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.
Highlights
Precursors of the S. cerevisiae mating pheromones, ␣-factor and a-factor, undergo posttranslational processing [1,2,3]
In Vitro Reconstitution of N-terminal Proteolysis (P13 P2) of the P1 a-Factor Precursor—We have previously established that the P1 a-factor precursor accumulates in vivo in a ste24⌬ strain, suggesting that Ste24p promotes the initial N-terminal (P13 P2) cleavage of the a-factor precursor [11]
Concluding Remarks—We have previously presented in vivo genetic data suggesting a role for Ste24p in the N-terminal (P13 P2) processing of the P1 a-factor precursor
Summary
Precursors of the S. cerevisiae mating pheromones, ␣-factor and a-factor, undergo posttranslational processing [1,2,3]. The first set of modifications (isoprenylation, proteolytic removal of the AAX tripeptide, and carboxylmethylation) are directed by the C-terminal CAAX motif [8, 9] These Cterminal modifications are common to all proteins bearing a CAAX sequence (e.g. Ras, G␥, and nuclear lamin precursors). Recent genetic studies have identified several components involved in the proteolytic processing of a-factor (Fig. 1B) These include the Ste24p ( called Afc1p) and Rce1p proteins that are required for the C-terminal CAAX processing of a-factor and other proteins bearing a CAAX motif [12]. The Axl1p and Ste23p proteins were shown to have some partial overlapping function for the final N-terminal cleavage event (P23 M) that yields mature a-factor [14]. The Ste24p-dependent N-terminal proteolytic activity was not examined in this study
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