The GTP hydrolysis defect of the Saccharomyces cerevisiae mutant G-protein Gpa1(G50V).
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 alpha subunit (G(alpha)), Gpa1p, 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 Gpa1(G50V) mutant protein in vitro by examining GTPgammaS 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 GTPgammaS binding and GDP exchange were not significantly altered. The yeast regulator of G protein Signalling (RGS) protein, Sst2p, was also expressed and purified, and found to have a significantly 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)(-) as efficiently as the WT Gpa1p. These biochemical results can explain many of the known gpa1(G50V) yeast cell phenotypes.
Kallal
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L
Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA 19107, USA. lkallal@hendrix.jci.tju.edu
Fishel
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R
D019943:Amino Acid Substitution; D001667:Binding, Competitive; D005656:Fungal Proteins / Q000235:genetics / Q000502:physiology; D020558:GTP Phosphohydrolases / Q000378:metabolism; D044385:GTP-Binding Protein alpha Subunits; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020690:GTPase-Activating Proteins; D017353:Gene Deletion; D005816:Genetic Complementation Test; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D020962:Heterotrimeric GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D006639:Histidine / Q000235:genetics; D006868:Hydrolysis; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D014357:Trypsin / Q000378:metabolism; D020710:RGS Proteins
D005656:Fungal Proteins; D020558:GTP Phosphohydrolases; D044385:GTP-Binding Protein alpha Subunits; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D020962:Heterotrimeric GTP-Binding Proteins; D006639:Histidine; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014357:Trypsin; C094301:SST2 protein, S cerevisiae; D020710:RGS Proteins; C084293:GPA1 protein, S cerevisiae