Regulation of ENA1 Na(+)-ATPase gene expression by the Ppz1 protein phosphatase is mediated by the calcineurin pathway

Abstract

Saccharomyces cerevisiae strains lacking the Ppz1 protein phosphatase are salt tolerant and display increased expression of the ENA1 Na(+)-ATPase gene, a major determinant for sodium extrusion, while cells devoid of the similar Ppz2 protein do not show these phenotypes. However, a ppz1 ppz2 mutant displays higher levels of ENA1 expression than the ppz1 strain. We show here that the increased activity of the ENA1 promoter in a ppz1 ppz2 mutant maps to two regions: one region located at -751 to -667, containing a calcineurin-dependent response element (CDRE), and one downstream region (-573 to -490) whose activity responds to intracellular alkalinization. In contrast, the increased ENA1 expression in a ppz1 mutant is mediated solely by an intact calcineurin/Crz1 signaling pathway, on the basis that (i) this effect maps to a single region that contains the CDRE and (ii) it is blocked by the calcineurin inhibitor FK506, as well as by deletion of the CNB1 or CRZ1 gene. The calcineurin dependence of the increased ENA1 expression of a ppz1 mutant would suggest that Ppz1 could negatively regulate calcineurin activity. In agreement with this notion, a ppz1 strain is calcium sensitive, and this mutation does not result in a decrease in the calcium hypertolerance of a cnb1 mutant. It has been shown that ENA1 can be induced by alkalinization of the medium and that a ppz1 ppz2 strain has a higher intracellular pH. However, we present several lines of evidence that show that the gene expression profile of a ppz1 mutant does not involve an alkalinization effect. In conclusion, we have identified a novel role for calcineurin, but not alkalinization, in the control of ENA1 expression in ppz1 mutants.

FIG. 1.

Effects of overexpression of Hal3 on ENA1 expression in different Ppz backgrounds. (Left) Strains JA100 (wild type [WT]), EDN75 (ppz1), JA103 (ppz2), and EDN76 (ppz1 ppz2) bearing plasmid pKC201 (2), which encodes the β-galactosidase reporter gene fused to the ENA1 promoter, were transformed with the high-copy-number plasmid YEplac181 carrying no insert (open bars) or the same plasmid carrying the entire HAL3 gene. Cells were grown as described in Materials and Methods, and β-galactosidase activity was measured. Data are means ± standard errors of the means from three to nine independent clones. (Right) The catalytic domains of both Ppz1 and Ppz2 were purified from E. coli as GST fusion proteins. Approximately 3 μg of purified protein was then used as an affinity resin to copurify Hal3 from crude yeast extracts. The material retained by the resin after extensive washing was then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and probed with antibodies specific for Hal3. The bottom panel shows the Ponceau S staining of the membrane, as a control for protein loading. The upper panel shows the result of Western blot analysis with Hal3-specific antibodies. Lane a, Hal3 purified by using the catalytic domain of Ppz2; lane b, Hal3 purified by using the catalytic domain of Ppz1). No Hal3 was detected by using GST alone as an affinity resin (lane c).

FIG. 2.

Mapping the activity of the ENA1 promoter in ppz1 and ppz1 ppz2 mutants. Strains JA100 (wild type), EDN75 (ppz1), and EDN76 (ppz1 ppz2) were transformed with the indicated constructs. (Bottom) Relevant regulatory elements described in the main text are schematically depicted. Rectangles represent the fragments of the ENA1 promoter contained in the constructs, and numbers indicate their relative nucleotide positions (from the initiating Met codon). (Top) Data represent the ratio of β-galactosidase activity between each mutant and the isogenic wild-type strain in the absence (open bars) or presence (solid bars) of 1.5 μg of FK506/ml. Each data point corresponds to the mean ± standard error of the mean from three to nine independent clones.

FIG. 3.

Regulation of ENA1 expression by Ppz1 requires the integrity of the calcineurin-Crz1 pathway. (Left) Strain DBY746 (wild type [WT]) and the indicated isogenic derivatives were transformed with plasmid pKC201, and β-galactosidase activity was measured. Data are means ± standard errors of the means from 6 to 12 independent clones. (Right) Strains DBY746 (wild type) (open bars), EDN2 (ppz1) (filled bars), and EDN85 (ppz1 ppz2) (hatched bars) were transformed with plasmid pLA (containing the −732-to-−711 CDRE found in the ENA1 promoter as the only regulatory element), pAMS366 (bearing the wild-type CDRE found in the FKS2 promoter), or pAMS364 (bearing a mutated, nonfunctional version of the CDRE in pAMS366). Data are means ± standard errors of the means of β-galactosidase activities measured from three to nine independent clones.

FIG. 4.

Effects on tolerance to calcium, lithium, and sodium ions of mutation of CNB1 and CRZ1 in a ppz1 background. (A) Strains DBY746 (wild type) (▵), EDN2 (ppz1) (▴), EDN85 (ppz1 ppz2) (▪), MAR15 (cnb1) (○), and MAR19 (ppz1 cnb1) (•) were tested for growth in liquid cultures at the indicated concentrations of CaCl₂. Data are expressed as the percentage of growth relative to that in cultures without added salt. Each data point is the mean ± standard error of the mean from four independent cultures. (B) Cultures (OD₆₆₀, 0.05 and 0.01) of the indicated strains were spotted onto YPD plates containing the indicated concentrations of LiCl or NaCl. Growth was monitored after incubation at 28°C for 60 h.

FIG. 5.

Lack of Ppz1 does not result in increased ENA1 expression in an Mck1 kinase-deficient background. The indicated strains were transformed with plasmid pKC201, and β-galactosidase activity was measured. Data are means ± standard errors of the means from six independent clones. WT, wild type.

FIG. 6.

Effects of calcineurin inhibition on the expression levels of different alkali-inducible genes in wild-type and Ppz-deficient backgrounds. Strains JA100 (wild type) (open bars), EDN75 (ppz1) (filled bars), JA103 (ppz2) (hatched bars), and EDN76 (ppz1 ppz2) (stippled bars) were transformed with plasmid pKC201, pPHO84, pPHO89, or pPHO12. Cultures were incubated in the presence (1.5 μg/ml) or absence of FK506 as described in Materials and Methods, and β-galactosidase activity was measured. Data are means ± standard errors of the means from 3 to 12 independent clones.

FIG. 7.

Sensitivities of Ppz-regulatable regions of the ENA1 promoter to different levels of alkalinization. The wild-type strain DBY746 was transformed with the indicated constructs bearing the regions of the ENA1 promoter depicted schematically at the bottom. Cells were exposed to the indicated pH for 60 min and then processed for β-galactosidase activity measurement. The responses of each construct are expressed as the ratio of expression between each pH tested and the lowest pH used in the experiment (6.3) (left graph) and as the percentage of the maximal response (right graph). Data are means ± standard errors of the means from three independent clones.

FIG. 8.

Effects of the absence of Ppz1 on the expression of ENA1 and on salt tolerance in a Trk-deficient background. (A) The wild-type (WT) strain DBY746 and its derivatives EDN2 (ppz1), ESV212 (trk1 trk2), and MAR37 (ppz1 trk1 trk2) were transformed with plasmid pKC201 and cultured in the presence or absence of FK506. β-Galactosidase activity was measured, and data are presented as means ± standard errors of the means from six independent clones. (B) Strains ESV212 (solid lines) and MAR37 (dashed lines) were tested for growth in liquid cultures at the indicated concentrations of LiCl or NaCl in the absence (solid symbols) or presence (open symbols) of 1.5 μg of FK506/ml. Data, expressed as percentages of the growth of cultures without added salt, are means ± standard errors of the means from three independent cultures. (C) The indicated strains were grown in the presence of 0.3 M NaCl, extracts were prepared as described in Materials and Methods, and the concentrations of potassium (open bars) and sodium (solid bars) were measured. Data, expressed as nanomoles per milligram of cells (fresh weight), are means ± standard errors of the means from two independent experiments, performed in duplicate.

FIG. 9.

Schematic depiction of the relationship of Hal3, Ppz1, and Ppz2 with the calcineurin-dependent pathway and the transcriptional regulation of the ENA1 gene. “X” represents the protein phosphatase(s) that dephosphorylates Rcn1, and “Y” represents the protein kinase that phosphorylates Crz1. Discontinuous lines indicate functional relationships whose mechanisms have yet to be clarified.