Response of the Saccharomyces cerevisiae Mpk1 mitogen-activated protein kinase pathway to increases in internal turgor pressure caused by loss of Ppz protein phosphatases

Abstract

The Mpk1 pathway of Saccharomyces cerevisiae is a key determinant of cell wall integrity. A genetic link between the Mpk1 kinase and the Ppz phosphatases has been reported, but the nature of this connection was unclear. Recently, the Ppz phosphatases were shown to be regulators of K+ and pH homeostasis. Here, we demonstrate that Ppz-deficient strains display increased steady-state K+ levels and sensitivity to increased KCl concentrations. Given these observations and the fact that K+ is the major determinant of intracellular turgor pressure, we reasoned that the connection between PPZ1 and -2 and MPK1 was due to the combination of increased internal turgor pressure in Ppz-deficient strains and cell wall instability observed in strains lacking MPK1. Accordingly, the MPK1 gene was up-regulated, the Mpk1 protein was overexpressed, and the phosphorylated active form was more abundant in Ppz-deficient strains. Moreover, the expression of genes previously identified as targets of the Mpk1 pathway are also up-regulated in strains lacking PPZ1 and -2. The transcriptional and posttranslational modifications of Mpk1 were not observed when the internal K+ concentration (and thus turgor pressure) was lowered by disrupting the TRK1 and -2 K+ transporter genes or when the cell wall was stabilized by the addition of sorbitol. Moreover, we present genetic evidence showing that both the Wsc1 and Mid2 branches of the Mpk1 pathway contribute to this response. Finally, despite its role in G1/S transition, increased levels of activated Mpk1 do not appear to be responsible for the cell cycle phenotype observed in Ppz-deficient strains.

FIG. 1.

Analysis of intracellular accumulation of K⁺, KCl toxicity, and cell size in strains lacking PPZ1 and -2. (A) The indicated strains (W303-1A, LY139, LY140, and WΔ3) were grown to mid-log phase in either rich or minimal medium, as indicated, and processed for HPLC analysis of K⁺ content as described previously (28). Data are averages of three separate measurements; error bars, standard deviations. Similar results were observed in three different experiments. (B) The indicated strains (W303-1A, LY139, LY140, WΔ3, DBY746, LY83, LY85, and LY102) were grown to saturation in selective media, serially diluted in sterile water, and spotted onto YPD plates containing the indicated amount of KCl or sorbitol. Images were taken after 2 to 3 days of incubation at 28°C. Identical results were obtained in two separate experiments. (C) The indicated strains (as in panel A) were grown to early-log phase, and cell size was determined as described in Materials and Methods. Data are averages from three independent colonies of each genotype. Similar results were observed in two separate experiments.

FIG. 2.

Northern and Western blot analyses of Mpk1 expression and phosphorylation. (A) The indicated strains (W303-1A, LY139, LY140, WΔ3, and LY190) were grown to mid-log phase in complete media supplemented where indicated with 1 M sorbitol (SORB). Northern blot analysis was performed as described in Materials and Methods by using the indicated probes. TBP1 was used as an internal loading control. (B) The same strains described above were grown to mid-log phase in complete media supplemented with the indicated amount of sorbitol, and immunoblots were prepared as described in Materials and Methods. The upper panel represents an immunoblot using an antiserum raised against Mpk1, and the center panel represents an immunoblot with a monoclonal antibody specific for the dually phosphorylated form of Mpk1 (Mpk1*). Similar results were observed in three separate experiments. The Ponceau S staining of the membrane is shown in the bottom panel as a control for protein loading.

FIG. 3.

Target genes of the Mpk1 pathway are up-regulated in Ppz-deficient strains. The indicated strains (W303-1A, LY139, LY140, WΔ3, and LY190) were grown to mid-log phase in complete media supplemented, where indicated, with 1 M sorbitol (SORB). Northern blot analysis was performed as described in Materials and Methods by using the indicated probes. TBP1 was used as an internal loading control.

FIG. 4.

The KCl sensitivity of the ppz1 ppz2 strain is exacerbated by the further disruption of MPK1. The indicated strains (W303-1A, LY190, LY139, and LY196) were grown to saturation in YPD plus 1 M sorbitol (SORB.), serially diluted in sterile 1 M sorbitol, and spotted onto YPD plates containing the indicated amount of KCl or sorbitol. Images were taken after 2 to 3 days of incubation at 28°C. Identical results were obtained in two separate experiments. Identical results were also observed in minimal media (data not shown).

FIG. 5.

Disruption of either WSC1 or MID2 exacerbates the lytic phenotype of the ppz1 mutant. The indicated strains (W303-1A, LY191, LY192, LY190, LY165, LY194, LY195, LY193, LY139, LY197, LY198, and LY196) were grown to saturation in YPD medium supplemented with 1 M sorbitol (Sorb.), serially diluted in sterile 1 M sorbitol, and spotted onto YPD plates containing the indicated amount of sorbitol. Images were taken after 2 to 3 days of incubation at 28 or 37°C. Identical results were obtained in two separate experiments.

FIG. 6.

The lytic phenotype of the ppz1 ppz2 mutant is relieved by further disruption of TRK1 and -2. The indicated strains (W303-1A, LY139, LY140, WΔ3, and LY190) were grown to saturation in rich media, serially diluted in sterile water, and spotted onto YPD plates. Images were taken after 2 to 3 days of incubation at 28 or 37°C. Identical results were obtained in two separate experiments.

FIG. 7.

Strains lacking PPZ1 and -2 continue to display accelerated α-factor recovery in the presence of sorbitol. (A) Strain LY139 was grown to mid-log phase in YPD medium containing the indicated amounts of sorbitol (SORB), processed as described in Materials and Methods. Nitrocellulose membranes were immunoblotted with the anti-Mpk1 antibody (upper panel) and the phosphospecific anti-Mpk1 antibody (lower panel). Similar results were observed in two different experiments. (B) The indicated strains (W303-1A and LY139) were grown to an optical density of 0.4 at 660 nm in complete medium containing the indicated concentration of sorbitol, and the assay was performed as described in Materials and Methods. Images of the plates were taken after 48 h. Identical results were observed in two separate experiments.