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 ,x subunit of the G protein involved in pheromone response in yeast. The scgl L~sass and scgl An°a91 mutations resulted in s e v e r e 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 SCG1 A~°a22 mutation resulted in pheromone response and mating defects. This effect is similar to the effect of the analogous Gas mutation, which results in a defect in stimulation of adenylate cyclase. The scgl was° mutation, which is analogous to the transforming mutation ras v"n2, 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 scgl V~a5° on a low-copy plasmid. Implications of these phenotypes are discussed.

Dual Lipid Modification Motifs in Gαand GγSubunits Are Required for Full Activity of the Pheromone Response Pathway inSaccharomyces cerevisiae

“…The pheromone response pathway is exquisitely sensitive to changes in Gpa1p expression (Cole et al, 1990;Kang et al, 1990). Studies of other GPA1 mutations have revealed different phenotypes when expressed on a centromere-based plasmid instead of the chromosomal locus (Miyajima et al, 1989;Kurjan et al, 1991). We found that haploid cells expressing the mutant allele as the sole copy of GPA1 were viable.…”

RGS Proteins and Signaling by Heterotrimeric G Proteins

“…Other genetic observations suggested a direct interaction between SST2 and GPA1. First, certain mutations in the G␣ gene could bypass the requirement for a fully active SST2 gene (30,31). Second, the recovery-promoting effect of G␣ overexpression was more efficient if residual SST2 function was present (32,33).…”

Sequence elements that contribute to the degradation of yeast Gα

“…We therefore tested the stability of the G␣ N388K mutant, whose phenotype is consistent with a defect in guaninenucleotide binding. G␣ N388K has been proposed to accumulate in the GTP-bound state, thereby preventing efficient interactions with G␤␥ (Kurjan et al 1991).…”

“…G␣ s exhibits an accelerated rate of degradation after activation of the signalling pathway. Interestingly, a mutation in G␣ s (R201C) that slows the rate of GTP hydrolysis is associated with increased degradation (Kurjan et al 1991). Similarly, the yeast G␣ N388K mutant, which may be primarily bound to GTP, is rapidly degraded.…”

“…G␣ N388K was constructed as follows: G␣ N388K was amplified using pYEp352-gpa1 Lys388 (Kurjan et al 1991) as a template and ligated into pKM1362-2 as above, to produce plasmid pKM1489. G␣ N388K is produced as an untagged protein and immunoprecipitated using G␣ antiserum.…”