Hrycyna C A, Clarke S
Department of Chemistry and Biochemistry, University of California, Los Angeles 90024-1569.
Mol Cell Biol. 1990 Oct;10(10):5071-6. doi: 10.1128/mcb.10.10.5071-5076.1990.
Membrane extracts of sterile Saccharomyces cerevisiae strains containing the a-specific ste14 mutation lack a farnesyl cysteine C-terminal carboxyl methyltransferase activity that is present in wild-type a and alpha cells. Other a-specific sterile strains with ste6 and ste16 mutations also have wild-type levels of the farnesyl cysteine carboxyl methyltransferase activity. This enzyme activity, detected by using a synthetic peptide sequence based on the C-terminus of a ras protein, may be responsible not only for the essential methylation of the farnesyl cysteine residue of a mating factor, but also for the methylation of yeast RAS1 and RAS2 proteins and possibly other polypeptides with similar C-terminal structures. We demonstrate that the farnesylation of the cysteine residue in the peptide is required for the methyltransferase activity, suggesting that methyl esterification follows the lipidation reaction in the cell. To show that the loss of methyltransferase activity is a direct result of the ste14 mutation, we transformed ste14 mutant cells with a plasmid complementing the mating defect of this strain and found that active enzyme was produced. Finally, we demonstrated that a similar transformation of cells possessing the wild-type STE14 gene resulted in sixfold overproduction of the enzyme. Although more complicated possibilities cannot be ruled out, these results suggest that STE14 is a candidate for the structural gene for a methyltransferase involved in the formation of isoprenylated cysteine alpha-methyl ester C-terminal structures.
含有a特异性ste14突变的无菌酿酒酵母菌株的膜提取物缺乏野生型a和α细胞中存在的法尼基半胱氨酸C末端羧基甲基转移酶活性。其他具有ste6和ste16突变的a特异性不育菌株也具有野生型水平的法尼基半胱氨酸羧基甲基转移酶活性。这种酶活性通过使用基于ras蛋白C末端的合成肽序列检测到，可能不仅负责交配因子法尼基半胱氨酸残基的必需甲基化，还负责酵母RAS1和RAS2蛋白以及可能其他具有相似C末端结构的多肽的甲基化。我们证明肽中半胱氨酸残基的法尼基化是甲基转移酶活性所必需的，这表明甲酯化在细胞中的脂化反应之后发生。为了表明甲基转移酶活性的丧失是ste14突变的直接结果，我们用补充该菌株交配缺陷的质粒转化ste14突变细胞，发现产生了活性酶。最后，我们证明具有野生型STE14基因的细胞的类似转化导致该酶过量产生六倍。尽管不能排除更复杂的可能性，但这些结果表明STE14是参与异戊烯基化半胱氨酸α-甲基酯C末端结构形成的甲基转移酶结构基因的候选者。
