Reconstitution of the Ste24p-dependent N-terminal proteolytic step in yeast a-factor biogenesis

The yeast mating pheromone a-factor precursor contains an N-terminal extens ion and a C-terminal CAAX motif within which multiple posttranslational pro cessing events occur. A recently discovered component in a-factor processin g is Ste24p/Afc1p, a multispanning endoplasmic reticulum membrane protein t hat contains an HEXXH metalloprotease motif, Our in vivo genetic characteri zation of this protein has demonstrated roles for Ste24p in both the N-term inal and C-terminal proteolytic processing of the a-factor precursor. Here, we present evidence that the N-terminal proteolysis of the a-factor precur sor P1 can be accurately reconstituted in vitro using yeast membranes. We s how that this activity is dependent on Ste24p and is abolished by mutation of the Ste24p HEXYH metalloprotease motif or by mutation of the a-factor P1 substrate at a residue adjacent to the N-terminal P1 cleavage site. We als o demonstrate that N-terminal proteolysis of the P1 a-factor precursor requ ires Zn2+ as a co-factor and can be inhibited by the addition of the metall oprotease inhibitor 1,10-orthophenanthroline, Our results are consistent wi th Ste24p itself being the P1-->P2 a-factor protease or a limiting activato r of this activity. Interestingly, we also show that the human Ste24 homolo g 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 sub strate in the absence of known human substrates, The results reported here, together with the previously reported in vitro reconstitution of Ste24p-de pendent CAAX processing, provide a system for examining the potential bifun ctional roles of yeast Ste24p and its homologs.