Comparison of the influence of small GTPases Arl1 and Ypt6 on yeast cells' tolerance to various stress factors.

The GTPases Arl1 and Ypt6 are involved in the intracellular transport of vesicles and their fusion with the trans-Golgi network. This work is focused on comparing the roles of these GTPases in the tolerance of Saccharomyces cerevisiae cells to an increased concentration of alkali metal cations and other stress factors. We studied the phenotypes of arl1 or ypt6 deletions in combination with the deletions of genes encoding alkali-metal-cation transporters (ena1-4, nha1, nhx1, and kha1). Salt sensitivity of the arl1 and ypt6 mutants was shown to be independent of the tested cation transporters and electrochemical membrane potential. Phenotype manifestations of ypt6 deletion were usually more prominent than those of arl1 (cells were more sensitive to KCl, NaCl, LiCl, hygromycin B, increased temperature, and increased pH). At suboptimal temperature, the growth inhibition of arl1 and ypt6 mutants was approximately the same, and low pH was the only condition where arl1 mutants grew even worse than ypt6 mutants. Overexpression of the ARL1 gene suppressed the phenotypes of ypt6 deletion; however, this did not work vice versa (additional copies of YPT6 could not replace ARL1). Our results suggest partially overlapping functions of the GTPases in resistance to various stress factors, with Ypt6 being more efficient under physiological conditions and Arl1 more versatile when overexpressed.

A genomewide screen for tolerance to cationic drugs reveals genes important for potassium homeostasis in Saccharomyces cerevisiae.

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.

Genetic interactions among the Arl1 GTPase and intracellular Na(+) /H(+) antiporters in pH homeostasis and cation detoxification.

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.

New applications of pHluorin--measuring intracellular pH of prototrophic yeasts and determining changes in the buffering capacity of strains with affected potassium homeostasis.

pHluorin 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.

Membrane hyperpolarization drives cation influx and fungicidal activity of amiodarone.

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 microm) elicit an immediate, dose-dependent hyperpolarization of the membrane. At higher doses (>3 microm), 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, tok1Delta, ena1-4Delta, or nha1Delta 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 pH-dependent. 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.

Applications of a microplate reader in yeast physiology research.

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.

Arabidopsis thaliana CHX17 gene complements the kha1 deletion phenotypes in Saccharomyces cerevisiae.

AtChx17p is a putative K(+)/H(+) exchanger from Arabidopsis thaliana, expressed in the roots and probably involved in K(+) acquisition and homeostasis. AtCHX17 cDNA complements the phenotypes of the kha1Delta mutation in S. cerevisiae cells: a growth defect at increased pH and hygromycin sensitivity. The localization of GFP-tagged AtChx17 protein in yeast cells is similar to that of ScKha1p: a bold dotted pattern inside the cells resembling the Golgi fluorescence markers. These results show that (a) the proteins AtChx17 and ScKha1 could have similar functions and (b) S. cerevisiae kha1 deletion mutants could serve for the heterologous expression and characterization of plant transporters. The results of this work are evidence that a S. cerevisiae strain with deletions of genes encoding alkali-metal-cation/H(+) antiporters (i.e. Nha1p, Nhx1p, Kha1p) could be an ideal tool for expression and functional analysis of any type of similar plant antiporters (plasma membrane, endosomal/prevacuolar and Golgi).

Measurements of plasma membrane potential changes in Saccharomyces cerevisiae cells reveal the importance of the Tok1 channel in membrane potential maintenance.

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.

Physiological characterization of Saccharomyces cerevisiae kha1 deletion mutants.

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 intracellular cation homeostasis and optimal pH control, similarly as the Nhx1p.