Nuclear magnetic resonance investigation of water transport through the plasma membrane of various yeast species.

A specific technique of nuclear magnetic resonance (NMR) spectroscopy, filter-exchange spectroscopy (FEXSY), was employed to investigate water transport through the plasma membrane in intact yeast cells. This technique allows water transport to be monitored directly, thus avoiding the necessity to subject the cells to any rapid change in the external conditions, e.g. osmotic shock. We established a sample preparation protocol, a data analysis procedure and verified the applicability of FEXSY experiments. We recorded the exchange rates in the temperature range 10-40°C for Saccharomyces cerevisiae. The resulting activation energy of 29 kJ mol-1 supports the hypothesis that water exchange is facilitated by water channels-aquaporins. Furthermore, we measured for the first time water exchange rates in three other phylogenetically unrelated yeast species (Schizosaccharomyces pombe, Candida albicans and Zygosaccharomyces rouxii) and observed remarkably different water exchange rates between these species. Findings of our work contribute to a better understanding of as fundamental a cell process as the control of water transport through the plasma membrane.

Potassium uptake systems of Candida krusei.

Candida krusei is a pathogenic yeast species that is phylogenetically outside both of the well-studied yeast groups, whole genome duplication and CUG. Like all other yeast species, it needs to accumulate high amounts of potassium cations, which are needed for proliferation and many other cell functions. A search in the sequenced genomes of nine C. krusei strains revealed the existence of two highly conserved genes encoding putative potassium uptake systems. Both of them belong to the TRK family, whose members have been found in all the sequenced genomes of species from the Saccharomycetales subclade. Analysis and comparison of the two C. krusei Trk sequences revealed all the typical features of yeast Trk proteins but also an unusual extension of the CkTrk2 hydrophilic N-terminus. The expression of both putative CkTRK genes in Saccharomyces cerevisiae lacking its own potassium importers showed that only CkTrk1 is able to complement the absence of S. cerevisiae's own transporters and provide cells with a sufficient amount of potassium. Interestingly, a portion of the CkTrk1 molecules were localized to the vacuolar membrane. The presence of CkTrk2 had no evident phenotype, due to the fact that this protein was not correctly targeted to the S. cerevisiae plasma membrane. Thus, CkTrk2 is the first studied yeast Trk protein to date that was not properly recognized and targeted to the plasma membrane upon heterologous expression in S. cerevisiae.

Osmotolerance of Dekkera bruxellensis and the role of two Stl glycerol-proton symporters.

Dekkera bruxellensis is important for lambic beer fermentation but is considered a spoilage yeast in wine fermentation. We compared two D. bruxellensis strains isolated from wine and found that they differ in some basic properties, including osmotolerance. The genomes of both strains contain two highly similar copies of genes encoding putative glycerol-proton symporters from the STL family that are important for yeast osmotolerance. Cloning of the two DbSTL genes and their expression in suitable osmosensitive Saccharomyces cerevisiae mutants revealed that both identified genes encode functional glycerol uptake systems, but only DbStl2 has the capacity to improve the osmotolerance of S. cerevisiae cells.

Three Candida albicans potassium uptake systems differ in their ability to provide Saccharomyces cerevisiae trk1trk2 mutants with necessary potassium.

Yeasts usually have one or two high-affinity potassium transporters. Two complete and one interrupted gene encoding three types of putative potassium uptake system exist in Candida albicans SC5314. As high intracellular potassium is essential for many yeast cell functions, the existence of three transporters with differing transport mechanisms (Trk uniporter, Hak cation-proton symporter, Acu ATPase) may help pathogenic C. albicans cells to acquire the necessary potassium in various organs and tissues of the host. When expressed in Saccharomyces cerevisiae lacking their own potassium uptake systems, all three putative transporters were able to provide cells with the ability to grow with low amounts of potassium over a broad range of external pH. Only CaTrk1 was properly recognized and secreted to the plasma membrane. Nevertheless, even the small number of CaHak1 and mainly CaAcu1 molecules which reached the plasma membrane resulted in an improved growth of cells in low potassium concentrations, suggesting a high affinity and capacity of the transporters. A single-point mutation restored the complete CaACU1 gene, and the resulting protein not only provided cells with the necessary potassium but also improved their tolerance to toxic lithium. In contrast to its known homologues, CaAcu1 did not seem to transport sodium.

Fluconazole affects the alkali-metal-cation homeostasis and susceptibility to cationic toxic compounds of Candida glabrata.

Candida glabrata is a salt-tolerant and fluconazole (FLC)-resistant yeast species. Here, we analyse the contribution of plasma-membrane alkali-metal-cation exporters, a cation/proton antiporter and a cation ATPase to cation homeostasis and the maintenance of membrane potential (ΔΨ). Using a series of single and double mutants lacking CNH1 and/or ENA1 genes we show that the inability to export potassium and toxic alkali-metal cations leads to a slight hyperpolarization of the plasma membrane of C. glabrata cells; this hyperpolarization drives more cations into the cells and affects cation homeostasis. Surprisingly, a much higher hyperpolarization of C. glabrata plasma membrane was produced by incubating cells with subinhibitory concentrations of FLC. FLC treatment resulted in a substantially increased sensitivity of cells to various cationic drugs and toxic cations that are driven into the cell by negative-inside plasma-membrane potential. The effect of the combination of FLC plus cationic drug treatment was enhanced by the malfunction of alkali-metal-cation transporters that contribute to the regulation of membrane potential and cation homeostasis. In summary, we show that the combination of subinhibitory concentrations of FLC and cationic drugs strongly affects the growth of C. glabrata cells.

Fluconazole treatment hyperpolarizes the plasma membrane of Candida cells.

Five pathogenic Candida species were compared in terms of their osmotolerance, tolerance to toxic sodium and lithium cations, and resistance to fluconazole. The species not only differed, in general, in their tolerance to high osmotic pressure (C. albicans and C. parapsilosis being the most osmotolerant) but exhibited distinct sensitivities to toxic sodium and lithium cations, with C. parapsilosis and C. tropicalis being very tolerant but C. krusei and C. dubliniensis sensitive to LiCl. The treatment of both fluconazole-susceptible (C. albicans and C. parapsilosis) and fluconazole-resistant (C. dubliniensis, C. krusei and C. tropicalis) growing cells with subinhibitory concentrations of fluconazole resulted in substantially elevated intracellular Na(+) levels. Using a diS-C3(3) assay, for the first time, to monitor the relative membrane potential (ΔΨ) of Candida cells, we show that the fluconazole treatment of growing cells of all five species results in a substantial hyperpolarization of their plasma membranes, which is responsible for an increased non-specific transport of toxic alkali metal cations and other cationic drugs (e.g., hygromycin B). Thus, the combination of relatively low doses of fluconazole and drugs, whose import into the tested Candida strains is driven by the cell membrane potential, might be especially potent in terms of its ability to inhibit the growth of or even kill various Candida species.