RESUMO
The number of alpha-factor binding sites on yeast MATa cells (8,000) and the equilibrium dissociation constant (6 X 10(-9) M) were determined from direct binding experiments. These values correct our previously reported estimates (D. D. Jennes, A. C. Burkholder, and L. H. Hartwell, Cell 35:521-529, 1983) that were based on indirect isotope dilution studies, and they lead to a revised rate constant for the association process (kon = 3 X 10(5) mol-1 s-1).
Assuntos
Peptídeos/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores de Peptídeos , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição , Cinética , Fator de Acasalamento , Feromônios/metabolismo , Receptores de Fator de AcasalamentoRESUMO
The STE2 gene of the yeast Saccharomyces cerevisiae encodes a component of the receptor for the oligopeptide pheromone alpha-factor. We have cloned and determined the nucleotide sequence of the STE2 gene. A sequence involved in the control of cell-type expression of the STE2 gene was found 5' of an open reading frame that could encode a protein of 431 amino acids. The predicted STE2 protein contains seven hydrophobic segments, suggesting that the alpha-factor receptor is an integral membrane protein. No extensive homology at the primary sequence level was detected between the predicted STE2 protein and other available protein sequences.
Assuntos
Genes Fúngicos , Feromônios , Receptores de Superfície Celular/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , DNA Fúngico/genética , Proteínas Fúngicas/genética , Teste de Complementação Genética , Fator de Acasalamento , Proteínas de Membrana/genética , Peptídeos/metabolismo , Feromônios/metabolismo , Conformação Proteica , Receptores de Superfície Celular/metabolismo , SolubilidadeRESUMO
The division cycle of yeast a cells is inhibited by alpha-factor. Haploid a cells were found to bind 35S-labeled alpha-factor, whereas haploid alpha cells and diploid a/alpha cells showed little binding. The association of alpha-factor with a cells was reversible upon dilution. Unlabeled alpha-factor competed for binding of 35S-alpha-factor; the concentration dependence for competition indicated 9 X 10(5) binding sites per cell with a dissociation constant (KD) of 3 X 10(-7) M. The rates of association (kon = 3 X 10(3) M-1 sec-1) and dissociation (koff = 9 X 10(-4) sec-1) were consistent with the equilibrium constant. The alpha-factor binding activity associated with five temperature-sensitive ste2 mutants was thermolabile, suggesting that the STE2 gene encodes the receptor for alpha-factor. In contrast, the binding activity of other temperature-sensitive mutants (ste4, ste5, ste7, ste11, and ste12) showed no thermolability.
