IRA2, an upstream negative regulator of RAS in yeast, is a RAS GTPase-activating protein.

Abstract

The ras GTPase-activating protein (GAP), identified and characterized in mammalian cells, stimulates the intrinsic GTPase activity of ras proteins. We have previously proposed that the IRA genes, negative regulators of RAS genes in Saccharomyces cerevisiae, encode yeast homologs of the mammalian GAP. In this paper, we present the following evidence that a product of the IRA2 gene exhibits GAP activity similar to that of the mammalian GAP protein. Previously, we have reported on the identification of two Saccharomyces cerevisiae genes, IRAI and IRA2, which encode proteins with domains homologous to the mammalian GAP protein (8, 9). Genetic results suggest that these genes fpnction as upstream negative regulators of yeast RAS genes (8,9). A mutation in either IRA] or IRA2 produces a set of phenotypes that are also observed in strains carrying an activated mutation of RAS2, such as RAS2vall9. These phenotypes include heat shock sensitivity, nitrogen starvation sensitivity, and sporulation deficiency.The following observations further support the idea that IRA proteins have functions similar to those of the mammalian GAP. First, expression of mammalian GAP suppresses the heat shock-sensitive phenotype of irq mutants (10, 11).Second, the fraction of RAS proteins in the GTP-bound form is increased in ira mutants (10).We report here that IRA2 indeed possesses a GAP activity. This provides evidence that IRA proteins are yeast homologs of the mammalian GAP proteins. Since IRA genes are upstream regulators of RAS genes, the mammalian GAP may also have an upstream regulatory function. MATERIALS AND METHODSStrains and Medium. The yeast strain TK161-R2V (MATa ura3 leu2 trpl his3 ade8 RAS2vl19) (12) was used as the host for transformation. This RAS2vall9 strain was chosen because the putative GAP activity of IRA2 may be positively feedback-regulated by cAMP-dependent protein phosphorylation (9). Transformants were grown in SDA medium, which is 2% dextrose/0.5% ammonium sulfate/0.17% yeast nitrogen base without amino acids and ammonium sulfate (Difco)/0.5% Casamino acid (Difco)/0.63 mg ofadenine sulfate per ml/0.25 mg of L-tryptophan per ml.Plasmids. The 5183-base-pair (bp) Pvu II/Pvu II fragment of IRA2 was placed into the plasmid pKT10 (9) *To whom reprint requests should be addressed. 468The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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