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) undergo a series of modifications involving isoprenylation of the Cys residue, proteolysis of AAX, and alpha-carboxyl methyl esterification of 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. Here we report the nucleotide sequence of STE14, which indicates that STE14 encodes a protein of 239 amino acids, predicted to contain multiple membrane-spanning segments. Mapping data indicate that STE14 resides on chromosome IV, tightly linked to ADE8. By analysis of ste14 null alleles, we demonstrated that MATa ste14 mutants are unable to mate but are viable and exhibit no apparent growth defects. Additional analysis of ste14 ras 1 and ste14 ras2 double mutants, which grow normally, reinforces our previous conclusion that RAS function is not significantly 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 localization of a-factor are normal, the ste14 null mutant exhibits a profound block in a-factor export. This observation suggests that the methyl group is likely to be a critical recognition determinant for the a-factor transporter, STE6, thus providing insight into the substrate specificity of STE6 and also supporting the hypothesis that carboxyl methylation can have a dramatic impact on protein-protein interactions.
Sapperstein
S
S
Department of Cell Biology and Anatomy, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Berkower
C
C
Michaelis
S
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D015825:Chromosomes, Fungal; D004271:DNA, Fungal / Q000378:metabolism; D005090:Exodeoxyribonucleases; D005800:Genes, Fungal; D000072235:Mating Factor; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism*; D010675:Pheromones / Q000378:metabolism; D011496:Protein Methyltransferases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D017384:Sequence Deletion; D014157:Transcription Factors
D004271:DNA, Fungal; D010455:Peptides; D010675:Pheromones; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide; D014157:Transcription Factors; D000072235:Mating Factor; D011496:Protein Methyltransferases; C056501:protein-S-isoprenylcysteine O-methyltransferase; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III