Jung V, Chen L, Hofmann S L, Wigler M, Powers S
Cold Spring Harbor Laboratory, New York 11724.
Mol Cell Biol. 1995 Mar;15(3):1333-42. doi: 10.1128/MCB.15.3.1333.
We have identified a gene, SHR5, in a screen for extragenic suppressors of the hyperactive RAS2Val-19 mutation in the budding yeast Saccharomyces cerevisiae. SHR5 was cloned, sequenced, and found to encode a 23-kDa protein not significantly homologous to other proteins in the current data bases. Genetic evidence arguing that Shr5 operates at the level of Ras is presented. We tested whether SHR5, like previously isolated suppressors of hyperactivated RAS2, acts by affecting the membrane attachment and/or posttranslational modification of Ras proteins. We found that less Ras protein is attached to the membrane in shr5 mutants than in wild-type cells and that the Ras proteins are markedly underpalmitoylated, suggesting that Shr5 is involved in palmitoylation of Ras proteins. However, shr5null mutants exhibit normal palmitoyltransferase activity measured in vitro. Further, shr5null mutations attenuate Ras function in cells containing mutant Ras2 proteins that are not palmitoylated or farnesylated. We conclude that SHR5 encodes a protein that participates in the membrane localization of Ras but also interacts in vivo with completely unprocessed and cytosolic Ras proteins.
我们在对出芽酵母酿酒酵母中RAS2Val - 19高活性突变的基因外抑制子进行筛选时，鉴定出了一个基因SHR5。对SHR5进行了克隆、测序，发现它编码一种23 kDa的蛋白质，与当前数据库中的其他蛋白质没有明显的同源性。本文提供了遗传学证据，证明Shr5在Ras水平发挥作用。我们测试了SHR5是否像之前分离出的RAS2高活性抑制子一样，通过影响Ras蛋白的膜附着和/或翻译后修饰来发挥作用。我们发现，与野生型细胞相比，shr5突变体中附着在膜上的Ras蛋白较少，并且Ras蛋白明显缺乏棕榈酰化，这表明Shr5参与了Ras蛋白的棕榈酰化过程。然而，shr5缺失突变体在体外测得的棕榈酰转移酶活性正常。此外，shr5缺失突变会减弱含有未进行棕榈酰化或法尼基化的突变Ras2蛋白的细胞中的Ras功能。我们得出结论，SHR5编码一种蛋白质，该蛋白质参与Ras的膜定位，但在体内也与完全未加工的胞质Ras蛋白相互作用。
