title: Saccharomyces cerevisiae a-factor mutants reveal residues critical for processing, activity, and export.
abstract: The Saccharomyces cerevisiae mating pheromone a-factor provides a paradigm for understanding the biogenesis of prenylated fungal pheromones. The biogenesis of a-factor involves multiple steps: (i) C-terminal CAAX modification (where C is cysteine, A is aliphatic, and X is any residue) which includes prenylation, proteolysis, and carboxymethylation (by Ram1p/Ram2p, Ste24p or Rce1p, and Ste14p, respectively); (ii) N-terminal processing, involving two sequential proteolytic cleavages (by Ste24p and Axl1p); and (iii) nonclassical export (by Ste6p). Once exported, mature a-factor interacts with the Ste3p receptor on MATalpha cells to stimulate mating. The a-factor biogenesis machinery is well defined, as is the CAAX motif that directs C-terminal modification; however, very little is known about the sequence determinants within a-factor required for N-terminal processing, activity, and export. Here we generated a large collection of a-factor mutants and identified residues critical for the N-terminal processing steps mediated by Ste24p and Axl1p. We also identified mutants that fail to support mating but do not affect biogenesis or export, suggesting a defective interaction with the Ste3p receptor. Mutants significantly impaired in export were also found, providing evidence that the Ste6p transporter recognizes sequence determinants as well as CAAX modifications. We also performed a phenotypic analysis of the entire set of isogenic a-factor biogenesis machinery mutants, which revealed information about the dependency of biogenesis steps upon one another, and demonstrated that export by Ste6p requires the completion of all processing events. Overall, this comprehensive analysis will provide a useful framework for the study of other fungal pheromones, as well as prenylated metazoan proteins involved in development and aging.
authors.affiliation: Department of Cell Biology, The Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205, USA.
authors.forename: Gregory
authors.initials: G
authors.lastname: Huyer
authors.forename: Amy
authors.initials: A
authors.lastname: Kistler
authors.forename: Franklin J
authors.initials: FJ
authors.lastname: Nouvet
authors.forename: Carolyn M
authors.initials: CM
authors.lastname: George
authors.forename: Meredith L
authors.initials: ML
authors.lastname: Boyle
authors.forename: Susan
authors.initials: S
authors.lastname: Michaelis
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D001692:Biological Transport; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism; D008074:Lipoproteins / Q000096:biosynthesis* / Q000502:physiology; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D010641:Phenotype; D010675:Pheromones; D043484:Proprotein Convertases; D011496:Protein Methyltransferases / Q000235:genetics / Q000378:metabolism; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D051964:Receptors, Mating Factor / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D014166:Transferases / Q000235:genetics / Q000378:metabolism
chemical_list: D018528:ATP-Binding Cassette Transporters; D006023:Glycoproteins; D008074:Lipoproteins; C118411:MFA1 protein, S cerevisiae; C095063:MFA2 protein, S cerevisiae; D008565:Membrane Proteins; D010675:Pheromones; D011498:Protein Precursors; D051964:Receptors, Mating Factor; C081863:STE3 protein, S cerevisiae; C489277:STE6 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014166:Transferases; D011496:Protein Methyltransferases; C498863:Ste14 protein, S cerevisiae; C070038:RAM1 protein, S cerevisiae; D010450:Endopeptidases; D043484:Proprotein Convertases; C105193:RCE1 protein, S cerevisiae; C090850:AXL1 protein, S cerevisiae; D008666:Metalloendopeptidases; C491251:STE24 protein, S cerevisiae