He B, Chen P, Chen S Y, Vancura K L, Michaelis S, Powers S
Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway 08854.
Proc Natl Acad Sci U S A. 1991 Dec 15;88(24):11373-7. doi: 10.1073/pnas.88.24.11373.
In the yeast Saccharomyces cerevisiae, mutations in either of two unlinked genes, RAM1 or RAM2, abolish the farnesyltransferase activity responsible for prenylation of Ras proteins and the a-factor mating pheromone. Here we report that the function of RAM1 and RAM2 genes is required for the membrane localization of Ras proteins and a-factor. The RAM2 gene was sequenced and can encode a 38-kDa protein. We examined the functional interaction of RAM2 and RAM1 by expressing the genes in Escherichia coli. Extracts derived from an E. coli strain that coexpressed RAM1 and RAM2 efficiently farnesylated a-factor peptide and Ras protein substrates. In contrast, extracts derived from E. coli strains that expressed either RAM gene alone were devoid of activity; however, when the latter extracts were mixed, protein farnesyltransferase activity was reconstituted. These results indicate that the yeast farnesyl-protein transferase is comprised of Ram1 and Ram2 polypeptides. Although Ram1 is a component of the enzyme, disruption of the RAM1 gene in yeast was not lethal, indicating that the Ram1-Ram2 farnesyltransferase is not essential for viability. In contrast, disruption of RAM2 was lethal, suggesting that Ram2 has an essential function in addition to its role with Ram1 in protein farnesylation.
在酿酒酵母中，两个不连锁的基因RAM1或RAM2中的任何一个发生突变，都会消除负责Ras蛋白和a因子交配信息素异戊二烯化的法尼基转移酶活性。在此我们报告，RAM1和RAM2基因的功能是Ras蛋白和a因子膜定位所必需的。对RAM2基因进行了测序，它可编码一种38 kDa的蛋白质。我们通过在大肠杆菌中表达这些基因，研究了RAM2和RAM1的功能相互作用。共表达RAM1和RAM2的大肠杆菌菌株提取物能有效地将法尼基基团连接到a因子肽和Ras蛋白底物上。相比之下，单独表达任一RAM基因的大肠杆菌菌株提取物都没有活性；然而，当将后一种提取物混合时，蛋白质法尼基转移酶活性得以重建。这些结果表明，酵母法尼基蛋白转移酶由Ram1和Ram2多肽组成。虽然Ram1是该酶的一个组分，但酵母中RAM1基因的破坏并不致命，这表明Ram1-Ram2法尼基转移酶对于细胞存活不是必需的。相比之下，RAM2的破坏是致命的，这表明Ram2除了与Ram1在蛋白质法尼基化中发挥作用外，还具有重要功能。
