The Transcriptional Response of the Yeast Na+-ATPase ENA1 Gene to Alkaline Stress Involves Three Main Signaling Pathways

Abstract

Adaptive response of the yeast Saccharomyces cerevisiae to environmental alkalinization results in remodeling of gene expression. A key target is the gene ENA1, encoding a Na ؉ -ATPase, whose induction by alkaline pH has been shown to involve calcineurin and the Rim101/Nrg1 pathway. Previous functional analysis of the ENA1 promoter revealed a calcineurin-independent pH responsive region (ARR2, 83 nucleotides). We restrict here this response to a small (42 nucleotides) ARR2 5-region, named MCIR (minimum calcineurin independent response), which contains a MIG element, able to bind Mig1,2 repressors. High pH-induced response driven from this region was largely abolished in snf1 cells and moderately reduced in a rim101 strain. Cells lacking Mig1 or Mig2 repressors had a near wild type response, but the double mutant presented a high level of expression upon alkaline stress. Deletion of NRG1 (but not of NRG2) resulted in increased expression. Induction from the MCIR region was marginal in a quadruple mutant lacking Nrg1,2 and Mig1,2 repressors. In vitro band shift experiments demonstrated binding of Nrg1 to the 5 end of the ARR2 region. Furthermore, we show that Nrg1 binds in vivo around the MCIR region under standard growth conditions, and that binding is largely abolished after high pH stress. Therefore, the calcineurin-independent response of the ENA1 gene is under the regulation of Rim101 (through Nrg1) and Snf1 (through Nrg1 and Mig2). Accordingly, induction by alkaline stress of the entire ENA1 promoter in a snf1 rim101 mutant in the presence of the calcineurin inhibitor FK506 is completely abolished. Thus, the transcriptional response to alkaline stress of the ENA1 gene integrates three different signaling pathways.

The exceptionality of stress response in Magnaporthe oryzae: a set of “salt stress-induced” genes unique to the rice blast fungus

“…We found that the genes MGG_01822 (MoHOG1) and MGG_10268 (MoPBS2) were significantly upregulated when subjected to salt stress, which is in accordance with functions published already regarding osmotic stress regulation either in yeast [9] or M. oryzae [4,10]. Further evidence is given by genes predicted to encode homologues to the yeast ATPase ENA2 (YDR039C), which is involved in salt tolerance [8,16] or even a group of genes involved in stress-induced transport mechanisms (Tab. S1).…”

Nsf1/Ypl230w participates in transcriptional activation during non-fermentative growth and in response to salt stress in Saccharomyces cerevisiae

“…The involvement of Snf1 and Nrg1 in the transcriptional regulation of ENA1, a gene that is known to respond to saline and alkaline stress (Alepuz et al, 1997;Lamb et al, 2001;Platara et al, 2006), was suggestive of saline-related transcriptional regulation of NSF1. Interestingly, the transcription of NSF1 has been reported to increase slightly when cells are treated with 0.8 M NaCl (Yoshimoto et al, 2002).…”

“…The transcription of ENA1 is activated under nonfermentable carbon conditions, saline stress and alkaline stress (Alepuz et al, 1997;Cyert, 2003;Lamb et al, 2001;Platara et al, 2006). To analyse the potential involvement of Nsf1 in the transcriptional activation of ENA1, we cultured pKC201 (ENA1-lacZ) transformants of the wild-type and nsf1D mutant strains in selective synthetic glucose media to exponential phase before shifting the cells to salt-stress conditions (see Methods).…”

“…The TOR (via Gln3/Ure2) (Crespo et al, 2001), Snf1 (via Mig1/Mig2 and Nrg1/Nrg2) (Platara et al, 2006), Rim101 (via Nrg1/Nrg2) (Lamb et al, 2001;Lamb & Mitchell, 2003;Platara et al, 2006) and the calcineurin pathways all converge on the ENA1 promoter (Cyert, 2003;Haro et al, 1991;Ruiz & Arino, 2007). The TOR and Snf1 pathways respond to nutrient availability, and the calcineurin pathway responds to intracellular ion concentrations, while Rim101 and Snf1 also regulate the yeast response to alkaline stress.…”

Assessing Differential Expression Measurements by Highly Parallel Pyrosequencing and DNA Microarrays: A Comparative Study

“…To this end, we have chosen a well-established perturbation model, the exposure of yeast cells to an abrupt increase in external pH. Previous DNA microarray-based analyses of the effect of this perturbation on gene expression remodeling have shown that it results in several hundred of genes being induced or repressed (Lamb and Mitchell, 2003;Lamb et al, 2001;Platara et al, 2006;Ruiz et al, 2008;Serrano et al, 2002Serrano et al, , 2006Viladevall et al, 2004), as a result of the modulation of diverse signaling pathways (for a recent review, see Arino, 2010). In this work we present a comparison of the transcriptional changes detected after short-term (10 min) exposure to high pH stress (pH = 8.0) for both wildtype and snf1 mutant strains.…”