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 G 1 /S transition, increased levels of activated Mpk1 do not appear to be responsible for the cell cycle phenotype observed in Ppz-deficient strains.

Intracellular pH and K؉ concentrations must be tightly controlled because they affect many cellular activities, including cell growth and death. The mechanisms of homeostasis of H ؉ and K ؉ are only partially understood. In the yeast Saccharomyces cerevisiae, proton efflux is mediated by the Pma1 H ؉ -ATPase. As this pump is electrogenic, the activity of the Trk1 and -2 K ؉ uptake system is crucial for sustained Pma1p operation. The coordinated activities of these two systems determine cell volume, turgor, membrane potential, and pH. Genetic evidence indicates that Trk1p is activated by the Hal4 and -5 kinases and inhibited by the Ppz1 and -2 phosphatases, which, in turn, are inhibited by their regulatory subunit, Hal3p. We show that Trk1p, present in plasma membrane "rafts," physically interacts with Ppz1p, that Trk1p is phosphorylated in vivo, and that its level of phosphorylation increases in ppz1 and -2 mutants. Interestingly, both the interaction with and inhibition of Ppz1p by Hal3p are pH dependent. These results are consistent with a model in which the Ppz1-Hal3 interaction is a sensor of intracellular pH that modulates H ؉ and K ؉ homeostasis through the regulation of Trk1p activity.

K؉ transport in living cells must be tightly controlled because it affects basic physiological parameters such as turgor, membrane potential, ionic strength, and pH. In yeast, the major high-affinity K ؉ transporter, Trk1, is inhibited by high intracellular K ؉ levels and positively regulated by two redundant "halotolerance" protein kinases, Sat4/Hal4 and Hal5. Here we show that these kinases are not required for Trk1 activity; rather, they stabilize the transporter at the plasma membrane under low K ؉ conditions, preventing its endocytosis and vacuolar degradation. High concentrations (0.2 M) of K ؉ , but not Na ؉ or sorbitol, transported by undefined low-affinity systems, maintain Trk1 at the plasma membrane in the hal4 hal5 mutant. Other nutrient transporters, such as Can1 (arginine permease), Fur4 (uracil permease), and Hxt1 (low-affinity glucose permease), are also destabilized in the hal4 hal5 mutant under low K ؉ conditions and, in the case of Can1, are stabilized by high K ؉ concentrations. Other plasma membrane proteins such as Pma1 (H ؉ -pumping ATPase) and Sur7 (an eisosomal protein) are not regulated by halotolerance kinases or by high K ؉ levels. This novel regulatory mechanism of nutrient transporters may participate in the quiescence/growth transition and could result from effects of intracellular K ؉ and halotolerance kinases on membrane trafficking and/or on the transporters themselves.