The CaaX proteases, Afc1p and Rce1p, have overlapping but distinct substrate specificities

Many proteins that contain a carboxyl-terminal CaaX sequence motif, includi ng Ras and yeast a-factor, undergo a series of sequential posttranslational processing steps. Following the initial prenylation of the cysteine, the t hree C-terminal amino acids are proteolytically removed, and the newly form ed prenylcysteine is carboxymethylated. The specific amino acids that compr ise the CaaX sequence influence whether the protein can be prenylated and p roteolyzed. In this study, we evaluated processing of a-factor variants wit h 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 substrat e specificity of the two known yeast CaaX proteases, Afc1p and Rce1p, was i nvestigated in vivo. Both Afc1p and Rce1p were able to proteolyze a-factor with A, V, L, I, C, or M at the a, position, V, L, I, C, or M at the a(2) p osition, or any amino acid at the,X position that was acceptable for prenyl ation of the cysteine. Eight additional a-factor variants with a(1) substit utions were proteolyzed by Rce1p but not by Afc1p. In contrast, Afc1p was a ble to proteolyze additional a-factor variants that Rce1p may not be able t o proteolyze. In vitro assays indicated that farnesylation was compromised or undetectable for 11 a-factor variants that produced no detectable halo i n the wild-type AFC1 RCE1 strain. The isolation of mutations in RCE1 that i mproved proteolysis of a-factor-CAMQ, indicated that amino acid substitutio ns E139K, F189L, and Q201R in Rce1p affected its substrate specificity.