NUCLEOTIDE-SEQUENCE OF THE YEAST STE14 GENE, WHICH ENCODES FARNESYLCYSTEINE CARBOXYL METHYLTRANSFERASE, AND DEMONSTRATION OF ITS ESSENTIAL ROLE IN A-FACTOR EXPORT

Eukaryotic proteins initially synthesized with a C-terminal CAAX motif (C is Cys, A is aliphatic, and X can be one of several amino acids) u ndergo a series of modifications involving isoprenylation of the Cys r esidue, proteolysis of AAX, and alpha-carboxyl methyl esterification o f the newly formed isoprenyl cysteine. We have previously demonstrated that STE14 encodes the enzyme which mediates carboxyl methylation of the Saccharomyces cerevisiae CAAX proteins a-factor, RAS1, and RAS2. H ere we report the nucleotide sequence of STE14, which indicates that S TE14 encodes a protein of 239 amino acids, predicted to contain multip le membrane-spanning segments. Mapping data indicate that STE14 reside s on chromosome IV, tightly linked to ADE8. By analysis of ste14 null alleles, we demonstrated that MATa ste14 mutants are unable to mate bu t are viable and exhibit no apparent growth defects. Additional analys is of ste14 rasl and st14 ras2 double mutants, which grow normally, re inforces our previous conclusion that RAS function is not significantl y influenced by its methylation status. We examine a-factor biogenesis in a ste14 null mutant by metabolic labeling and immunoprecipitation and demonstrate that although proteolytic processing and membrane loca lization of a-factor are normal, the ste14 null mutant exhibits a prof ound block in a-factor export. This observation suggests that the meth yl group is likely to be a critical recognition determinant for the a- factor transporter, STE6, thus providing insight into the substrate sp ecificity of STE6 and also supporting the hypothesis that carboxyl met hylation can have a dramatic impact on protein-protein interactions.