Acetic acid-induced stress is a common challenge in natural environments and industrial bioprocesses, significantly affecting the growth and metabolic performance of Saccharomyces cerevisiae. The adaptive response and tolerance to this stress involves the activation of a complex network of molecular pathways. This study aims to delve deeper into these mechanisms in S. cerevisiae, particularly focusing on the role of the Hrk1 kinase. Hrk1 is a key determinant of acetic acid tolerance, belonging to the NPR/Hal family, whose members are implicated in the modulation of the activity of plasma membrane transporters that orchestrate nutrient uptake and ion homeostasis. The influence of Hrk1 on S. cerevisiae adaptation to acetic acid-induced stress was explored by employing a physiological approach based on previous phosphoproteomics analyses. The results from this study reflect the multifunctional roles of Hrk1 in maintaining proton and potassium homeostasis during different phases of acetic acid-stressed cultivation. Hrk1 is shown to play a role in the activation of plasma membrane H+-ATPase, maintaining pH homeostasis, and in the modulation of plasma membrane potential under acetic acid stressed cultivation. Potassium (K+) supplementation of the growth medium, particularly when provided at limiting concentrations, led to a notable improvement in acetic acid stress tolerance of the hrk1Δ strain. Moreover, abrogation of this kinase expression is shown to confer a physiological advantage to growth under K+ limitation also in the absence of acetic acid stress. The involvement of the alkali metal cation/H+ exchanger Nha1, another proposed molecular target of Hrk1, in improving yeast growth under K+ limitation or acetic acid stress, is proposed.

• Keywords
• NPR/Hal family, Nha1, Pma1 activity, Saccharomyces cerevisiae, acetic acid tolerance, plasma membrane H+-ATPase, yeast kinases,
• Publication type
• Journal Article MeSH

The alteration of the fine-tuned balance of phospho/dephosphorylation reactions in the cell often results in functional disturbance. In the yeast Saccharomyces cerevisiae, the overexpression of Ser/Thr phosphatase Ppz1 drastically blocks cell proliferation, with a profound change in the transcriptomic and phosphoproteomic profiles. While the deleterious effect on growth likely derives from the alteration of multiple targets, the precise mechanisms are still obscure. Ppz1 is a negative effector of potassium influx. However, we show that the toxic effect of Ppz1 overexpression is unrelated to the Trk1/2 high-affinity potassium importers. Cells overexpressing Ppz1 exhibit decreased K+ content, increased cytosolic acidification, and fail to properly acidify the medium. These effects, as well as the growth defect, are counteracted by the deletion of NHA1 gene, which encodes a plasma membrane Na+, K+/H+ antiporter. The beneficial effect of a lack of Nha1 on the growth vanishes as the pH of the medium approaches neutrality, is not eliminated by the expression of two non-functional Nha1 variants (D145N or D177N), and is exacerbated by a hyperactive Nha1 version (S481A). All our results show that high levels of Ppz1 overactivate Nha1, leading to an excessive entry of H+ and efflux of K+, which is detrimental for growth.

• Keywords
• K+ transport, Nha1, Ppz1 phosphatase, Saccharomyces cerevisiae, cation homeostasis, intracellular pH,
• Publication type
• Journal Article MeSH

There are only a few antifungal drugs used systemically in treatment, and invasive fungal infections that are resistant to these drugs are an emerging problem in health care. In this study, we performed a high-copy-number genomic DNA (gDNA) library screening to find and characterize genes that reduce susceptibility to amphotericin B, caspofungin, and voriconazole in Saccharomyces cerevisiae We identified the PDR16 and PMP3 genes for amphotericin B, the RMD9 and SWH1 genes for caspofungin, and the MRS3 and TRI1 genes for voriconazole. The deletion mutants for PDR16 and PMP3 were drug susceptible, but the other mutants had no apparent susceptibility. Quantitative-PCR analyses suggested that the corresponding drugs upregulated expression of the PDR16, PMP3, SWH1, and MRS3 genes. To further characterize these genes, we also profiled the global expression patterns of the cells after treatment with the antifungals and determined the genes and paths that were up- or downregulated. We also cloned Candida albicans homologs of the PDR16, PMP3, MRS3, and TRI1 genes and expressed them in S. cerevisiae Heterologous expression of Candida homologs also provided reduced drug susceptibility to the budding yeast cells. Our analyses suggest the involvement of new genes in antifungal drug resistance.

• Keywords
• amphotericin B, antifungal agents, caspofungin, drug resistance, genomics, multidrug resistance, voriconazole,
• MeSH
• Amphotericin B pharmacology   MeSH
• Antifungal Agents pharmacology   MeSH
• Candida albicans drug effects   genetics   metabolism   MeSH
• Drug Resistance, Fungal genetics   MeSH
• Caspofungin pharmacology   MeSH
• Microbial Sensitivity Tests MeSH
• Saccharomyces cerevisiae Proteins genetics   metabolism   MeSH
• Saccharomyces cerevisiae drug effects   genetics   metabolism   MeSH
• Saccharomycetales drug effects   genetics   metabolism   MeSH
• Voriconazole pharmacology   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Amphotericin B MeSH
• Antifungal Agents MeSH
• Caspofungin MeSH
• Saccharomyces cerevisiae Proteins MeSH
• Voriconazole MeSH

Candida glabrata is a haploid yeast that is considered to be an emergent pathogen since it is the second most prevalent cause of candidiasis. Contrary to most yeasts, this species carries only one plasma membrane potassium transporter named CgTrk1. We show in this work that the activity of this transporter is regulated at the posttranslational level, and thus Trk1 contributes to potassium uptake under very different external cation concentrations. In addition to its function in potassium uptake, we report a diversity of physiological effects related to this transporter. CgTRK1 contributes to proper cell size, intracellular pH and membrane-potential homeostasis when expressed in Saccharomyces cerevisiae. Moreover, lithium influx experiments performed both in C. glabrata and S. cerevisiae indicate that the salt tolerance phenotype linked to CgTrk1 can be related to a high capacity to discriminate between potassium and lithium (or sodium) during the transport process. In summary, we show that CgTRK1 exerts a diversity of pleiotropic physiological roles and we propose that the corresponding protein may be an attractive pharmacological target for the development of new antifungal drugs.

• Keywords
• Candida glabrata, Membrane potential, Potassium transport, Saccharomyces cerevisiae, Salt tolerance, Trk1,
• MeSH
• Cell Membrane metabolism   MeSH
• Candida glabrata genetics   metabolism   MeSH
• Potassium metabolism   MeSH
• Fungal Proteins genetics   metabolism   MeSH
• Homeostasis MeSH
• Hydrogen-Ion Concentration MeSH
• Cation Transport Proteins genetics   metabolism   MeSH
• Gene Expression Regulation, Fungal MeSH
• Sodium metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Potassium MeSH
• Fungal Proteins MeSH
• Cation Transport Proteins MeSH
• Sodium MeSH

Saccharomyces species, which are mostly used in the food and beverage industries, are known to differ in their fermentation efficiency and tolerance of adverse fermentation conditions. However, the basis of their difference has not been fully elucidated, although their genomes have been sequenced and analyzed. Five strains of four Saccharomyces species (S. cerevisiae, S. kudriavzevii, S. bayanus, and S. paradoxus), when grown in parallel in laboratory conditions, exhibit very similar basic physiological parameters such as membrane potential, intracellular pH, and the degree to which they are able to quickly activate their Pma1 H+-ATPase upon glucose addition. On the other hand, they differ in their ability to proliferate in media with a very low concentration of potassium, in their osmotolerance and tolerance to toxic cations and cationic drugs in a growth-medium specific manner, and in their capacity to survive anhydrobiosis. Overall, S. cerevisiae (T73 more than FL100) and S. paradoxus are the most robust, and S. kudriavzevii the most sensitive species. Our results suggest that the difference in stress survival is based on their ability to quickly accommodate their cell size and metabolism to changing environmental conditions and to adjust their portfolio of available detoxifying transporters.

• Keywords
• Intracellular pH, Membrane potential, Saccharomyces, Stress tolerance,
• MeSH
• Fermentation MeSH
• Fungal Proteins genetics   metabolism   MeSH
• Stress, Physiological MeSH
• Glucose metabolism   MeSH
• Proton-Translocating ATPases genetics   metabolism   MeSH
• Saccharomyces classification   genetics   growth & development   physiology   MeSH
• Publication type
• Journal Article MeSH
• Comparative Study MeSH
• Names of Substances
• Fungal Proteins MeSH
• Glucose MeSH
• Proton-Translocating ATPases MeSH

According to the common view, weak acid uncouplers increase proton conductance of biological (and phospholipid bilayer) membranes, thus effecting H+ fluxes driven by their electrochemical gradients. Under certain conditions, however, uncouplers can induce unexpected effects opposite to the dissipation of H+ gradients. Results are presented here demonstrating CCCP-induced proton influx into Saccharomyces cerevisiae cytosol driven by the electrochemical potentials of CCCP and its CCCP- anions, independent of electrochemical H+-gradient. Another view of week acid uncouplers' action is proposed that is logically consistent with these observations.

• Keywords
• CCCP, Electrochemical H+ gradient, Mode of action, Saccharomyces cerevisiae, Weak acid uncouplers, pHluorin,
• MeSH
• Biological Transport drug effects   MeSH
• Carbonyl Cyanide m-Chlorophenyl Hydrazone pharmacology   MeSH
• Hydrogen-Ion Concentration drug effects   MeSH
• Membrane Potentials * drug effects   MeSH
• Protons * MeSH
• Uncoupling Agents pharmacology   MeSH
• Saccharomyces cerevisiae metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Carbonyl Cyanide m-Chlorophenyl Hydrazone MeSH
• Protons * MeSH
• Uncoupling Agents MeSH

Yeast cells exhibit a negative surface potential due to negative charges at the cell membrane surface. Consequently, local concentrations of cations at the periplasmic membrane surface may be significantly increased compared to their bulk environment. However, in cell suspensions only bulk concentrations of cations can be measured directly. Here we present a novel method enabling the assessment of local pH at the periplasmic membrane surface which can be directly related to the underlying cell surface potential. In this proof of concept study using Saccharomyces cerevisiae cells with episomally expressed pH reporter, pHluorin, intracellular acidification induced by the addition of the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) was measured using synchronously scanned fluorescence spectroscopy (SSF). The analysis of titration curves revealed that the pH at the periplasmic surface of S. cerevisiae cells was about two units lower than the pH of bulk medium. This pH difference was significantly decreased by increasing the ionic strength of the bulk medium. The cell surface potential was estimated to amount to -130 mV. Comparable results were obtained also with another protonophore, pentachlorophenol (PCP).

• Keywords
• Cell surface potential, Cytosolic pH, Periplasmic pH, Saccharomyces cerevisiae, Yeast, pHluorin,
• MeSH
• Spectrometry, Fluorescence methods   MeSH
• Carbonyl Cyanide m-Chlorophenyl Hydrazone MeSH
• Hydrogen-Ion Concentration * MeSH
• Membrane Potentials * MeSH
• Methods MeSH
• Periplasm chemistry   MeSH
• Saccharomyces cerevisiae chemistry   cytology   MeSH
• Green Fluorescent Proteins MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Carbonyl Cyanide m-Chlorophenyl Hydrazone MeSH
• PHluorin MeSH Browser
• Green Fluorescent Proteins MeSH

The maintenance of potassium homeostasis is crucial for all types of cells, including Candida glabrata. Three types of plasma-membrane systems mediating potassium influx with different transport mechanisms have been described in yeasts: the Trk1 uniporter, the Hak cation-proton symporter and the Acu ATPase. The C. glabrata genome contains only one gene encoding putative system for potassium uptake, the Trk1 uniporter. Therefore, its importance in maintaining adequate levels of intracellular potassium appears to be critical for C. glabrata cells. In this study, we first confirmed the potassium-uptake activity of the identified gene's product by heterologous expression in a suitable S. cerevisiae mutant, further we generated a corresponding deletion mutant in C. glabrata and analysed its phenotype in detail. The obtained results show a pleiotropic effect on the cell physiology when CgTRK1 is deleted, affecting not only the ability of trk1Δ to grow at low potassium concentrations, but also the tolerance to toxic alkali-metal cations and cationic drugs, as well as the membrane potential and intracellular pH. Taken together, our results find the sole potassium uptake system in C. glabrata cells to be a promising target in the search for its specific inhibitors and in developing new antifungal drugs.

• MeSH
• Cell Membrane metabolism   MeSH
• Candida glabrata metabolism   physiology   MeSH
• Potassium metabolism   MeSH
• Homeostasis physiology   MeSH
• Ion Transport physiology   MeSH
• Cations metabolism   MeSH
• Membrane Potentials physiology   MeSH
• Cation Transport Proteins metabolism   MeSH
• Gene Expression Regulation, Fungal physiology   MeSH
• Saccharomyces cerevisiae metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Potassium MeSH
• Cations MeSH
• Cation Transport Proteins MeSH
• Trk1 protein, Candida albicans MeSH Browser

We investigated the impact of the deletions of genes from the final steps in the biosynthesis of ergosterol (ERG6, ERG2, ERG3, ERG5, ERG4) on the physiological function of the Saccharomyces cerevisiae plasma membrane by a combination of biological tests and the diS-C3(3) fluorescence assay. Most of the erg mutants were more sensitive than the wild type to salt stress or cationic drugs, their susceptibilities were proportional to the hyperpolarization of their plasma membranes. The different sterol composition of the plasma membrane played an important role in the short-term and long-term processes that accompanied the exposure of erg strains to a hyperosmotic stress (effect on cell size, pH homeostasis and survival of yeasts), as well as in the resistance of cells to antifungal drugs. The pleiotropic drug-sensitive phenotypes of erg strains were, to a large extent, a result of the reduced efficiency of the Pdr5 efflux pump, which was shown to be more sensitive to the sterol content of the plasma membrane than Snq2p. In summary, the erg4Δ and erg6Δ mutants exhibited the most compromised phenotypes. As Erg6p is not involved in the cholesterol biosynthetic pathway, it may become a target for a new generation of antifungal drugs.

• MeSH
• ATP-Binding Cassette Transporters genetics   metabolism   MeSH
• Antifungal Agents pharmacology   MeSH
• Biosynthetic Pathways genetics   MeSH
• Cell Membrane chemistry   physiology   MeSH
• Ergosterol biosynthesis   chemistry   MeSH
• Fluconazole pharmacology   MeSH
• Microscopy, Fluorescence MeSH
• Hydrogen-Ion Concentration MeSH
• Membrane Potentials physiology   MeSH
• Methyltransferases genetics   metabolism   MeSH
• Drug Resistance, Multiple, Fungal drug effects   genetics   physiology   MeSH
• Molecular Structure MeSH
• Mutation MeSH
• Saccharomyces cerevisiae Proteins genetics   metabolism   MeSH
• Saccharomyces cerevisiae chemistry   genetics   physiology   MeSH
• Salt Tolerance genetics   physiology   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• ATP-Binding Cassette Transporters MeSH
• Antifungal Agents MeSH
• delta 24-sterol methyltransferase MeSH Browser
• Ergosterol MeSH
• Fluconazole MeSH
• Methyltransferases MeSH
• PDR5 protein, S cerevisiae MeSH Browser
• Saccharomyces cerevisiae Proteins MeSH
• SNQ2 protein, S cerevisiae MeSH Browser

Three different transport systems exist to accumulate a sufficient amount of potassium cations in yeasts. The most common of these are Trk-type transporters, which are used by all yeast species. Though most yeast species employ two different types of transporters, we only identified one gene encoding a potassium uptake system (Trk-type) in the genome of the highly osmotolerant yeast Zygosaccharomyces rouxii, and our results showed that ZrTrk1 is its major (and probably only) specific potassium uptake system. When expressed in Saccharomyces cerevisiae, the product of the ZrTRK1 gene is localized to the plasma membrane and its presence efficiently complements the phenotypes of S. cerevisiae trk1∆ trk2∆ cells. Deletion of the ZrTRK1 gene resulted in Z. rouxii cells being almost incapable of growth at low K(+) concentrations and it changed some cell physiological parameters in a way that differs from S. cerevisiae. In contrast to S. cerevisiae, Z. rouxii cells without the TRK1 gene contained less potassium than the control cells and their plasma membrane was significantly hyperpolarized compared with those of the parental strain when grown in the presence of 100 mM KCl. On the other hand, subsequent potassium starvation led to a substantial depolarization which is again different from S. cerevisiae. Plasma-membrane hyperpolarization did not prevent the efflux of potassium from Z. rouxii trk1Δ cells during potassium starvation, and the activity of ZrPma1 is less affected by the absence of ZrTRK1 than in S. cerevisiae. The use of a newly constructed Z. rouxii-specific plasmid for the expression of pHluorin showed that the intracellular pH of the Z. rouxii wild type and the trk1∆ mutant is not significantly different. Together with the fact that Z. rouxii cells contain a significantly lower amount of intracellular potassium than identically grown S. cerevisiae cells, our results suggest that this highly osmotolerant yeast species maintain its intracellular pH and potassium homeostasis in way(s) partially distinct from S. cerevisiae.

• MeSH
• Adaptation, Biological MeSH
• Biological Transport MeSH
• Cell Membrane physiology   MeSH
• Gene Deletion MeSH
• DNA, Fungal genetics   metabolism   MeSH
• Potassium metabolism   MeSH
• Genes, Fungal * MeSH
• Homeostasis MeSH
• Homologous Recombination MeSH
• Hydrogen-Ion Concentration MeSH
• Membrane Potentials MeSH
• Cation Transport Proteins genetics   metabolism   MeSH
• Gene Expression Regulation, Fungal * MeSH
• Saccharomyces cerevisiae Proteins genetics   metabolism   MeSH
• Saccharomyces cerevisiae genetics   physiology   MeSH
• Amino Acid Sequence MeSH
• Sequence Homology MeSH
• Sequence Alignment MeSH
• Zygosaccharomyces genetics   physiology   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Fungal MeSH
• Potassium MeSH
• Cation Transport Proteins MeSH
• Saccharomyces cerevisiae Proteins MeSH
• TRK1 protein, S cerevisiae MeSH Browser