RESUMEN
Msn2p and the partially redundant factor Msn4p are key regulators of stress-responsive gene expression in Saccharomyces cerevisiae. They are required for the transcription of a number of genes coding for proteins with stress-protective functions. Both Msn2p and Msn4p are Cys2His2 zinc finger proteins and bind to the stress response element (STRE). In vivo footprinting studies show that the occupation of STREs is enhanced in stressed cells and dependent on the presence of Msn2p and Msn4p. Both factors accumulate in the nucleus under stress conditions, such as heat shock, osmotic stress, carbon-source starvation, and in the presence of ethanol or sorbate. Stress-induced nuclear localization was found to be rapid, reversible, and independent of protein synthesis. Nuclear localization of Msn2p and Msn4p was shown to be correlated inversely to cAMP levels and protein kinase A (PKA) activity. A region with significant homologies shared between Msn2p and Msn4p is sufficient to confer stress-regulated localization to a SV40-NLS-GFP fusion protein. Serine to alanine or aspartate substitutions in a conserved PKA consensus site abolished cAMP-driven nuclear export and cytoplasmic localization in unstressed cells. We propose stress and cAMP-regulated intracellular localization of Msn2p to be a key step in STRE-dependent transcription and in the general stress response.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Saccharomyces cerevisiae , Factores de Transcripción/metabolismo , Dedos de Zinc , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Transporte Biológico/efectos de los fármacos , Compartimento Celular/efectos de los fármacos , AMP Cíclico/farmacología , Cisteína , ADN de Hongos/metabolismo , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Histidina , Datos de Secuencia Molecular , Unión Proteica , Saccharomyces cerevisiae , Homología de Secuencia de Aminoácido , Transducción de Señal , Factores de TiempoRESUMEN
Expression of invertase in the yeast Saccharomyces cerevisiae is greatly delayed when derepression occurs in a medium that lacks a usable carbon source. The delay is not a consequence of defects in the transcription of the SUC2 gene but is due to the impossibility of translating the normal levels of mRNA generated under derepressing conditions. The inhibition of translation in the absence of glucose has to be considered when reporter genes such as E. coli lacZ are used to measure transcription in conditions of carbon source starvation.
Asunto(s)
Glicósido Hidrolasas/genética , Biosíntesis de Proteínas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transcripción Genética , Northern Blotting , División Celular , Medios de Cultivo , Proteínas Fúngicas/biosíntesis , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Genes Reporteros , Glucosa/metabolismo , Glicósido Hidrolasas/biosíntesis , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , beta-Fructofuranosidasa , beta-Galactosidasa/biosíntesis , beta-Galactosidasa/genéticaRESUMEN
The MSN2 and MSN4 genes encode homologous and functionally redundant Cys2His2 zinc finger proteins. A disruption of both MSN2 and MSN4 genes results in a higher sensitivity to different stresses, including carbon source starvation, heat shock and severe osmotic and oxidative stresses. We show that MSN2 and MSN4 are required for activation of several yeast genes such as CTT1, DDR2 and HSP12, whose induction is mediated through stress-response elements (STREs). Msn2p and Msn4p are important factors for the stress-induced activation of STRE dependent promoters and bind specifically to STRE-containing oligonucleotides. Our results suggest that MSN2 and MSN4 encode a DNA-binding component of the stress responsive system and it is likely that they act as positive transcription factors.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción , Transcripción Genética , Secuencia de Bases , Calor , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Presión Osmótica , Estrés Oxidativo , Unión Proteica , Saccharomyces cerevisiae/genética , Dedos de ZincRESUMEN
An epidermal-growth-factor(EGF)-receptor preparation isolated by calmodulin-affinity chromatography from rat liver plasma membranes is able to phosphorylate calmodulin. Calmodulin phosphorylation was enhanced 3-8-fold by EGF, was dependent on the presence of a polycation or basic protein and was inhibited by micromolar concentrations of Ca2+. Phosphate incorporation into calmodulin occurs predominantly on tyrosine residues. Partial proteolysis of phosphocalmodulin by thrombin identifies Tyr99, located in the third calcium-binding domain of calmodulin, as the phosphorylated residue. Stoichiometric measurements show a 32P/calmodulin molar ratio of approximately 1 when optimal phosphorylation conditions are used.