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

Carbocyanine dye diS-C3(3) was repeatedly employed in monitoring the plasma membrane potential of yeast and other living cells. Four methods of measuring and evaluating probe fluorescence signal were used in different studies, based on following fluorescence parameters: fluorescence intensity emitted within a certain spectral interval, F(580)/F(560) fluorescence emission ratio, wavelength of emission spectrum maximum, and the ratio of respective fluorescence intensities corresponding to the diS-C3(3) bound to cytosolic macromolecules and remaining dissolved in the aqueous cell medium (i.e., unbound, or free). Here we show that data corresponding to the three latter spectral assessments of diS-C3(3) accumulation in cells is mutually convertible, which means that their alternative use cannot lead to ambiguities in the interpretation of the results of biological experiments. On the other hand, experiments based on the effortless measurements of fluorescence intensities should be interpreted cautiously because controversial results can be obtained, depending on the particular choice of cell-to-dye concentration ratio and emission wavelength.

• Keywords
• Fluorescent probe, Plasma membrane potential, Saccharomyces cerevisiae, Spectral analysis, Yeast,
• MeSH
• Fluorescent Dyes chemistry   MeSH
• Spectrometry, Fluorescence methods   MeSH
• Carbocyanines chemistry   MeSH
• Membrane Potentials * MeSH
• Saccharomyces cerevisiae chemistry   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 3,3'-dipropylthiacarbocyanine MeSH Browser
• Fluorescent Dyes MeSH
• Carbocyanines MeSH

Recently we introduced a fluorescent probe technique that makes possible to convert changes of equilibrium fluorescence spectra of 3,3'-dipropylthiadicarbocyanine, diS-C3(3), measured in yeast cell suspensions under defined conditions into underlying membrane potential differences, scaled in millivolts (Plasek et al. in J Bioenerg Biomembr 44: 559-569, 2012). The results presented in this paper disclose measurements of real early changes of plasma membrane potential induced by the increase of extracellular K(+), Na(+) and H(+) concentration in S. cerevisiae with and without added glucose as energy source. Whereas the wild type and the ∆tok1 mutant cells exhibited similar depolarization curves, mutant cells lacking the two Trk1,2 potassium transporters revealed a significantly decreased membrane depolarization by K(+), particularly at lower extracellular potassium concentration [K(+)]out. In the absence of external energy source plasma membrane depolarization by K(+) was almost linear. In the presence of glucose the depolarization curves exhibited an exponential character with increasing [K(+)]out. The plasma membrane depolarization by Na(+) was independent from the presence of Trk1,2 transporters. Contrary to K(+), Na(+) depolarized the plasma membrane stronger in the presence of glucose than in its absence. The pH induced depolarization exhibited a fairly linear relationship between the membrane potential and the pHo of cell suspensions, both in the wild type and the Δtrk1,2 mutant strains, when cells were energized by glucose. In the absence of glucose the depolarization curves showed a biphasic character with enhanced depolarization at lower pHo values.

• MeSH
• Cell Membrane metabolism   MeSH
• Potassium metabolism   MeSH
• Fluorescent Dyes chemistry   MeSH
• Fluorometry MeSH
• Cations, Monovalent metabolism   MeSH
• Hydrogen-Ion Concentration MeSH
• Membrane Potentials drug effects   MeSH
• Saccharomyces cerevisiae drug effects   metabolism   MeSH
• Sodium metabolism   MeSH
• Hydrogen metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Potassium MeSH
• Fluorescent Dyes MeSH
• Cations, Monovalent MeSH
• Sodium MeSH
• Hydrogen MeSH

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

The fluorescent dye 3,3'-dipropylthiadicarbocyanine, diS-C(3)(3), is a suitable probe to monitor real changes of plasma membrane potential in yeast cells which are too small for direct membrane potential measurements with microelectrodes. A method presented in this paper makes it possible to convert changes of equilibrium diS-C(3)(3) fluorescence spectra, measured in yeast cell suspensions under certain defined conditions, into underlying membrane potential differences, scaled in the units of millivolts. Spectral analysis of synchronously scanned diS-C(3)(3) fluorescence allows to assess the amount of dye accumulated in cells without otherwise necessary sample taking and following separation of cells from the medium. Moreover, membrane potential changes can be quantified without demanding calibration protocols. The applicability of this approach was demonstrated on the depolarization of Rhodotorula glutinis yeast cells upon acidification of cell suspensions and/or by increasing extracellular K(+) concentration.

• MeSH
• Fluorescent Dyes chemistry   MeSH
• Carbocyanines chemistry   MeSH
• Membrane Potentials physiology   MeSH
• Rhodotorula cytology   physiology   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• 3,3'-dipropylthiacarbocyanine MeSH Browser
• Fluorescent Dyes MeSH
• Carbocyanines MeSH

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.

• MeSH
• Amiodarone pharmacology   MeSH
• Fluorescence MeSH
• Immunoprecipitation MeSH
• Ion Transport MeSH
• Humans MeSH
• Membrane Proteins * MeSH
• Saccharomyces cerevisiae drug effects   physiology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Amiodarone MeSH
• Membrane Proteins * MeSH