title: Reconstitution of the Ste24p-dependent N-terminal proteolytic step in yeast a-factor biogenesis.
abstract: The yeast mating pheromone a-factor precursor contains an N-terminal extension and a C-terminal CAAX motif within which multiple posttranslational processing events occur. A recently discovered component in a-factor processing is Ste24p/Afc1p, a multispanning endoplasmic reticulum membrane protein that contains an HEXXH metalloprotease motif. Our in vivo genetic characterization of this protein has demonstrated roles for Ste24p in both the N-terminal and C-terminal proteolytic processing of the a-factor precursor. Here, we present evidence that the N-terminal proteolysis of the a-factor precursor P1 can be accurately reconstituted in vitro using yeast membranes. We show that this activity is dependent on Ste24p and is abolished by mutation of the Ste24p HEXXH metalloprotease motif or by mutation of the a-factor P1 substrate at a residue adjacent to the N-terminal P1 cleavage site. We also demonstrate that N-terminal proteolysis of the P1 a-factor precursor requires Zn(2+) as a co-factor and can be inhibited by the addition of the metalloprotease inhibitor 1,10-orthophenanthroline. Our results are consistent with Ste24p itself being the P1-->P2 a-factor protease or a limiting activator of this activity. Interestingly, we also show that the human Ste24 homolog expressed in yeast can efficiently promote the N-terminal processing of a-factor in vivo and in vitro, thus establishing a-factor as a surrogate substrate in the absence of known human substrates. The results reported here, together with the previously reported in vitro reconstitution of Ste24p-dependent CAAX processing, provide a system for examining the potential bifunctional roles of yeast Ste24p and its homologs.
authors.affiliation: Department of Cell Biology and Anatomy, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
authors.forename: W K
authors.initials: WK
authors.lastname: Schmidt
authors.forename: A
authors.initials: A
authors.lastname: Tam
authors.forename: S
authors.initials: S
authors.lastname: Michaelis
mesh_terms: D015107:4-Butyrolactone / Q000031:analogs & derivatives* / Q000378:metabolism; D000595:Amino Acid Sequence; D005656:Fungal Proteins / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity; D015032:Zinc / Q000494:pharmacology
chemical_list: D005656:Fungal Proteins; D008565:Membrane Proteins; D011498:Protein Precursors; D029701:Saccharomyces cerevisiae Proteins; C025035:A-factor (Streptomyces); D008666:Metalloendopeptidases; C491251:STE24 protein, S cerevisiae; D015032:Zinc; D015107:4-Butyrolactone