Kim J H, Powers S
Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854.
Mol Cell Biol. 1991 Aug;11(8):3894-904. doi: 10.1128/mcb.11.8.3894-3904.1991.
A high-copy-number plasmid genomic library was screened for genes that when overexpressed down-regulate Ras protein activity in Saccharomyces cerevisiae. We report on the structure and characterization of one such gene, RPI1, which potentially encodes a novel 46-kDa negative regulator of the Ras-cyclic AMP pathway. Three lines of evidence suggest that the RPI1 gene product operates upstream to negatively regulate the activity of normal but not mutationally activated Ras proteins: (i) overexpressed RPI1 lowers cyclic AMP levels in wild-type yeast cells but not in yeast cells carrying the RAS2Val-19 mutation, (ii) overexpressed RPI1 suppresses the heat shock sensitivity phenotype induced by overexpression of normal RAS2 but does not suppress the same phenotype induced by RAS2Val-19, and (iii) disruption of RPI1 results in a heat shock sensitivity phenotype which can be suppressed by mutations that lower normal Ras activity. Thus, RPI1 appears to encode an inhibitor of Ras activity that shares a common feature with Ras GTPase-activating proteins in that it fails to down-regulate activated RAS2Val-19 function. We present evidence that the down-regulatory effect of RPI1 requires the presence of one of the two Ras GTPase activators, IRA1 and IRA2.
对一个高拷贝数质粒基因组文库进行筛选，以寻找在酿酒酵母中过表达时能下调Ras蛋白活性的基因。我们报告了一个这样的基因RPI1的结构和特性，它可能编码一种新型的46 kDa的Ras-环磷酸腺苷途径的负调控因子。有三条证据表明RPI1基因产物在正常而非突变激活的Ras蛋白活性的负调控中起上游作用：（i）过表达的RPI1降低野生型酵母细胞中的环磷酸腺苷水平，但不降低携带RAS2Val-19突变的酵母细胞中的环磷酸腺苷水平；（ii）过表达的RPI1抑制正常RAS2过表达诱导的热休克敏感性表型，但不抑制RAS2Val-19诱导的相同表型；（iii）RPI1的破坏导致一种热休克敏感性表型，这种表型可被降低正常Ras活性的突变所抑制。因此，RPI1似乎编码一种Ras活性抑制剂，它与Ras GTP酶激活蛋白有一个共同特征，即它不能下调激活的RAS2Val-19功能。我们提供的证据表明，RPI1的下调作用需要两种Ras GTP酶激活剂之一IRA1和IRA2的存在。
