Characterization of the Calcium-mediated Response to Alkaline Stress in Saccharomyces cerevisiae

Abstract

Exposure of the yeast Saccharomyces cerevisiae to alkaline stress resulted in adaptive changes that involved remodeling the gene expression. Recent evidence suggested that the calcium-activated protein phosphatase calcineurin could play a role in alkaline stress signaling. By using an aequorin luminescence reporter, we showed that alkaline stress resulted in a sharp and transient rise in cytoplasmic calcium. This increase was largely abolished by addition of EGTA to the medium or in cells lacking Mid1 or Cch1, components of the high affinity cell membrane calcium channel. Under these circumstances, the alkaline response of different calcineurin-sensitive transcriptional promoters was also blocked. Therefore, exposure to alkali resulted in entry of calcium from the external medium, and this triggered a calcineurin-mediated response. The involvement of calcineurin and Crz1/Tcn1, the transcription factor activated by the phosphatase, in the transcriptional response triggered by alkalinization has been globally assessed by DNA microarray analysis in a time course experiment using calcineurin-deficient (cnb1) and crz1 mutants. We found that exposure to pH 8.0 increased at least 2-fold the mRNA levels of 266 genes. In many cases (60%) the response was rather early (peak after 10 min). The transcriptional response of 27 induced genes (10%) was reduced or fully abolished in cnb1 cells. In general, the response of crz1 mutants was similar to that of calcineurin-deficient cells. By analysis of a systematic deletion library, we found 48 genes whose mutation resulted in increased sensitivity to the calcineurin inhibitor FK506. Twenty of these mutations (42%) also provoked alkaline pH sensitivity. In conclusion, our results demonstrated that calcium signaling and calcineurin activation represented a significant component of the yeast response to environmental alkalinization.Calcium-mediated signaling mechanisms are used by virtually every eukaryotic cell to regulate a wide variety or cellular processes, including gene expression. Transient increases in cytosolic calcium results in activation of diverse enzymes, such as the protein phosphatase calcineurin. Calcineurin is a heterodimer of catalytic subunit and regulatory subunits. In the yeast Saccharomyces cerevisiae, the catalytic subunit is encoded by two genes, CNA1 and CNA2 (1), whereas a single gene, CNB1, encodes the regulatory subunit (2). Cells lacking the catalytic subunits, or the regulatory subunit, are deficient in calcineurin activity.Exposure of yeast cells to a number of signals, such as ␣-factor (3, 4), glucose (5), sphingosine (6), and certain stress conditions (7-9), triggers a rise in cytoplasmic calcium. This increase in calcium can be a consequence of external calcium influx or release from internal stores, such as the vacuole, and results in activation of calcineurin. For instance, hyperosmotic shock has been reported to provoke calcium release from vacuolar stores (8) through Yvc1, a member of the transient receptor potential channel family, and to trig...

Signaling Alkaline pH Stress in the Yeast Saccharomyces cerevisiae through the Wsc1 Cell Surface Sensor and the Slt2 MAPK Pathway

“…Interestingly, the gene encoding the alternative 1,3-␤-glucan synthase subunit (GSC2/FKS2) is induced by high pH, whereas the gas1 mutant (lacking ␤-1,3-glucanosyltransferase activity) is sensitive to high pH. In contrast, CHS1 and CHS3, which encode two of the three chitin synthases in yeast and which are responsible for most of this catalytic activity, are induced by exposure to Congo red, but not to high pH (10,34). These results reinforce the notion that Wsc1 acts as a sensor of high pH and suggest that alkalinization of the environment may cause cell wall damage by affecting glucan integrity or organization.…”

“…Transcriptional analyses were performed using DNA microarrays containing PCR-amplified fragments from 6014 S. cerevisiae open reading frames (10,26). Fluorescent Cy3-and Cy5-labeled cDNA probes were prepared from 8 g of purified total RNA by the indirect dUTP labeling method using a CyScribe post-labeling kit (Amersham Biosciences).…”

“…Correlation between the transcriptional responses to high pH stress and cell wall-damaging drugs or specific mutations. A table was generated with the expression levels of genes induced by the indicated treatments or in specific mutants, which was crossed with the list of genes induced by severe alkaline stress as defined in a previous work (10). This produced a final list of 29 genes.…”

“…Activation of the calcineurin/Crz1 pathway has relevance in high pH adaptation, as deduced from the alkali-sensitive phenotype of cnb1 mutants. About 10% of the alkali-induced transcriptional response is partially or fully dependent on the activation of this pathway (8,10). Analysis of the transcriptional profile after alkaline stress also reveals that alkalinization of the medium probably affects the availability of different nutrients, such as phosphate, and that of specific cations, such as copper and iron (6,8,12,13).…”

“…In addition to the Rim101 pathway, adaptation to external high pH involves the activation of the calcium-activated phosphatase calcineurin (8 -11). It has been shown that exposure to alkali triggers a strong and transient intracellular calcium burst (10), which results in activation of calcineurin and the calcineurin-regulated transcription factor Crz1/Tcn1. Activation of the calcineurin/Crz1 pathway has relevance in high pH adaptation, as deduced from the alkali-sensitive phenotype of cnb1 mutants.…”