title: The CaaX proteases, Afc1p and Rce1p, have overlapping but distinct substrate specificities.
abstract: Many proteins that contain a carboxyl-terminal CaaX sequence motif, including Ras and yeast a-factor, undergo a series of sequential posttranslational processing steps. Following the initial prenylation of the cysteine, the three C-terminal amino acids are proteolytically removed, and the newly formed prenylcysteine is carboxymethylated. The specific amino acids that comprise the CaaX sequence influence whether the protein can be prenylated and proteolyzed. In this study, we evaluated processing of a-factor variants with all possible single amino acid substitutions at either the a(1), the a(2), or the X position of the a-factor Ca(1)a(2)X sequence, CVIA. The substrate specificity of the two known yeast CaaX proteases, Afc1p and Rce1p, was investigated in vivo. Both Afc1p and Rce1p were able to proteolyze a-factor with A, V, L, I, C, or M at the a(1) position, V, L, I, C, or M at the a(2) position, or any amino acid at the X position that was acceptable for prenylation of the cysteine. Eight additional a-factor variants with a(1) substitutions were proteolyzed by Rce1p but not by Afc1p. In contrast, Afc1p was able to proteolyze additional a-factor variants that Rce1p may not be able to proteolyze. In vitro assays indicated that farnesylation was compromised or undetectable for 11 a-factor variants that produced no detectable halo in the wild-type AFC1 RCE1 strain. The isolation of mutations in RCE1 that improved proteolysis of a-factor-CAMQ, indicated that amino acid substitutions E139K, F189L, and Q201R in Rce1p affected its substrate specificity.
authors.affiliation: Molecular and Cell Biology Department, University of California, Berkeley, California 94720, USA.
authors.forename: C E
authors.initials: CE
authors.lastname: Trueblood
authors.forename: V L
authors.initials: VL
authors.lastname: Boyartchuk
authors.forename: E A
authors.initials: EA
authors.lastname: Picologlou
authors.forename: D
authors.initials: D
authors.lastname: Rozema
authors.forename: C D
authors.initials: CD
authors.lastname: Poulter
authors.forename: J
authors.initials: J
authors.lastname: Rine
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D029681:Arabidopsis Proteins; D001665:Binding Sites; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D008666:Metalloendopeptidases; D008969:Molecular Sequence Data; D009154:Mutation; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D043484:Proprotein Convertases; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity
chemical_list: D029681:Arabidopsis Proteins; D010940:Plant Proteins; D029701:Saccharomyces cerevisiae Proteins; D011494:Protein Kinases; C090861:AFC1 protein, Arabidopsis; D010450:Endopeptidases; D043484:Proprotein Convertases; C105193:RCE1 protein, S cerevisiae; D008666:Metalloendopeptidases