Abstract
The Ras converting enzyme (RCE) promotes a proteolytic activity that is required for the maturation of Ras, the yeast a-factor mating pheromone, and certain other proteins whose precursors bear a C-terminal CAAX tetrapeptide motif. Despite the physiological importance of RCE, the enzymatic mechanism of this protease remains undefined. In this study, we have evaluated the substrate specificity of RCE orthologs from yeast (Rce1p), worm, plant, and human and have determined the importance of conserved residues toward enzymatic activity. Our findings indicate that RCE orthologs have conserved substrate specificity, cleaving CVIA, CTLM, and certain other CAAX motifs, but not the CASQ motif, when these motifs are placed in the context of the yeast a-factor precursor. Our mutational studies of residues conserved between the orthologs indicate that an alanine substitution at His194 completely inactivates yeast Rce1p enzymatic activity, whereas a substitution at Glu156 or His248 results in marginal activity. We have also determined that residues Glu157, Tyr160, Phe190, and Asn252 impact the substrate selectivity of Rce1p. Computational methods predict that residues influencing Rce1p function are all near or within hydrophobic segments. Combined, our data indicate that yeast Rce1p function requires residues that are invariably conserved among an extended family of prokaryotic and eukaryotic enzymes and that these residues are likely to lie within or immediately adjacent to the transmembrane segments of this membrane-localized enzyme.
Highlights
Tumor growth [5, 6]
Other established proteases with multiple membrane spans include the STE24 CAAX protease that has a partially overlapping role with Ras converting enzyme (RCE), the presenilins that are involved in A production and Notch signaling, S2P that is involved in production of the sterol-response element-binding protein, SPP that is involved in clearance of signal sequences, and rhomboid that is involved in the production of Drosophila epidermal growth factor-␣ [17, 18]
We have evaluated the ability of the CAAX proteases from budding yeast (S. cerevisiae), plant (Arabidopsis thaliana), worm (Caenorhabditis elegans), and humans to cleave three distinct CAAX motifs that have been proposed to be nonspecific (CVIA), RCE-specific (CTLM), or STE24-specific (CASQ) (Fig. 1) [39]
Summary
Tumor growth [5, 6]. Inhibition of the proteolytic step in CAAX protein modification may have similar anti-cancer potential [4, 7]. Despite the relatively low degree of primary sequence conservation, all Rce1p orthologs examined to date can substitute for yeast Rce1p in the maturation of the yeast a-factor mating pheromone [11, 12] These observations suggest that the RCE family may have conserved substrate specificity. Certain amino acids have been identified that are reportedly critical for RCE activity These include cysteine, glutamate, and histidine residues [14]. We show that the enzymatic activity of yeast Rce1p requires a glutamate and a pair of histidine residues that are invariably conserved among RCE orthologs but not a conserved cysteine that has been proposed to be a catalytic residue
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