The GTP hydrolysis defect of theSaccharomyces cerevisiae mutant G-protein Gpa1G50V

✍ Scribed by Kallal, Lorena; Fishel, Richard

• Book ID: 101224501
• Publisher: John Wiley and Sons
• Year: 2000
• Tongue: English
• Weight: 227 KB
• Volume: 16
• Category: Article
• ISSN: 0749-503X
• DOI: 10.1002/(sici)1097-0061(20000330)16:5<387::aid-yea525>3.0.co;2-u

✦ Synopsis

The Saccharomyces cerevisiae haploid cell response to pheromone involves two seven-transmembrane-domain pheromone receptors that couple to a heterotrimeric G protein. The G50V mutation in the G protein a subunit (G a ), Gpa1p, is analogous to the p21 ras transforming mutation GlypVal 12, and has been extensively examined for the phenotypes it produces in yeast cells. Here we have characterized the Gpa1 G50V mutant protein in vitro by examining GTPcS binding, GDP exchange, GTP occupancy and guanosine triphosphatase (GTPase) activity. Compared to wild-type (WT) Gpa1p, Gpa1 G50V p was found to have a moderately reduced GTPase activity and increased GTP occupancy, while GTPcS binding and GDP exchange were not signi®cantly altered. The yeast regulator of G protein Signalling (RGS) protein, Sst2p, was also expressed and puri®ed, and found to have a signi®cantly reduced ability to stimulate the initial rate of GTP hydrolysis of Gpa1 G50V p compared to its effect on WT Gpa1p. Probing conformational transitions by a protease sensitivity assay suggested that Gpa1 G50V p did not bind the transition state mimetic GDP/AlF 4

x as ef®ciently as the WT Gpa1p. These biochemical results can explain many of the known gpa1 G50V yeast cell phenotypes.