dodecapeptide that is initially synthesized as a larger precursor containing 36 or 38 amino acids. We have previously shown that the maturation of a-factor occurs by an ordered biogenesis pathway involving 1) three C-terminal modification steps, 2) two N-terminal proteolytic processing events, and 3) a nonclassical export mechanism mediated by the ATP-binding-cassette (ABC) transporter Ste6p. In the present study, we demonstrate that an unexpected and abundant a-factor-related peptide (AFRP) exists in the culture fluid of MATa cells and that its biogenesis is integrally related to that of mature a-factor itself. We show by purification followed by mass spectrometry that AFRP corresponds to the C-terminal 7 amino acids (VFWDPAC) of mature a-factor (YIIKGVFWDPAC), including both the farnesyl- and carboxylmethylcysteine modifications. The formation and export of AFRP displays three striking features. First, we show that AFRP is produced intracellularly and that mutants (ste24 and axl1) that cannot produce mature a-factor due to an N-terminal processing defect are nevertheless normal for AFRP production. Thus, AFRP is not derived from mature a-factor but, instead, from the P1 form of the a-factor precursor. Second, fusion constructs with foreign amino acids substituted for authentic a-factor residues still yield AFRP-sized molecules; however, the composition of these corresponds to the altered residues instead of to AFRP residues. Thus, AFRP may be generated by a sequence-dependent but length-specific proteolytic activity. Third, a-factor and AFRP use distinct cellular machinery for their secretion. Whereas a-factor export is Ste6p-dependent, AFRP is secreted normally even in a ste6 deletion mutant. Thus, AFRP may exit the cell by another ATP-binding-cassette transporter, a different type of transporter altogether, or possibly by diffusion. Taken together, these studies indicate that the biogenesis of AFRP involves novel mechanisms and machinery, distinct from those used to generate mature a-factor. Because AFRP neither stimulates nor inhibits mating or a-factor halo activity, its function remains an intriguing question.
authors.affiliation: Department of Cell Biology and Anatomy, Johns Hopkins School of Medicine, Baltimore, Maryland 21205, USA.
authors.forename: P
authors.initials: P
authors.lastname: Chen
authors.forename: J D
authors.initials: JD
authors.lastname: Choi
authors.forename: R
authors.initials: R
authors.lastname: Wang
authors.forename: R J
authors.initials: RJ
authors.lastname: Cotter
authors.forename: S
authors.initials: S
authors.lastname: Michaelis
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000378:metabolism / Q000502:physiology*; D000595:Amino Acid Sequence; D001692:Biological Transport; D005656:Fungal Proteins / Q000378:metabolism / Q000502:physiology*; D006023:Glycoproteins; D008074:Lipoproteins / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000378:metabolism; D008666:Metalloendopeptidases / Q000378:metabolism; D008745:Methylation; D008969:Molecular Sequence Data; D010675:Pheromones; D017368:Protein Prenylation; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins
chemical_list: D018528:ATP-Binding Cassette Transporters; D005656:Fungal Proteins; D006023:Glycoproteins; D008074:Lipoproteins; C095063:MFA2 protein, S cerevisiae; D008565:Membrane Proteins; D010675:Pheromones; D011993:Recombinant Fusion Proteins; C489277:STE6 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C090850:AXL1 protein, S cerevisiae; D008666:Metalloendopeptidases; C491251:STE24 protein, S cerevisiae