RESUMO
In Saccharomyces cerevisiae the transcription of the ENA1 gene is modulated by multiple transduction pathways that respond to osmotic, ionic and nutrient stresses. We have investigated the molecular mechanisms involved in ENA1 induction by the calcium-calcineurin-activated transcription factor Crzl/Tcn1. We found in the ENA1 promoter a calcium-responsive, Crzl-dependent upstream activating region (UASENA1) located between -713 bp and 826 bp relative to the translation start. This region contains two separate control elements: the upstream element (5'-GAATGGCTG-3') between -813 and -821 binds Crzlp with lower affinity and mostly contributes to basal ENA1 expression, whereas the downstream element (5'-GGGTGGCTG-3') between 727 and 719 binds Crz1p with higher affinity and is a major determinant of the induction response to calcium.
Assuntos
Adenosina Trifosfatases/genética , Proteínas de Transporte de Cátions , Regiões Promotoras Genéticas/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Transativadores/genética , Sequência de Bases , Calcineurina/genética , Proteínas de Ligação a DNA , Regulação Fúngica da Expressão Gênica , Dados de Sequência Molecular , ATPase Trocadora de Sódio-Potássio , Fatores de Transcrição , Ativação TranscricionalRESUMO
Four putative yeast transcription factors (Hal6-9p) have been identified which upon overexpression in multicopy plasmids increase sodium and lithium tolerance. This effect is mediated, at least in part, by increased expression of the Enalp Na+/Li+ extrusion pump. Hal6p and Hal7p are bZIP proteins and their gene disruptions affected neither salt tolerance nor ENA1 expression. Hal8p and Hal9p are putative zinc fingers and their gene disruptions decreased both salt tolerance and ENA1 expression. Therefore, Hal8p and Hal9p, but not Hal6p and Hal7p, qualify as transcriptional activators of ENA1 under physiological conditions. Hal8p seems to mediate the calcineurin-dependent part of ENA1 expression.
Assuntos
Proteínas de Transporte de Cátions , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Lítio/farmacologia , Proteínas de Saccharomyces cerevisiae , Sódio/farmacologia , Fatores de Transcrição/genética , Adaptação Fisiológica , Adenosina Trifosfatases/genética , Sequência de Aminoácidos , Divisão Celular , Mapeamento Cromossômico , Meios de Cultura , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Zíper de Leucina , Dados de Sequência Molecular , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , ATPase Trocadora de Sódio-Potássio , Fatores de Transcrição/metabolismoRESUMO
We have isolated a novel yeast gene, HAL1, which upon overexpression improves growth under salt stress. In addition, disruption of this gene decreases salt tolerance. Therefore HAL1 constitutes a rate-limiting determinant for halotolerance. It encodes a polar protein of 32 kDa located in the yeast cytoplasm and unrelated to sequences in data banks. The expression of this gene is increased by high concentrations of either NaCl, KCl or sorbitol. On the other hand, the growth advantage obtained by overexpression of HAL1 is specific for NaCl stress. In cells overexpressing HAL1, sodium toxicity seems to be counteracted by an increased accumulation of potassium. The HAL1 protein could interact with the transport systems which determine intracellular K+ homeostasis. The HAL1 gene and encoded protein are conserved in plants, being induced in these organisms by salt stress and abscisic acid. These results suggest that yeast serves as a convenient model system for the molecular biology of plant salt tolerance.
Assuntos
Proteínas Fúngicas/genética , Genes Fúngicos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Sais/farmacologia , Sequência de Aminoácidos , Sequência de Bases , Transporte Biológico , Divisão Celular/efeitos dos fármacos , Clonagem Molecular , Citoplasma/metabolismo , DNA Fúngico , Proteínas Fúngicas/biossíntese , Regulação Fúngica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular , Dados de Sequência Molecular , Mutagênese , Plantas/efeitos dos fármacos , Plantas/genética , Cloreto de Potássio/farmacologia , RNA Mensageiro/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/fisiologia , Homologia de Sequência do Ácido Nucleico , Cloreto de Sódio/farmacologia , Sorbitol/farmacologiaRESUMO
The function of the amino- and carboxyl-terminal domains of the yeast plasma membrane H+-ATPase have been investigated by constructing deletions in vitro and selectively expressing the mutant enzymes in vivo. The first 27 amino acids are dispensable but deletion of a further 33 amino acids greatly decreases the appearance of the enzyme in the plasma membrane. Membrane localization is also prevented by carboxyl-terminal deletions which include the last hydrophobic stretch, but the last 46 amino acids of the ATPase are not required. Removal of the last 11 amino acids produces an enzyme in glucose-starved cells with the kinetic parameters of the wild-type ATPase activated by glucose fermentation. This region seems to constitute a regulatory domain.
