Mutagenesis of Ste18, a putative G gamma subunit in the Saccharomyces cerevisiae pheromone response pathway.
The yeast STE18 gene product has sequence and functional similarity to the gamma subunits of G proteins. The cloned STE18 gene was subjected to a saturation mutagenesis using doped oligonucleotides. The populations of mutant genes were screened for two classes of STE18 mutations, those that allowed for increased mating of a strain containing a defective STE4 gene (compensators) and those that inhibited mating even in the presence of a functional STE18 gene (dominant negatives). Three amino acid substitutions that enhanced mating in a specific STE4 (G beta) point mutant background were identified. These compensatory mutations were allele specific and had no detectable phenotype of their own; they may define residues that mediate an association between the G beta and G gamma subunits or in the association of the G beta gamma subunit with other components of the signalling pathway. Several dominant negative mutations were also identified, including two C terminal truncations. These mutant proteins were unable to function in signal transduction by themselves, but they prevented signal transduction mediated by pheromone, as well as the constitutive signalling which is present in cells defective in the GPA1 (G alpha) gene. These mutant proteins may sequester G beta or some other component of the signalling machinery in a nonfunctional complex.
Whiteway
M
M
Biotechnology Research Institute, National Research Council of Canada, Montréal, Que.
Dignard
D
D
Thomas
D Y
DY
D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000502:physiology; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D000072235:Mating Factor; D016296:Mutagenesis; D010455:Peptides / Q000502:physiology*; D010675:Pheromones / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012098:Reproduction; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology
D005656:Fungal Proteins; D010455:Peptides; D010675:Pheromones; D011993:Recombinant Fusion Proteins; D000072235:Mating Factor; D019204:GTP-Binding Proteins