Biochemical and genetic analysis of dominant-negative mutations affecting a yeast G-protein γ subunit

Heterotrimeric guanine nucleotide-binding proteins (G proteins) consisting of α, β, and γ subunits mediate signalling between cell surface receptors and intracellular effectors in eukaryotic cells. To define signalling functions of G(γ) subunits (STE18 gene product) involved in pheromone response and mating in the yeast Saccharomyces cerevisiae, we isolated and characterized dominant-negative STE18 alleles. We obtained dominant-negative mutations that disrupt C-terminal sequences required for prenylation of G(γ) precursors (CAAX box) and that affect residues in the N-terminal half of Ste18p. Overexpression of mutant G(γ) subunits in wild-type cells blocked signal transduction; this effect was suppressed upon overexpression of G(β) subunits. Mutant G(γ) subunits may therefore sequester G(β) subunits into nonproductive G(βγ) dimers. Because mutant G(γ) subunits blocked the constitutive signal resulting from disruption of the G(α) subunit gene (GPA1), they are defective in functions required for downstream signalling. Ste18p bearing a C107Y substitution in the CAAX box displayed reduced electrophoretic mobility, consistent with a prenylation defect. G(γ) subunits carrying N-terminal substitutions had normal electrophoretic mobilities, suggesting that these proteins were prenylated. G(γ) subunits bearing substitutions in their N-terminal region or C-terminal CAAX box (C107Y) supported receptor-G protein coupling in vitro, whereas C-terminal truncations caused partial defects in receptor coupling.