Biochemical and genetic analysis of dominant-negative mutations affecting a yeast G-protein gamma subunit.
Mol Cell Biol
; 14(7): 4571-8, 1994 Jul.
Article
em En
| MEDLINE
| ID: mdl-8007961
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Saccharomyces cerevisiae
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Mutagênese Insercional
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Proteínas de Ligação ao GTP
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Proteínas Heterotriméricas de Ligação ao GTP
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Proteínas de Saccharomyces cerevisiae
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Subunidades gama da Proteína de Ligação ao GTP
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Genes Dominantes
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Genes Fúngicos
Idioma:
En
Revista:
Mol Cell Biol
Ano de publicação:
1994
Tipo de documento:
Article