The GTP hydrolysis defect of the Saccharomyces cerevisiae mutant G-proteinGpa1(G50V)

The Saccharomyces cerevisiae haploid cell response to pheromone involves tw o seven-transmembrane-domain pheromone receptors that couple to a heterotri meric G protein. The G50V mutation in the G protein alpha subunit (G(alpha) ), Gpalp, is analogous to the p21(ras) transforming mutation Gly --> Val 12 , and has been extensively examined for the phenotypes it produces in yeast cells. Here we have characterized the Gpal(G50V) mutant protein in vitro b y examining GTP gamma S binding, GDP exchange, GTP occupancy and guanosine triphosphatase (GTPase) activity. Compared to wild-type (WT) Gpalp, Gpal(G5 0V)p was found to have a moderately reduced GTPase activity and increased G TP occupancy, while GTP gamma S binding and GDP exchange were not significa ntly altered. The yeast regulator of G protein Signalling (RGS) protein, Ss t2p, was also expressed and purified, and found to have a significantly red uced ability to stimulate the initial rate of GTP hydrolysis of Gpal(G50V)p compared to its effect on WT Gpalp. Probing conformational transitions by a protease sensitivity assay suggested that Gpal(G50V)p did not bind the tr ansition state mimetic GDP/AIF(4)(-) as efficiently as the WT Gpalp. These biochemical results can explain many of the known gpal(G50V) yeast cell phe notypes. Copyright (C) 2000 John Wiley & Sons, Ltd.