Summary Maintenance of intracellular K+ homeostasis is one of the crucial requisites for the survival of yeast cells. In Saccharomyces cerevisiae , the high K + content corresponds to a steady state between simultaneous influx and efflux across the plasma membrane. One of the transporters formerly believed to extrude K + from the yeast cells (besides Ena1-4p and Nha1p) was named Kha1p and presumed as a putative plasma membrane K + /H + antiporter. We prepared kha1 and tok1-kha1 deletion strains in the B31 and MAB 2d background. Both the strains contain the ena1-4 and nha1 deletions; that means they lack the main active sodium and potassium efflux systems. MAB 2d has additional trk1 and trk2 deletions, i.e. is impaired in active K + uptake as well. We performed a large physiological study with these strains to specify the phenotype of kha1 deletion. In our experiments, no difference in K + content or efflux was observed in strains lacking the KHA1 gene compared with control strains. Two main phenotype manifestations of the kha1 deletion were growth defect on high external pH and hygromycin sensitivity. The correlation between these phenotypes and the kha1 deletion was confirmed by plasmid complementation. Fluorescence microscopy of green fluorescent protein (GFP)-tagged Kha1p showed that this antiporter is localized preferentially intracellularly (in contrast to the plasma membrane Na + /H + antiporter Nha1p). Based on these findings, Kha1p is probably not localized in plasma membrane and does not mediate efflux of alkali metal cations from cells, but is important for the regulation of intracelular cation homeostasis and optimal pH control, similarly as the Nhx1p.

Cationic amphipathic drugs, such as amiodarone, interact preferentially with lipid membranes to exert their biological effect. In the yeast Saccharomyces cerevisiae, toxic levels of amiodarone trigger a rapid influx of Ca 2؉ that can overwhelm cellular homeostasis and lead to cell death. To better understand the mechanistic basis of antifungal activity, we assessed the effect of the drug on membrane potential. We show that low concentrations of amiodarone (0.1-2 M) elicit an immediate, dose-dependent hyperpolarization of the membrane. At higher doses (>3 M), hyperpolarization is transient and is followed by depolarization, coincident with influx of Ca 2؉ and H ؉ and loss in cell viability. Proton and alkali metal cation transporters play reciprocal roles in membrane polarization, depending on the availability of glucose. Diminishment of membrane potential by glucose removal or addition of salts or in pma1, tok1⌬, ena1-4⌬, or nha1⌬ mutants protected against drug toxicity, suggesting that initial hyperpolarization was important in the mechanism of antifungal activity. Furthermore, we show that the link between membrane hyperpolarization and drug toxicity is pHdependent. We propose the existence of pH-and hyperpolarization-activated Ca 2؉ channels in yeast, similar to those described in plant root hair and pollen tubes that are critical for cell elongation and growth. Our findings illustrate how membrane-active compounds can be effective microbicidals and may pave the way to developing membrane-selective agents.

Potassium homeostasis is crucial for living cells. In the yeast Saccharomyces cerevisiae, the uptake of potassium is driven by the electrochemical gradient generated by the Pma1 H ؉ -ATPase, and this process represents a major consumer of the gradient. We considered that any mutation resulting in an alteration of the electrochemical gradient could give rise to anomalous sensitivity to any cationic drug independently of its toxicity mechanism. Here, we describe a genomewide screen for mutants that present altered tolerance to hygromycin B, spermine, and tetramethylammonium. Two hundred twenty-six mutant strains displayed altered tolerance to all three drugs (202 hypersensitive and 24 hypertolerant), and more than 50% presented a strong or moderate growth defect at a limiting potassium concentration (1 mM). Functional groups such as protein kinases and phosphatases, intracellular trafficking, transcription, or cell cycle and DNA processing were enriched. Essentially, our screen has identified a substantial number of genes that were not previously described to play a direct or indirect role in potassium homeostasis. A subset of 27 representative mutants were selected and subjected to diverse biochemical tests that, in some cases, allowed us to postulate the basis for the observed phenotypes.

K+ is one of the cations (besides protons) whose transport across the plasma membrane is believed to contribute to the maintenance of membrane potential. To ensure K+ transport, Saccharomyces cerevisiae cells possess several types of active and passive transporters mediating the K+ influx and efflux, respectively. A diS-C3(3) assay was used to compare the contributions of various potassium transporters to the membrane potential changes of S. cerevisiae cells in the exponential growth phase. Altogether, the contributions of six K+ transporters to the maintenance of a stable membrane potential were tested. As confirmed by the observed hyperpolarization of trk1 trk2 deletion strains, the diS-C3(3) assay is a suitable method for comparative studies of the membrane potential of yeast strains differing in the presence/absence of one or more cation transporters. We have shown that the presence of the Tok1 channel strongly influences membrane potential: deletion of the TOK1 gene results in significant plasma membrane depolarization, whereas strains overexpressing the TOK1 gene are hyperpolarized. We have also proved that plasma membrane potential is not the only parameter determining the hygromycin B sensitivity of yeast cells, and that the role of intracellular transporters in protecting against its toxic effects must also be considered.

AbstractpHluorin is a pH-sensitive variant of green fluorescent protein for measuring intracellular pH (pH in ) in living cells. We constructed a new pHluorin plasmid with the dominant selection marker KanMX. This plasmid allows pH measurements in cells without auxotrophic mutations and/or grown in chemically indefinite media. We observed differing values of pH in for three prototrophic wild-types. The new construct was also used to determine the pH in in strains differing in the activity of the plasma membrane Pma1 H + -ATPase and the influence of glucose on pH in . We describe in detail pHluorin measurements performed in a microplate reader, which require much less hands-on time and much lower cell culture volumes compared to standard cuvettes measurements. We also utilized pHluorin in a new method of measuring the buffering capacity of yeast cell cytosol in vivo, shown to be ca. 52 mM/pH for wild-type yeast and moderately decreased in mutants with affected potassium transport.

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Microplate readers have been useful assistants of researchers for several decades. This work is focused on the applications of a simple absorbance microplate reader in yeast physiology research, and its advantages and limitations in comparison with alternative methods are discussed. The two main procedures involved are measuring growth curves and monitoring the pH changes of medium using two different pH indicators. We suggest mathematical formulas for converting absorbance data into pH values. With a microplate reader as many as 96 samples can be simultaneously analyzed, while medium consumption is minimized to 100 microL per sample. The results can be observed in 24-48 h (for growth curves) or in 1-3 h (for pH changes) with minimal hands-on time required.

The roles of intracellular GTPase Arl1 and organellar cation/H(+) antiporters (Kha1 and Nhx1) in Saccharomyces cerevisiae tolerance to various stress factors were investigated and interesting new phenotypes of strains devoid of these proteins were found. The role of Arl1 GTPase in their tolerance to various cations is not caused by an altered plasma-membrane potential. Besides the known sensitivity of arl1 mutants to high temperature, we discovered their sensitivity to low temperature. We found for the first time that in the absence of Arl1p, Kha1p increases potassium, sodium and lithium tolerance, and can thus be categorized as an antiporter with broad substrate specificity. Kha1p also participates in the detoxification of undesired chemical compounds, pH regulation and growth at nonoptimal temperatures. Cells with the combined deletions of all three genes have considerable difficulty growing under nonoptimal conditions. We conclude that Arl1p, Kha1p and Nhx1p collaborate in survival strategies at nonoptimal pH, temperatures and cation concentrations, but work independent of each other.