Mutations in the guanine nucleotide-binding domains of a yeast G alpha protein confer a constitutive or uninducible state to the pheromone response pathway

Abstract

Several domains of guanine nucleotide-binding proteins are conserved and form the guanine nucleotide-binding pocket. Mutations in these domains in EF-Tu, ras, and Gas have been shown to result in informative phenotypes. We made several analogous changes in SCG1, which encodes the alpha subunit of the G protein involved in pheromone response in yeast. The scg1Lys388 and scg1Ala391 mutations resulted in severe growth and cell morphology defects; this phenotype is similar to the null phenotype and results from constitutive activation of the pheromone response pathway. On the basis of the model for the action of the yeast G protein, the effect of these mutations is consistent with the effect of analogous mutations in ras, which result in a transforming phenotype. The SCG1Ala322 mutation resulted in pheromone response and mating defects. This effect is similar to the effect of the analogous G alpha s mutation, which results in a defect in stimulation of adenylate cyclase. The scg1Val50 mutation, which is analogous to the transforming mutation rasVal12, resulted in multiple effects, including defects in growth, cell morphology, and mating. Some of our results and interpretations are different from previously published results of others for the same mutation in SCG1; specifically, our gene replacement of this mutation resulted in high basal activation of the pheromone response pathway, consistent with a GTPase defect, which was not seen previously with scg1Val50 on a low-copy plasmid. Implications of these phenotypes are discussed.