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Severe alkaline pH stress induces a glucose starvation-related transcriptional response in S. cerevisiae

ABSTRACT: Alkaline pH stress invokes in S. cerevisiae a potent and fast transcriptional response that includes many genes repressed by glucose. Certain mutants in the glucose-sensing and response pathways, such as those lacking the Snf1 kinase, are sensitive to alkalinization. We show that addition of glucose to the medium improves growth of wild type cells at high pH, fully abolish the snf1 alkali-sensitive phenotype and attenuates high pH-induced Snf1 phosphorylation at Thr210. The elm1 mutant, lacking one of the three upstream Snf1 kinases (tos3, elm1 and sak1), is markedly alkali sensitive, whereas the phenotype of the tos3 elm1 sak1 strain is even stronger than that of snf1 cells and it is not fully rescued by glucose supplementation. DNA microarray analysis reveals that about 75% of genes induced at short term by high pH are also induced by glucose scarcity. Snf1 mediates, in full or in part, the activation of a significant subset (38%) of short-term alkali-induced genes, including those coding high-affinity hexose transporters and phosphorylating enzymes. Induction of genes encoding enzymes involved in glycogen (but not trehalose) metabolism is largely dependent of the presence of Snf1. Therefore, the function of Snf1 in adaptation to glucose scarcity appears crucial for alkaline pH tolerance. Incorporation of micromolar amounts of iron and copper to a glucose-supplemented medium result in an additive effect and allows near normal growth at high pH, thus indicating that these three nutrients are key limiting factors for growth in an alkaline environment.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE25697 | GEO | 2012/03/02

SECONDARY ACCESSION(S): PRJNA135851

REPOSITORIES: GEO

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Project description:Exposure of Saccharomyces cerevisiae to alkaline pH represents a stress condition that generates a compensatory reaction. Here we examine a possible role of the protein kinase-A (PKA) pathway in this response. The phenotypic analysis reveals that mutations that activate the PKA pathway (ira1 ira2, bcy1) tend to cause sensitivity to alkaline pH, whereas its deactivation develops tolerance to this stress. We observe that alkalinization causes a transient decrease in cAMP, the main regulator of the pathway. Alkaline pH causes rapid nuclear localization of the PKA-regulated Msn2 transcription factor which, together with Msn4, mediates a general stress response by binding to STRE sequences in many promoters. Consequently, a synthetic STRE-LacZ reporter shows a rapid induction in response to alkaline stress. An msn2 msn4 mutant is sensitive to alkaline pH, and transcriptomic analysis reveals that after 10 minutes of alkaline stress, the expression of many induced genes (47%) depends, at least in part, on the presence of Msn2 and Msn4. Taken together, these results demonstrate that inhibition of the PKA pathway by alkaline pH represents a substantial part of the adaptive response to this kind of stress and that this response involves Msn2/Msn4-mediated gene remodeling. However, the relevance of attenuation of PKA in high pH tolerance is not restricted to regulation of Msn2 function. Eight samples were analyzed: WT and the MCY5278 mutant strain, lacking both Msn2 and Msn4, in the presence of 20 mM KOH (pH 8) and in the presence of 20 mM KCl (non-induced conditions) for 10 and 30 min of stress. 2 biological replicates were analyzed for each condition, and dye-swapping was carried out for each comparison of samples. We compared the expression profiles of: 1) WT +KOH vs. WT +KCl after 10 min 2) msn2 msn4 mutant +KOH vs. msn2 msn4 +KCl after 10 min 3)WT +KOH vs. WT +KCl after 30 min 4) msn2 msn4 mutant +KOH vs. msn2 msn4 +KCl after 30 min Total number of chips analyzed: 16.

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Severe alkaline pH stress induces a glucose starvation-related transcriptional response in S. cerevisiae

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