RESUMEN
The GAL3 gene plays a critical role in galactose induction of the GAL genes that encode galactose- metabolizing enzymes in Saccharomyces cerevisiae. Defects in GAL3 result in a long delay in GAL gene induction, and overproduction of Gal3p causes constitutive expression of GAL. Here we demonstrate that concomitant overproduction of the negative regulator, Gal80p, and Gal3p suppresses this constitutive GAL expression. This interplay between Gal80p and Gal3p is direct, as tagged Gal3p coimmunoprecipitated with Gal80p. The amount of coprecipitated Gal80p increased when GAL80 yeast cells were grown in the presence of galactose. When both GAL80 and GAL3 were overexpressed, the amount of coprecipitated Gal80p was not affected by galactose. Tagged gal3 mutant proteins bound to purified Gal80p, but only poorly in comparison with the wild type, suggesting that formation of the Gal80p-Gal3p complex depends on the normal function of Gal3p. Gal3p appeared larger in Western blots (immunoblots) than predicted by the published nucleic acid sequence. Reexamination of the DNA sequence of GAL3 revealed several mistakes, including an extension at the 3' end of another predicted 97 amino acids.
Asunto(s)
Proteínas Fúngicas/metabolismo , Galactosa/metabolismo , Proteínas Represoras/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Western Blotting , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/química , Galactoquinasa/biosíntesis , Galactoquinasa/química , Regulación Fúngica de la Expresión Génica , Modelos Biológicos , Datos de Secuencia Molecular , Mutagénesis , Plásmidos , Unión Proteica , Estructura Secundaria de Proteína , Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Lugares Marcados de Secuencia , Transducción de Señal , Factores de Transcripción/biosíntesis , Factores de Transcripción/química , Activación Transcripcional , UDPglucosa 4-Epimerasa/metabolismoRESUMEN
CD8 deficiency is an autosomal recessive form of severe combined immunodeficiency diseases characterized by the absence of CD8(+) T lymphocytes and impaired T cell functions. We identified two novel mis-sense mutations in the zap70 genes of a CD8-deficiency patient. One mutation (P80Q) affects a residue in an SH2 domain and another (M572L) in the kinase subdomain XI. Both mutations cause a degradation of ZAP70 protein in a temperature-sensitive manner through an ATP-dependent and proteasome-independent pathway. We further demonstrated that Cdc37, a protein kinase-specific chaperone, bound to M572L but not P80Q mutant and restored the expression of the M572L mutant when overexpressed. The restoration of M572L mutant by Cdc37 required the function of HSP90. These results indicate that Cdc37 in conjunction with HSP90 functions as a molecular chaperone for a temperature-sensitive kinase domain mutant of ZAP70.