Overexpression of RPI1, a novel inhibitor of the yeast Ras-cyclic AMP pathway, down-regulates normal but not mutationally activated Ras function

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, RP11, which potentially encodes a novel 46-kDa negative regulator of the Ras-cyclic AMP pathway. Three lines of evidence suggest that the RP11 gene product operates upstream to negatively regulate the activity of normal but not mutationally activated Ras proteins: (i) overexpressed RP11 lowers cyclic AMP levels in wild-type yeast cells but not in yeast cells carrying the RAS2^Val-19 mutation, (ii) overexpressed RP11 suppresses the heat shock sensitivity phenotype induced by overexpression of normal RAS2 but does not suppress the same phenotype induced by RAS2^Val-19, and (iii) disruption of RP11 results in a heat shock sensitivity phenotype which can be suppressed by mutations that lower normal Ras activity. Thus, RP11 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 RAS2^Val-19 function. We present evidence that the down-regulatory effect of RP11 requires the presence of one of the two Ras GTPase activators, IRA1 and IRA2.