Biochemical and genetic analysis of dominant-negative mutations affecting a yeast G-protein gamma subunit.
Heterotrimeric guanine nucleotide-binding proteins (G proteins) consisting of alpha, beta, and gamma subunits mediate signalling between cell surface receptors and intracellular effectors in eukaryotic cells. To define signalling functions of G gamma 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 gamma precursors (CAAX box) and that affect residues in the N-terminal half of Ste18p. Overexpression of mutant G gamma subunits in wild-type cells blocked signal transduction; this effect was suppressed upon overexpression of G beta subunits. Mutant G gamma subunits may therefore sequester G beta subunits into nonproductive G beta gamma dimers. Because mutant G gamma subunits blocked the constitutive signal resulting from disruption of the G alpha 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 gamma subunits carrying N-terminal substitutions had normal electrophoretic mobilities, suggesting that these proteins were prenylated. G gamma 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.
Grishin
A V
AV
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110.
Weiner
J L
JL
Blumer
K J
KJ
D000483:Alleles; D000595:Amino Acid Sequence; D044388:GTP-Binding Protein gamma Subunits; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000235:genetics*; D015870:Gene Expression / Q000187:drug effects; D005799:Genes, Dominant; D005800:Genes, Fungal; D020962:Heterotrimeric GTP-Binding Proteins; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010675:Pheromones / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D014158:Transcription, Genetic
D044388:GTP-Binding Protein gamma Subunits; D046911:Macromolecular Substances; D010675:Pheromones; C084294:STE18 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D019204:GTP-Binding Proteins; D020962:Heterotrimeric GTP-Binding Proteins