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.

G-quadruplexes form in guanine-rich regions of DNA and the presence of these structures at telomeres prevents the activity of telomerase in vitro. Ligands such as the cationic porphyrin TMPyP4 stabilise G-quadruplexes and are therefore under investigation for their potential use as anti-cancer drugs. In order to investigate the mechanism of action of TMPyP4 in vivo, we carried out a genome-wide screen in the budding yeast Saccharomyces cerevisiae. We found that deletion of key pentose phosphate pathway (PPP) genes increased the sensitivity of yeast to the presence of TMPyP4. The PPP plays an important role in the oxidative stress response and sensitivity to TMPyP4 also increased when genes involved in the oxidative stress response, CCS1 and YAP1, were deleted. For comparison we also report genome wide-screens using hydrogen peroxide, which causes oxidative stress, RHPS4, another G-quadruplex binder and hydroxyurea, an S phase poison. We found that a number of TMPyP4-sensitive strains are also sensitive to hydrogen peroxide in a genome-wide screen. Overall our results suggest that treatment with TMPyP4 results in light-dependent oxidative stress response in budding yeast, and that this, rather than G-quadruplex binding, is the major route to cytotoxicity. Our results have implications for the usefulness and mechanism of action of TMPyP4.

We have investigated the effects of alterations in potassium homeostasis on cell cycle progression and genome stability in Saccharomyces cerevisiae. Yeast strains lacking the PPZ1 and PPZ2 phosphatase genes, which aberrantly accumulate potassium, are sensitive to agents causing replicative stress or DNA damage and present a cell cycle delay in the G 1 ⁄ S phase. A synthetic slow growth phenotype was identified in a subset of DNA repair mutants upon inhibition of Ppz activity. Moreover, we observe that this slow growth phenotype observed in cdc7 ts mutants with reduced Ppz activity is reverted by disrupting the TRK1 potassium transporter gene. As over-expression of a mammalian potassium transporter leads to similar phenotypes, we conclude that these defects can be attributed to potassium accumulation. As we reported previously, internal potassium accumulation activates the Slt2 MAP kinase pathway. We show that the removal of SLT2 in ppz1 ppz2 mutants ameliorates sensitivity to agents causing replication stress and DNA damage, whereas over-activation of the pathway leads to similar cell cyclerelated defects. Taken together, these results are consistent with inappropriate potassium accumulation reducing DNA replication efficiency, negatively influencing DNA integrity and leading to the requirement of mismatch repair, the MRX complex, or homologous recombination pathways for normal growth.

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