73 results on '"Sychrova H"'
Search Results
2. Determination of a specific region of the purine–cytosine permease involved in the recognition of its substrates
- Author
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Bloch, J. C., primary, Sychrova, H., additional, Souciet, J. L., additional, Jund, R., additional, and Chevallier, M. R., additional
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- 1992
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Catalog
3. The Zygosaccharomyces rouxii strain CBS732 contains only one copy of the HOG1 and the SOD2 genes
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Kinclova, O., Potier, S., and Sychrova, H.
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- 2001
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4. Biogenesis of Candida albicans Can1 permease expressed in Saccharomyces cerevisiae
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Matijekova, A. and Sychrova, H.
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- 1997
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5. Characterization of the NHA1 gene encoding a Na^+/H^+-antiporter of the yeast Saccharomyces cerevisiae
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Prior, C., Potier, S., Souciet, J.-L., and Sychrova, H.
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- 1996
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6. Functional comparison of plasma-membrane Na+/H+ antiporters from two pathogenic Candida species
- Author
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Sychrova Hana and Krauke Yannick
- Subjects
Microbiology ,QR1-502 - Abstract
Abstract Background The virulence of Candida species depends on many environmental conditions. Extracellular pH and concentration of alkali metal cations belong among important factors. Nevertheless, the contribution of transporters mediating the exchange of alkali metal cations for protons across the plasma membrane to the cell salt tolerance and other physiological properties of various Candida species has not been studied so far. Results The tolerance/sensitivity of four pathogenic Candida species to alkali metal cations was tested and the role of one of the cation transporters in that tolerance (presumed to be the plasma-membrane Na+/H+ antiporter) was studied. The genes encoding these antiporters in the most and least salt sensitive species, C. dubliniensis and C. parapsilosis respectively, were identified, cloned and functionally expressed in the plasma membranes of Saccharomyces cerevisiae cells lacking their own cation exporters. Both CpCnh1 and CdCnh1 antiporters had broad substrate specificity and transported Na+, K+, Li+, and Rb+. Their activity in S. cerevisiae cells differed; CpCnh1p provided cells with a much higher salt tolerance than the CdCnh1 antiporter. The observed difference in activity was confirmed by direct measurements of sodium and potassium efflux mediated by these antiporters. Conclusion We have cloned two genes encoding putative Na+/H+ antiporters in C. parapsilosis and C. dubliniensis, and characterized the transport properties of encoded proteins. Our results show that the activity of plasma-membrane Na+/H+ antiporters is one of the factors determining the tolerance of pathogenic Candida species to high external concentrations of alkali metal cations. more...
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- 2008
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7. Distinct regions of its first intracellular loop contribute to the proper localization, transport activity and substrate-affinity adjustment of the main yeast K + importer Trk1.
- Author
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Papouskova K, Zimmermannova O, and Sychrova H
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- Biological Transport, Saccharomyces cerevisiae Proteins metabolism, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins chemistry, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae genetics, Cation Transport Proteins metabolism, Cation Transport Proteins genetics, Cation Transport Proteins chemistry, Potassium metabolism
- Abstract
Trk1 is the main K
+ importer of Saccharomyces cerevisiae. Its proper functioning enables yeast cells to grow in environments with micromolar amounts of K+superscript> . Although the structure of Trk1 has not been experimentally determined, the transporter is predicted to be composed of four MPM (transmembrane segment - pore loop - transmembrane segment) motifs which are connected by intracellular loops. Of those, in particular the first loop (IL1) is unique in its length; it forms more than half of the entire protein. The deletion of the majority of IL1 does not abolish the transport activity of Trk1. However IL1 is thought to be involved in the modulation of the transporter's functioning. In this work, we prepared a series of internally shortened versions of Trk1 that lacked various parts of IL1, and we studied their properties in S. cerevisiae cells without chromosomal copies of TRK genes. Using this approach, we were able to determine that both N- and C-border regions of IL1 are necessary for the proper localization of Trk1. Moreover, the N-border part of IL1 is also important for the functioning of Trk1, as its absence resulted in a decrease in the transporter's substrate affinity. In addition, in the internal part of IL1, we newly identified a stretch of amino-acid residues that are indispensable for retaining the transporter's maximum velocity, and another region whose deletion affected the ability of Trk1 to adjust its affinity in response to external levels of K + ., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.) more...- Published
- 2024
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8. The superior growth of Kluyveromyces marxianus at very low potassium concentrations is enabled by the high-affinity potassium transporter Hak1.
- Author
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Papouskova K, Akinola J, Ruiz-Castilla FJ, Morrissey JP, Ramos J, and Sychrova H
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- Fungal Proteins genetics, Fungal Proteins metabolism, Gene Knockout Techniques, Hydrogen-Ion Concentration, Gene Expression Regulation, Fungal, Membrane Potentials, Kluyveromyces genetics, Kluyveromyces metabolism, Kluyveromyces growth & development, Potassium metabolism, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae growth & development, Cation Transport Proteins genetics, Cation Transport Proteins metabolism
- Abstract
The non-conventional yeast Kluyveromyces marxianus has recently emerged as a promising candidate for many food, environment, and biotechnology applications. This yeast is thermotolerant and has robust growth under many adverse conditions. Here, we show that its ability to grow under potassium-limiting conditions is much better than that of Saccharomyces cerevisiae, suggesting a very efficient and high-affinity potassium uptake system(s) in this species. The K. marxianus genome contains two genes for putative potassium transporters: KmHAK1 and KmTRK1. To characterize the products of the two genes, we constructed single and double knock-out mutants in K. marxianus and also expressed both genes in an S. cerevisiae mutant, that lacks potassium importers. Our results in K. marxianus and S. cerevisiae revealed that both genes encode efficient high-affinity potassium transporters, contributing to potassium homeostasis and maintaining plasma-membrane potential and cytosolic pH. In K. marxianus, the presence of HAK1 supports growth at low K+ much better than that of TRK1, probably because the substrate affinity of KmHak1 is about 10-fold higher than that of KmTrk1, and its expression is induced ~80-fold upon potassium starvation. KmHak1 is crucial for salt stress survival in both K. marxianus and S. cerevisiae. In co-expression experiments with ScTrk1 and ScTrk2, its robustness contributes to an increased tolerance of S. cerevisiae cells to sodium and lithium salt stress., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.) more...
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- 2024
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9. Shaping microbiology for 75 years: highlights of research published in Microbiology . Part 2 - Communities and evolution.
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Brockhurst M, Cavet J, Diggle SP, Grainger D, Mangenelli R, Sychrova H, Martin-Verstraete I, Welch M, Palmer T, and Thomas GH
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- Microbiology, Soil Microbiology, Bacteria
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- 2023
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10. Shaping microbiology for 75 years: highlights of research published in Microbiology . Part 1 - Physiology and growth.
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Brockhurst M, Cavet J, Diggle SP, Grainger D, Mangenelli R, Sychrova H, Martin-Verstraete I, Welch M, Palmer T, and Thomas GH
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- Microbiology, Publications, Biological Phenomena
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- 2023
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11. Allosteric links between the hydrophilic N-terminus and transmembrane core of human Na + /H + antiporter NHA2.
- Author
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Velázquez D, Průša V, Masrati G, Yariv E, Sychrova H, Ben-Tal N, and Zimmermannova O
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- Humans, Amino Acid Sequence, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Protons, Sodium-Hydrogen Exchangers chemistry, Sodium-Hydrogen Exchangers genetics
- Abstract
The human Na
+ /H+ antiporter NHA2 (SLC9B2) transports Na+ or Li+ across the plasma membrane in exchange for protons, and is implicated in various pathologies. It is a 537 amino acids protein with an 82 residues long hydrophilic cytoplasmic N-terminus followed by a transmembrane part comprising 14 transmembrane helices. We optimized the functional expression of HsNHA2 in the plasma membrane of a salt-sensitive Saccharomyces cerevisiae strain and characterized in vivo a set of mutated or truncated versions of HsNHA2 in terms of their substrate specificity, transport activity, localization, and protein stability. We identified a highly conserved proline 246, located in the core of the protein, as being crucial for ion selectivity. The replacement of P246 with serine or threonine resulted in antiporters with altered substrate specificity that were not only highly active at acidic pH 4.0 (like the native antiporter), but also at neutral pH. P246T/S versions also exhibited increased resistance to the HsNHA2-specific inhibitor phloretin. We experimentally proved that a putative salt bridge between E215 and R432 is important for antiporter function, but also structural integrity. Truncations of the first 50-70 residues of the N-terminus doubled the transport activity of HsNHA2, while changes in the charge at positions E47, E56, K57, or K58 decreased the antiporter's transport activity. Thus, the hydrophilic N-terminal part of the protein appears to allosterically auto-inhibit cation transport of HsNHA2. Our data also show this in vivo approach to be useful for a rapid screening of SNP's effect on HsNHA2 activity., (© 2022 The Protein Society.) more...- Published
- 2022
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12. K+-specific importers Trk1 and Trk2 play different roles in Ca2+ homeostasis and signalling in Saccharomyces cerevisiae cells.
- Author
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Zimmermannova O, Felcmanova K, Sacka L, Colinet AS, Morsomme P, and Sychrova H
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- Calcium pharmacology, Cation Transport Proteins genetics, Cinnamates pharmacology, Hygromycin B analogs & derivatives, Hygromycin B pharmacology, Potassium Chloride pharmacology, Saccharomyces cerevisiae drug effects, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins genetics, Calcium metabolism, Cation Transport Proteins metabolism, Homeostasis, Potassium metabolism, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins metabolism, Signal Transduction
- Abstract
The maintenance of K+ and Ca2+ homeostasis is crucial for many cellular functions. Potassium is accumulated in cells at high concentrations, while the cytosolic level of calcium, to ensure its signalling function, is kept at low levels and transiently increases in response to stresses. We examined Ca2+ homeostasis and Ca2+ signalling in Saccharomyces cerevisiae strains lacking plasma-membrane K+ influx (Trk1 and Trk2) or efflux (Tok1, Nha1 and Ena1-5) systems. The lack of K+ exporters slightly increased the cytosolic Ca2+, but did not alter the Ca2+ tolerance or Ca2+-stress response. In contrast, the K+-importers Trk1 and Trk2 play important and distinct roles in the maintenance of Ca2+ homeostasis. The presence of Trk1 was vital mainly for the growth of cells in the presence of high extracellular Ca2+, whilst the lack of Trk2 doubled steady-state intracellular Ca2+ levels. The absence of both K+ importers highly increased the Ca2+ response to osmotic or CaCl2 stresses and altered the balance between Ca2+ flux from external media and intracellular compartments. In addition, we found Trk2 to be important for the tolerance to high KCl and hygromycin B in cells growing on minimal media. All the data describe new interconnections between potassium and calcium homeostasis in S. cerevisiae., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS.) more...
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- 2021
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13. Regulation and activity of CaTrk1, CaAcu1 and CaHak1, the three plasma membrane potassium transporters in Candida albicans.
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Ruiz-Castilla FJ, Bieber J, Caro G, Michán C, Sychrova H, and Ramos J
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- Candida albicans genetics, Cation Transport Proteins genetics, Cell Membrane genetics, Fungal Proteins genetics, Humans, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Candida albicans metabolism, Cation Transport Proteins metabolism, Cell Membrane metabolism, Fungal Proteins metabolism, Potassium metabolism
- Abstract
Wild-type cells of Candida albicans, the most common human fungal pathogen, are able to grow at very low micromolar concentrations of potassium in the external milieu. One of the reasons behind that behaviour is the existence of three different types of K
+ transporters in their plasma membrane: Trk1, Acu1 and Hak1. This work shows that the transporters are very differently regulated at the transcriptional level upon exposure to saline stress, pH alterations or K+ starvation. We propose that different transporters take the lead in the diverse environmental conditions, Trk1 being the "house-keeping" one, and Acu1/Hak1 dominating upon K+ limiting conditions. Heterologous expression of the genes coding for the three transporters in a Saccharomyces cerevisiae strain lacking its endogenous potassium transporters showed that all of them mediated cation transport but with very different efficiencies. Moreover, expression of the transporters in S. cerevisiae also affected other physiological characteristics such as sodium and lithium tolerance, membrane potential or intracellular pH, being, in general, CaTrk1 the most effective in keeping these parameters close to the usual wild-type physiological levels., (Copyright © 2020 Elsevier B.V. All rights reserved.) more...- Published
- 2021
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14. C5 conserved region of hydrophilic C-terminal part of Saccharomyces cerevisiae Nha1 antiporter determines its requirement of Erv14 COPII cargo receptor for plasma-membrane targeting.
- Author
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Papouskova K, Moravcova M, Masrati G, Ben-Tal N, Sychrova H, and Zimmermannova O
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- Antiporters genetics, Antiporters metabolism, COP-Coated Vesicles genetics, COP-Coated Vesicles metabolism, Cation Transport Proteins metabolism, Cell Membrane metabolism, Endoplasmic Reticulum metabolism, Membrane Proteins physiology, Protein Transport, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins physiology, Sodium metabolism, Membrane Proteins metabolism, Saccharomyces cerevisiae Proteins metabolism
- Abstract
Erv14, a conserved cargo receptor of COPII vesicles, helps the proper trafficking of many but not all transporters to the yeast plasma membrane, for example, three out of five alkali-metal-cation transporters in Saccharomyces cerevisiae. Among them, the Nha1 cation/proton antiporter, which participates in cell cation and pH homeostasis, is a large membrane protein (985 aa) possessing a long hydrophilic C-terminus (552 aa) containing six conserved regions (C1-C6) with unknown function. A short Nha1 version, lacking almost the entire C-terminus, still binds to Erv14 but does not need it to be targeted to the plasma membrane. Comparing the localization and function of ScNha1 variants shortened at its C-terminus in cells with or without Erv14 reveals that only ScNha1 versions possessing the complete C5 region are dependent on Erv14. In addition, our broad evolutionary conservation analysis of fungal Na
+ /H+ antiporters identified new conserved regions in their C-termini, and our experiments newly show C5 and other, so far unknown, regions of the C-terminus, to be involved in the functionality and substrate specificity of ScNha1. Taken together, our results reveal that also relatively small hydrophilic parts of some yeast membrane proteins underlie their need to interact with the Erv14 cargo receptor., (© 2020 John Wiley & Sons Ltd.) more...- Published
- 2021
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15. A new pH sensor localized in the Golgi apparatus of Saccharomyces cerevisiae reveals unexpected roles of Vph1p and Stv1p isoforms.
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Deschamps A, Colinet AS, Zimmermannova O, Sychrova H, and Morsomme P
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- Chemical Engineering, Hydrogen-Ion Concentration, Isoenzymes chemistry, Saccharomyces cerevisiae enzymology, Biosensing Techniques instrumentation, Golgi Apparatus chemistry, Saccharomyces cerevisiae chemistry, Saccharomyces cerevisiae Proteins chemistry, Vacuolar Proton-Translocating ATPases chemistry
- Abstract
The gradual acidification of the secretory pathway is conserved and extremely important for eukaryotic cells, but until now there was no pH sensor available to monitor the pH of the early Golgi apparatus in Saccharomyces cerevisiae. Therefore, we developed a pHluorin-based sensor for in vivo measurements in the lumen of the Golgi. By using this new tool we show that the cis- and medial-Golgi pH is equal to 6.6-6.7 in wild type cells during exponential phase. As expected, V-ATPase inactivation results in a near neutral Golgi pH. We also uncover that surprisingly Vph1p isoform of the V-ATPase is prevalent to Stv1p for Golgi acidification. Additionally, we observe that during changes of the cytosolic pH, the Golgi pH is kept relatively stable, mainly thanks to the V-ATPase. Eventually, this new probe will allow to better understand the mechanisms involved in the acidification and the pH control within the secretory pathway. more...
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- 2020
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16. The activity of Saccharomyces cerevisiae Na + , K + /H + antiporter Nha1 is negatively regulated by 14-3-3 protein binding at serine 481.
- Author
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Smidova A, Stankova K, Petrvalska O, Lazar J, Sychrova H, Obsil T, Zimmermannova O, and Obsilova V
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- Binding Sites, Cell Proliferation, Saccharomyces cerevisiae cytology, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins metabolism, 14-3-3 Proteins metabolism, Saccharomyces cerevisiae chemistry, Serine metabolism
- Abstract
Na
+ /H+ antiporters are involved in ensuring optimal intracellular concentrations of alkali-metal cations and protons in most organisms. In Saccharomyces cerevisiae, the plasma-membrane Na+ , K+ /H+ antiporter Nha1 mediates Na+ and K+ efflux, which is important for cell growth in the presence of salts. Nha1 belongs among housekeeping proteins and, due to its ability to export K+ , it has many physiological functions. The Nha1 transport activity is regulated through its long, hydrophilic and unstructured C-terminus (554 of 985 aa). Although Nha1 has been previously shown to interact with the yeast 14-3-3 isoform (Bmh2), the binding site remains unknown. In this work, we identified the residues through which Nha1 interacts with the 14-3-3 protein. Biophysical characterization of the interaction between the C-terminal polypeptide of Nha1 and Bmh proteins in vitro revealed that the 14-3-3 protein binds to phosphorylated Ser481 of Nha1, and the crystal structure of the phosphopeptide containing Ser481 bound to Bmh1 provided the structural basis of this interaction. Our data indicate that 14-3-3 binding induces a disorder-to-order transition of the C-terminus of Nha1, and in vivo experiments showed that the mutation of Ser481 to Ala significantly increases cation efflux activity via Nha1, which renders cells sensitive to low K+ concentrations. Hence, 14-3-3 binding is apparently essential for the negative regulation of Nha1 activity, which should be low under standard growth conditions, when low amounts of toxic salts are present and yeast cells need to accumulate high amounts of K+ ., (Copyright © 2019 Elsevier B.V. All rights reserved.) more...- Published
- 2019
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17. Trk1, the sole potassium-specific transporter in Candida glabrata, contributes to the proper functioning of various cell processes.
- Author
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Caro G, Bieber J, Ruiz-Castilla FJ, Michán C, Sychrova H, and Ramos J
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- Candida glabrata metabolism, Cation Transport Proteins metabolism, Cell Membrane metabolism, Fungal Proteins metabolism, Gene Expression Regulation, Fungal, Homeostasis, Hydrogen-Ion Concentration, Potassium metabolism, Sodium metabolism, Candida glabrata genetics, Cation Transport Proteins genetics, Fungal Proteins genetics
- Abstract
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. more...
- Published
- 2019
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18. Potassium uptake systems of Candida krusei.
- Author
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Elicharova H, Herynkova P, Zimmermannova O, and Sychrova H
- Subjects
- Candida classification, Candida genetics, Candida growth & development, Cation Transport Proteins genetics, Fungal Proteins genetics, Genetic Complementation Test, Genetic Variation, Genome, Fungal genetics, Ion Transport, Phylogeny, Recombinant Proteins genetics, Recombinant Proteins metabolism, Saccharomyces cerevisiae classification, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae growth & development, Saccharomyces cerevisiae metabolism, Saccharomycetales classification, Saccharomycetales genetics, Candida metabolism, Cation Transport Proteins metabolism, Fungal Proteins metabolism, Potassium metabolism
- Abstract
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., (© 2019 John Wiley & Sons, Ltd.) more...
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- 2019
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19. Monovalent cation transporters at the plasma membrane in yeasts.
- Author
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Ariño J, Ramos J, and Sychrova H
- Subjects
- Cations, Monovalent metabolism, Cell Membrane genetics, Cell Membrane metabolism, Gene Expression Regulation, Fungal, Homeostasis, Ion Transport, Potassium metabolism, Potassium Channels genetics, Potassium Channels metabolism, Proton-Translocating ATPases, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins genetics, Sodium metabolism, Sodium-Hydrogen Exchangers genetics, Sodium-Hydrogen Exchangers metabolism, Sodium-Potassium-Exchanging ATPase genetics, Sodium-Potassium-Exchanging ATPase metabolism, Cation Transport Proteins genetics, Cation Transport Proteins metabolism, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins metabolism
- Abstract
Maintenance of proper intracellular concentrations of monovalent cations, mainly sodium and potassium, is a requirement for survival of any cell. In the budding yeast Saccharomyces cerevisiae, monovalent cation homeostasis is determined by the active extrusion of protons through the Pma1 H
+ -ATPase (reviewed in another chapter of this issue), the influx and efflux of these cations through the plasma membrane transporters (reviewed in this chapter), and the sequestration of toxic cations into the vacuoles. Here, we will describe the structure, function, and regulation of the plasma membrane transporters Trk1, Trk2, Tok1, Nha1, and Ena1, which play a key role in maintaining physiological intracellular concentrations of Na+ , K+ , and H+ , both under normal growth conditions and in response to stress., (© 2018 John Wiley & Sons, Ltd.) more...- Published
- 2019
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20. Expression of heterologous transporters in Saccharomyces kudriavzevii: A strategy for improving yeast salt tolerance and fermentation performance.
- Author
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Dibalova-Culakova H, Alonso-Del-Real J, Querol A, and Sychrova H
- Subjects
- Cation Transport Proteins genetics, Fermentation, Fructose metabolism, Glycerol metabolism, Yeast, Dried, Cation Transport Proteins metabolism, Ion Transport physiology, Osmotic Pressure physiology, Saccharomyces metabolism, Salt Tolerance physiology
- Abstract
S. kudriavzevii has potential for fermentations and other biotechnological applications, but is sensitive to many types of stress. We tried to increase its tolerance and performance via the expression of various transporters from different yeast species. Whereas the overexpression of Z. rouxii fructose uptake systems (ZrFfz1 and ZrFsy1) or a glycerol importer (ZrStl1) did not improve the ability of S. kudriavzevii to consume fructose and survive osmotic stress, the expression of alkali-metal-cation exporters (ScEna1, ScNha1, YlNha2) improved S. kudriavzevii salt tolerance, and that of ScNha1 also the fermentation performance. The level of improvement depended on the type and activity of the transporter suggesting that the natural sensitivity of S. kudriavzevii cells to salts is based on a non-optimal functioning of its own transporters., (Copyright © 2018. Published by Elsevier B.V.) more...
- Published
- 2018
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21. Yeast Kch1 and Kch2 membrane proteins play a pleiotropic role in membrane potential establishment and monovalent cation homeostasis regulation.
- Author
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Felcmanova K, Neveceralova P, Sychrova H, and Zimmermannova O
- Subjects
- Candida albicans genetics, Candida albicans growth & development, Cation Transport Proteins genetics, Gene Deletion, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae growth & development, Saccharomyces cerevisiae Proteins genetics, Candida albicans physiology, Cation Transport Proteins metabolism, Cations, Monovalent metabolism, Homeostasis, Membrane Potentials, Saccharomyces cerevisiae physiology, Saccharomyces cerevisiae Proteins metabolism
- Abstract
The Kch1 and Kch2 plasma-membrane proteins were identified in Saccharomyces cerevisiae as being essential for the activation of a high-affinity Ca2+ influx system. We searched for Kch proteins roles in the maintenance of cation homeostasis and tested the effect of kch1 and/or kch2 deletions on various physiological parameters. Compared to wild-type, kch1 kch2 mutant cells were smaller, relatively hyperpolarised, grew better under limited K+ conditions and exhibited altered growth in the presence of monovalent cations. The absence of Kch1 and Kch2 did not change the intracellular pH in cells growing at low potassium or the tolerance of cells to divalent cations, high concentration of sorbitol or extreme external pH. The overexpression of KCH1 only increased the intracellular pH in the presence of elevated K+ in media. None of the phenotypes associated with the deletion of KCH1 and KCH2 in wild type were observed in a strain lacking KCH genes and main K+ uptake systems Trk1 and Trk2. The role of the Kch homologue in cation homeostasis was also tested in Candida albicans cells. Our data demonstrate that Kch proteins significantly contribute to the maintenance of optimal cation homeostasis and membrane potential in S. cerevisiae but not in C. albicans., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.) more...
- Published
- 2017
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22. Lack of cortical endoplasmic reticulum protein Ist2 alters sodium accumulation in Saccharomyces cerevisiae cells.
- Author
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Papouskova K, Andrsova M, and Sychrova H
- Subjects
- Cations metabolism, Gene Deletion, Homeostasis, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae growth & development, Saccharomyces cerevisiae Proteins genetics, Endoplasmic Reticulum enzymology, Endoplasmic Reticulum metabolism, Saccharomyces cerevisiae enzymology, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins metabolism, Sodium metabolism
- Abstract
The maintenance of intracellular alkali-metal-cation homeostasis is a fundamental property of all living organisms, including the yeast Saccharomyces cerevisiae. Several transport systems are indispensable to ensure proper alkali-metal-cation levels in the yeast cytoplasm and organelles. Ist2 is an endoplasmic reticulum (ER)-resident protein involved, together with other tethering proteins, in the formation of contacts between the plasma and ER membranes. As IST2 gene deletion was shown to influence yeast growth in the presence of sodium, we focused on the roles of Ist2 in the cell response to the presence of various concentrations of alkali metal cations, and its interactions with characterised plasma membrane alkali-metal-cation transporters. Most importantly, we show that, in BY4741 background, the lack of Ist2 results in the accumulation of higher amounts of sodium when the cells are exposed to the presence of this cation, demonstrating the importance of Ist2 for the maintenance of low intracellular levels of toxic sodium. As the function and localisation of alkali-metal-cation exporters is not affected in ist2Δ cells, IST2 deletion results in an increased non-specific uptake of sodium to cells. Moreover, the deletion of IST2 influences relative cell membrane potential, pHin and the growth of cells in the presence of a limiting K+ concentration., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.) more...
- Published
- 2017
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23. Characterization of the Candida albicans Amino Acid Permease Family: Gap2 Is the Only General Amino Acid Permease and Gap4 Is an S -Adenosylmethionine (SAM) Transporter Required for SAM-Induced Morphogenesis.
- Author
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Kraidlova L, Schrevens S, Tournu H, Van Zeebroeck G, Sychrova H, and Van Dijck P
- Abstract
Amino acids are key sources of nitrogen for growth of Candida albicans . In order to detect and take up these amino acids from a broad range of different and changing nitrogen sources inside the host, this fungus must be able to adapt via its expression of genes for amino acid uptake and further metabolism. We analyzed six C. albicans putative general amino acid permeases based on their homology to the Saccharomyces cerevisiae Gap1 general amino acid permease. We generated single- and multiple-deletion strains and found that, based on growth assays and transcriptional or posttranscriptional regulation, Gap2 is the functional orthologue to Sc Gap1, with broad substrate specificity. Expression analysis showed that expression of all GAP genes is under control of the Csy1 amino acid sensor, which is different from the situation in S. cerevisiae , where the expression of ScGAP1 is not regulated by Ssy1. We show that Gap4 is the functional orthologue of Sc Sam3, the only S -adenosylmethionine (SAM) transporter in S. cerevisiae , and we report that Gap4 is required for SAM-induced morphogenesis. IMPORTANCE Candida albicans is a commensal organism that can thrive in many niches in its human host. The environmental conditions at these different niches differ quite a bit, and this fungus must be able to sense these changes and adapt its metabolism to them. Apart from glucose and other sugars, the uptake of amino acids is very important. This is underscored by the fact that the C. albicans genome encodes 6 orthologues of the Saccharomyces. cerevisiae general amino acid permease Gap1 and many other amino acid transporters. In this work, we characterize these six permeases and we show that C. albicans Gap2 is the functional orthologue of Sc Gap1 and that C. albicans Gap4 is an orthologue of Sc Sam3, an S -adenosylmethionine (SAM) transporter. Furthermore, we show that Gap4 is required for SAM-induced morphogenesis, an important virulence factor of C. albicans . more...
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- 2016
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24. Zygosaccharomyces rouxii Trk1 is an efficient potassium transporter providing yeast cells with high lithium tolerance.
- Author
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Zimmermannova O, Salazar A, Sychrova H, and Ramos J
- Subjects
- Cation Transport Proteins genetics, Cytosol chemistry, Gene Expression, Hydrogen-Ion Concentration, Saccharomyces cerevisiae drug effects, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Cation Transport Proteins metabolism, Drug Tolerance, Lithium metabolism, Lithium toxicity, Potassium metabolism, Zygosaccharomyces drug effects, Zygosaccharomyces metabolism
- Abstract
Zygosaccharomyces rouxii is an osmotolerant yeast growing in the presence of high concentrations of salts and/or sugars. The maintenance of intracellular potassium homeostasis is essential for osmostress adaptation. Zygosaccharomyces rouxii is endowed with only one typical potassium transporter (ZrTrk1). We characterized ZrTrk1 activity and its contribution to various physiological parameters in detail. Our results show that ZrTrk1 is a high-affinity K(+) transporting system efficiently discriminating between K(+) and Li(+) and indicate the presence of another, currently unknown K(+) importing system with a low affinity in Z. rouxii cells. Upon ZrTrk1 heterologous expression in Saccharomyces cerevisiae, it confers cells with a remarkably high lithium tolerance (even to wild-type strains) due to preventing Li(+) influx into cells, and is able to complement a plasma-membrane hyperpolarization and cell sensitivity to cationic compounds caused by the lack of endogenous K(+) transporters. Intracellular pH measurements with pHluorin, whose coding sequence was integrated into the genome, showed that the expression of ZrTrk1 also complements a decrease in intracellular pH in S. cerevisiae trk1Δ trk2Δ cells. Our data corroborate a tight connection between potassium and proton transporters in yeasts and provide new insights into Z. rouxii cation homeostasis and the basis of its high osmotolerance., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.) more...
- Published
- 2015
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25. A hydrophobic filter confers the cation selectivity of Zygosaccharomyces rouxii plasma-membrane Na+/H+ antiporter.
- Author
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Kinclova-Zimmermannova O, Falson P, Cmunt D, and Sychrova H
- Subjects
- Amino Acid Sequence, Cations metabolism, Fungal Proteins genetics, Hydrophobic and Hydrophilic Interactions, Lithium metabolism, Models, Molecular, Molecular Sequence Data, Point Mutation, Potassium metabolism, Protein Conformation, Sodium metabolism, Sodium-Hydrogen Exchangers genetics, Substrate Specificity, Zygosaccharomyces genetics, Fungal Proteins chemistry, Fungal Proteins metabolism, Sodium-Hydrogen Exchangers chemistry, Sodium-Hydrogen Exchangers metabolism, Zygosaccharomyces chemistry, Zygosaccharomyces metabolism
- Abstract
Na(+)/H(+) antiporters may recognize all alkali-metal cations as substrates but may transport them selectively. Plasma-membrane Zygosaccharomyces rouxii Sod2-22 antiporter exports Na(+) and Li(+), but not K(+). The molecular basis of this selectivity is unknown. We combined protein structure modeling, site-directed mutagenesis, phenotype analysis and cation efflux measurements to localize and characterize the cation selectivity region. A three-dimensional model of the ZrSod2-22 transmembrane domain was generated based on the X-ray structure of the Escherichia coli NhaA antiporter and primary sequence alignments with homologous yeast antiporters. The model suggested a close proximity of Thr141, Ala179 and Val375 from transmembrane segments 4, 5 and 11, respectively, forming a hydrophobic hole in the putative cation pathway's core. A series of mutagenesis experiments verified the model and showed that structural modifications of the hole resulted in altered cation selectivity and transport activity. The triple ZrSod2-22 mutant T141S-A179T-V375I gained K(+) transport capacity. The point mutation A179T restricted the antiporter substrate specificity to Li(+) and reduced its transport activity, while serine at this position preserved the native cation selectivity. The negative effect of the A179T mutation can be eliminated by introducing a second mutation, T141S or T141A, in the preceding transmembrane domain. Our experimental results confirm that the three residues found through modeling play a central role in the determination of cation selectivity and transport activity in Z. rouxii Na(+)/H(+) antiporter and that the cation selectivity can be modulated by repositioning a single local methyl group., (Copyright © 2015 Elsevier Ltd. All rights reserved.) more...
- Published
- 2015
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26. Vcx1 and ESCRT components regulate intracellular pH homeostasis in the response of yeast cells to calcium stress.
- Author
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Papouskova K, Jiang L, and Sychrova H
- Subjects
- Endosomal Sorting Complexes Required for Transport genetics, Gene Deletion, Hydrogen-Ion Concentration, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae growth & development, Antiporters metabolism, Calcium metabolism, Endosomal Sorting Complexes Required for Transport metabolism, Homeostasis, Saccharomyces cerevisiae drug effects, Saccharomyces cerevisiae physiology, Saccharomyces cerevisiae Proteins metabolism, Stress, Physiological
- Abstract
Endosomal sorting complexes required for transport (ESCRTs) are involved in the formation of multivesicular bodies and sorting of targeted proteins to the yeast vacuole. The deletion of seven genes encoding components of the ESCRT machinery render Saccharomyces cerevisiae cells sensitive to high extracellular CaCl2 concentrations as well as to low pH in media. In this work, we focused on intracellular pH (pHin) homeostasis of these mutants. None of the studied ESCRT mutants exhibited an altered pHin level compared to the wild type under standard growth conditions. Nevertheless, 60 min of CaCl2 treatment resulted in a more significant drop in pHin levels in these mutants than in the wild type, suggesting that pHin homeostasis is affected in ESCRT mutants upon the addition of calcium. Similarly, CaCl2 treatment caused a bigger pHin decrease in cells lacking the vacuolar Ca(2+)/H(+) antiporter Vcx1 which indicates a role for this protein in the maintenance of proper pHin homeostasis when cells need to cope with a high CaCl2 concentration in media. Importantly, ESCRT gene deletions in the vcx1Δ strain did not result in an increase in the CaCl2-invoked drop in the pHin levels of cells, which demonstrates a genetic interaction between VCX1 and studied ESCRT genes., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.) more...
- Published
- 2015
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27. The role of glycerol transporters in yeast cells in various physiological and stress conditions.
- Author
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Duskova M, Borovikova D, Herynkova P, Rapoport A, and Sychrova H
- Subjects
- Biological Transport, Cell Membrane metabolism, Membrane Proteins genetics, Membrane Transport Proteins genetics, Phenotype, Saccharomyces cerevisiae cytology, Saccharomyces cerevisiae Proteins genetics, Glycerol metabolism, Membrane Proteins metabolism, Membrane Transport Proteins metabolism, Mutation, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae physiology, Saccharomyces cerevisiae Proteins metabolism, Stress, Physiological
- Abstract
Small and uncharged glycerol is an important molecule for yeast metabolism and osmoadaptation. Using a series of S. cerevisiae BY4741-derived mutants lacking genes encoding a glycerol exporter (Fps1p) and/or importer (Stl1p) and/or the last kinase of the HOG pathway (Hog1p), we studied their phenotypes and various physiological characteristics with the aim of finding new roles for glycerol transporters. Though the triple mutant hog1Δ stl1Δ fps1Δ was viable, it was highly sensitive to various stresses. Our results showed that the function of both Stl1p and Fps1p transporters contributes to the cell ability to survive during the transfer into the state of anhydrobiosis, and that the deletion of FPS1 decreases the cell's tolerance of hyperosmotic stress. The deletion of STL1 results in a slight increase in cell size and in a substantial increase in intracellular pH. Taken together, our results suggest that the fluxes of glycerol in both directions across the plasma membrane exist in yeast cells simultaneously, and the export or import predominates according to the actual specific conditions., (© FEMS 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.) more...
- Published
- 2015
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28. Fluconazole affects the alkali-metal-cation homeostasis and susceptibility to cationic toxic compounds of Candida glabrata.
- Author
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Elicharova H and Sychrova H
- Subjects
- Candida glabrata drug effects, Candida glabrata genetics, Candida glabrata growth & development, Cell Membrane drug effects, Cell Membrane genetics, Cell Membrane metabolism, Fungal Proteins genetics, Fungal Proteins metabolism, Homeostasis, Metals, Alkali toxicity, Sodium-Hydrogen Exchangers genetics, Sodium-Hydrogen Exchangers metabolism, Sodium-Potassium-Exchanging ATPase genetics, Sodium-Potassium-Exchanging ATPase metabolism, Antifungal Agents pharmacology, Candida glabrata metabolism, Cations metabolism, Fluconazole pharmacology, Metals, Alkali metabolism
- Abstract
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., (© 2014 The Authors.) more...
- Published
- 2014
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29. Potassium uptake system Trk2 is crucial for yeast cell viability during anhydrobiosis.
- Author
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Borovikova D, Herynkova P, Rapoport A, and Sychrova H
- Subjects
- Biological Transport, Cation Transport Proteins genetics, Desiccation, Homeostasis, Microbial Viability, Potassium analysis, Saccharomyces cerevisiae physiology, Saccharomyces cerevisiae Proteins genetics, Sequence Deletion, Cation Transport Proteins metabolism, Potassium metabolism, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins metabolism
- Abstract
Yeasts grow at very different potassium concentrations, adapting their intracellular cation levels to changes in the external environment. Potassium homeostasis is maintained with the help of several transporters mediating the uptake and efflux of potassium with various affinities and mechanisms. In the model yeast Saccharomyces cerevisiae, two uptake systems, Trk1 and Trk2, are responsible for the accumulation of a relatively high intracellular potassium content (200-300 mM) and the efflux of surplus potassium is mediated by the Tok1 channel and active exporters Ena ATPase and Nha1 cation/proton antiporter. Using a series of deletion mutants, we studied the role of individual potassium transporters in yeast cell resistance to dehydration. The Trk2 transporter (whose role in S. cerevisiae physiology was not clear) is important for cell viability in the stationary phase of growth and, moreover, it plays a crucial role in the yeast survival of dehydration/rehydration treatments. Mutants lacking the TRK2 gene accumulated significantly lower amounts of potassium ions in the stationary culture growth phase, and these lower amounts correlated with decreased resistance to dehydration/rehydration stress. Our results showed Trk2 to be the major potassium uptake system in stationary cells, and potassium content to be a crucial parameter for desiccation survival., (© 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.) more...
- Published
- 2014
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30. Fluconazole treatment hyperpolarizes the plasma membrane of Candida cells.
- Author
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Elicharova H and Sychrova H
- Subjects
- Cytosol chemistry, Lithium toxicity, Osmotic Pressure, Sodium toxicity, Antifungal Agents pharmacology, Candida drug effects, Cell Membrane drug effects, Cell Membrane physiology, Fluconazole pharmacology, Membrane Potentials drug effects
- Abstract
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. more...
- Published
- 2013
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31. Human NKCC2 cation–Cl– co-transporter complements lack of Vhc1 transporter in yeast vacuolar membranes.
- Author
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Petrezselyova S, Dominguez A, Herynkova P, Macias JF, and Sychrova H
- Subjects
- Cation Transport Proteins metabolism, Cations metabolism, Cell Membrane metabolism, Chlorides metabolism, Gene Deletion, Humans, Ion Transport, Lithium Chloride metabolism, Phenotype, Potassium Chloride metabolism, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins genetics, Sodium Chloride metabolism, Sodium-Hydrogen Exchangers metabolism, Sodium-Potassium-Exchanging ATPase metabolism, Solute Carrier Family 12, Member 1 genetics, Substrate Specificity, Symporters genetics, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins metabolism, Solute Carrier Family 12, Member 1 metabolism, Symporters metabolism, Vacuoles metabolism
- Abstract
Cation–chloride co-transporters serve to transport Cl– and alkali metal cations. Whereas a large family of these exists in higher eukaryotes, yeasts only possess one cation–chloride co-transporter, Vhc1, localized to the vacuolar membrane. In this study, the human cation–chloride co-transporter NKCC2 complemented the phenotype of VHC1 deletion in Saccharomyces cerevisiae and its activity controlled the growth of salt-sensitive yeast cells in the presence of high KCl, NaCl and LiCl. A S. cerevisiae mutant lacking plasma-membrane alkali–metal cation exporters Nha1 and Ena1-5 and the vacuolar cation–chloride co-transporter Vhc1 is highly sensitive to increased concentrations of alkali–metal cations, and it proved to be a suitable model for characterizing the substrate specificity and transport activity of human wild-type and mutated cation–chloride co-transporters., (Copyright © 2013 John Wiley & Sons, Ltd.) more...
- Published
- 2013
32. Salt and oxidative stress tolerance in Debaryomyces hansenii and Debaryomyces fabryi.
- Author
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Michán C, Martínez JL, Alvarez MC, Turk M, Sychrova H, and Ramos J
- Subjects
- Cell Division drug effects, Cell Membrane physiology, Cytoplasm chemistry, Humans, Membrane Fluidity drug effects, Membrane Potentials drug effects, Potassium analysis, Reactive Oxygen Species analysis, Saccharomycetales chemistry, Saccharomycetales growth & development, Sodium analysis, Osmotic Pressure, Oxidative Stress, Saccharomycetales drug effects, Saccharomycetales physiology, Salts toxicity, Stress, Physiological
- Abstract
We report the characterization of five strains belonging to the halotolerant highly related Debaryomyces hansenii/fabryi species. The analysis performed consisted in studying tolerance properties, membrane characteristics, and cation incell amounts. We have specifically investigated (1) tolerance to different chemicals, (2) tolerance to osmotic and salt stress, (3) tolerance and response to oxidative stress, (4) reactive oxygen species (ROS) content, (5) relative membrane potential, (6) cell volume, (7) K(+) and Na(+) ion content, and (8) membrane fluidity. Unexpectedly, no direct relationship was found between one particular strain, Na(+) content and its tolerance to NaCl or between its ROS content and its tolerance to H(2)O(2). Results show that, although in general, human origin D. fabryi strains were more resistant to oxidative stress and presented shorter doubling times and smaller cell volume than food isolated D. hansenii ones, strains belonging to the same species can be significantly different. Debaryomyces fabryi CBS1793 strain highlighted for its extremely tolerant behavior when exposed to the diverse stress factors studied., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.) more...
- Published
- 2013
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33. Vhc1, a novel transporter belonging to the family of electroneutral cation-Cl(-) cotransporters, participates in the regulation of cation content and morphology of Saccharomyces cerevisiae vacuoles.
- Author
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Petrezselyova S, Kinclova-Zimmermannova O, and Sychrova H
- Subjects
- Chlorides chemistry, DNA chemistry, Electrochemistry methods, Genotype, Hydrogen-Ion Concentration, Ion Transport, Membrane Potentials, Microscopy, Confocal methods, Microscopy, Fluorescence methods, Nigericin pharmacology, Oligonucleotides chemistry, Open Reading Frames, Osmotic Pressure, Phylogeny, Recombination, Genetic, Saccharomyces cerevisiae Proteins physiology, Sorbitol chemistry, Symporters physiology, Cations chemistry, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins chemistry, Symporters chemistry, Vacuoles chemistry
- Abstract
Cation-Cl(-) cotransporters (CCCs) are integral membrane proteins which catalyze the coordinated symport of Cl(-) with Na(+) and/or K(+) ions in plant and mammalian cells. Here we describe the first Saccharomyces cerevisiae CCC protein, encoded by the YBR235w open reading frame. Subcellular localization studies showed that this yeast CCC is targeted to the vacuolar membrane. Deletion of the YBR235w gene in a salt-sensitive strain (lacking the plasma-membrane cation exporters) resulted in an increased sensitivity to high KCl, altered vacuolar morphology control and decreased survival upon hyperosmotic shock. In addition, deletion of the YBR235w gene in a mutant strain deficient in K(+) uptake produced a significant growth advantage over the parental strain under K(+)-limiting conditions, and a hypersensitivity to the exogenous K(+)/H(+) exchanger nigericin. These results strongly suggest that we have identified a novel yeast vacuolar ion transporter mediating a K(+)-Cl(-) cotransport and playing a role in vacuolar osmoregulation. Considering its identified function, we propose to refer to the yeast YBR235w gene as VHC1 (vacuolar protein homologous to CCC family 1)., (Copyright © 2012 Elsevier B.V. All rights reserved.) more...
- Published
- 2013
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34. Role of individual phosphorylation sites for the 14-3-3-protein-dependent activation of yeast neutral trehalase Nth1.
- Author
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Veisova D, Macakova E, Rezabkova L, Sulc M, Vacha P, Sychrova H, Obsil T, and Obsilova V
- Subjects
- Enzyme Activation, Mutation, Phosphorylation, Proteolysis, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Trehalase genetics, 14-3-3 Proteins metabolism, Saccharomyces cerevisiae enzymology, Trehalase metabolism
- Abstract
Trehalases are important highly conserved enzymes found in a wide variety of organisms and are responsible for the hydrolysis of trehalose that serves as a carbon and energy source as well as a universal stress protectant. Emerging evidence indicates that the enzymatic activity of the neutral trehalase Nth1 in yeast is enhanced by 14-3-3 protein binding in a phosphorylation-dependent manner through an unknown mechanism. In the present study, we investigated in detail the interaction between Saccharomyces cerevisiae Nth1 and 14-3-3 protein isoforms Bmh1 and Bmh2. We determined four residues that are phosphorylated by PKA (protein kinase A) in vitro within the disordered N-terminal segment of Nth1. Sedimentation analysis and enzyme kinetics measurements show that both yeast 14-3-3 isoforms form a stable complex with phosphorylated Nth1 and significantly enhance its enzymatic activity. The 14-3-3-dependent activation of Nth1 is significantly more potent compared with Ca2+-dependent activation. Limited proteolysis confirmed that the 14-3-3 proteins interact with the N-terminal segment of Nth1 where all phosphorylation sites are located. Site-directed mutagenesis in conjunction with the enzyme activity measurements in vitro and the activation studies of mutant forms in vivo suggest that Ser60 and Ser83 are sites primarily responsible for PKA-dependent and 14-3-3-mediated activation of Nth1. more...
- Published
- 2012
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35. Cnh1 Na(+) /H(+) antiporter and Ena1 Na(+) -ATPase play different roles in cation homeostasis and cell physiology of Candida glabrata.
- Author
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Krauke Y and Sychrova H
- Subjects
- Candida glabrata genetics, Candida glabrata metabolism, Fungal Proteins genetics, Gene Deletion, Gene Expression Regulation, Fungal, Genetic Complementation Test, Potassium metabolism, Saccharomyces cerevisiae metabolism, Sodium metabolism, Sodium-Hydrogen Exchangers genetics, Substrate Specificity, Candida glabrata physiology, Cations metabolism, Fungal Proteins metabolism, Homeostasis, Sodium-Hydrogen Exchangers metabolism
- Abstract
Yeasts tightly regulate their intracellular concentrations of alkali metal cations. In Saccharomyces cerevisiae, the Nha1 Na(+) /H(+) -antiporter and Ena1 Na(+) -ATPase, mediate the efflux of toxic sodium and surplus potassium. We report the characterization of Candida glabrata CgCnh1 and CgEna1 homologues. Their substrate specificity and transport properties were compared upon expression in S. cerevisiae, and their function characterized directly in C. glabrata. The CgCnh1 antiporter and the CgEna1 ATPase transport both potassium and sodium when expressed in S. cerevisiae. CgEna1p fully complements the lack of S. cerevisiae own Na(+) -ATPases but the activity of the CgCnh1 antiporter is lower than that of ScNha1p. Candida glabrata deletion mutants and analyses of their phenotypes revealed that though both transporters have a broad substrate specificity, their function in C. glabrata cells is not the same. Their differing physiological roles are also reflected in their regulation of expression, CgENA1 is highly upregulated by an increased osmotic pressure or sodium concentration, whereas CgCNH1 is expressed constitutively. The Cnh1 antiporter is involved in the regulation of potassium content and the Ena1 ATPase in sodium detoxification of C. glabrata cells. This situation differs from S. cerevisiae, where the Nha1 antiporter and Ena ATPases both participate together in Na(+) detoxification and in the regulation of K(+) homeostasis., (© 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.) more...
- Published
- 2011
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36. Monovalent cations regulate expression and activity of the Hak1 potassium transporter in Debaryomyces hansenii.
- Author
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Martínez JL, Sychrova H, and Ramos J
- Subjects
- Culture Media chemistry, Gene Expression Profiling, Hydrogen-Ion Concentration, Saccharomycetales metabolism, Sodium metabolism, Cations, Monovalent metabolism, Gene Expression Regulation, Fungal, Potassium metabolism, Saccharomycetales physiology, Symporters biosynthesis
- Abstract
Debaryomyces hansenii was able to grow in a medium containing residual amounts of K(+), indicating the activity of high affinity K(+) transporters. Transcriptional regulation analysis of the genes encoding the two potassium uptake systems in D. hansenii revealed that while DhTRK1 is not regulated at transcriptional level, expression of DhHAK1 required starvation in the absence of K(+) and Na(+) and was not affected by changes in membrane potential. Rb(+) transport in cells expressing DhHAK1 was activated by external Na(+) or acidic pH and inhibited by high pH. We propose a K(+)-H(+) symporter that, under certain conditions may work as a K(+)-Na(+) transporter, as the mechanism driving K(+) influx mediated by DhHak1p., (Copyright © 2010 Elsevier Inc. All rights reserved.) more...
- Published
- 2011
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37. 2-DE based proteomic analysis of Saccharomyces cerevisiae wild and K+ transport-affected mutant (trk1,2) strains at the growth exponential and stationary phases.
- Author
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Curto M, Valledor L, Navarrete C, Gutiérrez D, Sychrova H, Ramos J, and Jorrin J
- Subjects
- Biological Transport genetics, Electrophoresis, Gel, Two-Dimensional, Mutation, Rubidium metabolism, Saccharomyces cerevisiae enzymology, Saccharomyces cerevisiae growth & development, Saccharomyces cerevisiae Proteins analysis, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Cation Transport Proteins genetics, Potassium metabolism, Proteomics methods, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins genetics
- Abstract
By using a 2-DE based workflow, the proteome of wild and potassium transport mutant trk1,2 under optimal growth potassium concentration (50mM) has been analyzed. At the exponential and stationary phases, both strains showed similar growth, morphology potassium content, and Vmax of rubidium transport, the only difference found being the Km values for this potassium analogue transport, higher for the mutant (20mM) than for the wild (3-6mM) cells. Proteins were buffer-extracted, precipitated, solubilized, quantified, and subjected to 2-DE analysis in the 5-8 pH range. More differences in protein content (37-64mgg(-1) cell dry weight) and number of resolved spots (178-307) were found between growth phases than between strains. In all, 164 spots showed no differences between samples and a total of 105 were considered to be differential after ANOVA test. 171 proteins, corresponding to 71 unique gene products have been identified, this set being dominated by cytosolic species and glycolitic enzymes. The ranking of the more abundant spots revealed no differences between samples and indicated fermentative metabolism, and active cell wall biosynthesis, redox homeostasis, biosynthesis of amino acids, coenzymes, nucleotides, and RNA, and protein turnover, apart from cell division and growth. PCA analysis allowed the separation of growth phases (PC1 and 2) and strains at the stationary phase (PC3 and 4), but not at the exponential one. These results are also supported by clustering analysis. As a general tendency, a number of spots newly appeared at the stationary phase in wild type, and to a lesser extent, in the mutant. These up-accumulated spots corresponded to glycolitic enzymes, indicating a more active glucose catabolism, accompanied by an accumulation of methylglyoxal detoxification, and redox-homeostasis enzymes. Also, more extensive proteolysis was observed at the stationary phase with this resulting in an accumulation of low Mr protein species., (Copyright © 2010 Elsevier B.V. All rights reserved.) more...
- Published
- 2010
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38. Four pathogenic Candida species differ in salt tolerance.
- Author
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Krauke Y and Sychrova H
- Subjects
- Candida metabolism, Candida albicans pathogenicity, Candida albicans physiology, Candida glabrata pathogenicity, Candida glabrata physiology, Lithium Chloride analysis, Lithium Chloride pharmacology, Potassium Chloride analysis, Potassium Chloride pharmacology, Salts, Sodium Chloride analysis, Sodium Chloride pharmacology, Species Specificity, Virulence, Candida pathogenicity, Candida physiology, Salt Tolerance physiology
- Abstract
The virulence of Candida species depends on many environmental conditions, including extracellular pH and concentration of alkali metal cations. Tests of the tolerance/sensitivity of four pathogenic Candida species (C. albicans, C. dubliniensis, C. glabrata, and C. parapsilosis) to alkali metal cations under various growth conditions revealed significant differences among these species. Though all of them can be classified as rather osmotolerant yeast species, they exhibit different levels of tolerance to different salts. C. parapsilosis and C. albicans are the most salt-tolerant in general; C. dubliniensis is the least tolerant on rich YPD media and C. glabrata on acidic (pH 3.5) minimal YNB medium. C. dubliniensis is relatively salt-sensitive in spite of its ability to maintain as high intracellular K(+)/Na(+) ratio as its highly salt-tolerant relative C. albicans. On the other hand, C. parapsilosis can grow in the presence of very high external NaCl concentrations in spite of its high intracellular Na(+) concentrations (and thus lower K(+)/Na(+) ratio) and thus resembles salt-tolerant (halophilic) Debaryomyces hansenii. more...
- Published
- 2010
- Full Text
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39. Saccharomyces cerevisiae BY4741 and W303-1A laboratory strains differ in salt tolerance.
- Author
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Petrezselyova S, Zahradka J, and Sychrova H
- Subjects
- Biological Transport, Cations, Cell Membrane metabolism, Cell Membrane physiology, Gene Expression Regulation, Fungal, Homeostasis, Hygromycin B pharmacology, Lithium metabolism, Lithium pharmacology, Membrane Potentials, Mutation, Potassium metabolism, Potassium pharmacology, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae growth & development, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins metabolism, Sodium metabolism, Sodium pharmacology, Species Specificity, Saccharomyces cerevisiae classification, Saccharomyces cerevisiae physiology, Salt Tolerance physiology
- Abstract
Saccharomyces cerevisiae yeast cells serve as a model to elucidate the bases of salt tolerance and potassium homeostasis regulation in eukaryotic cells. In this study, we show that two widely used laboratory strains, BY4741 and W303-1A, differ not only in cell size and volume but also in their relative plasma-membrane potential (estimated with a potentiometric fluorescent dye diS-C3(3) and as Hygromycin B sensitivity) and tolerance to alkali-metal cations. W303-1A cells and their mutant derivatives lacking either uptake (trk1 trk2) or efflux (nha1) systems for alkali-metal cations are more tolerant to toxic sodium and lithium cations but also more sensitive to higher external concentrations of potassium than BY4741 cells and their mutants. Moreover, our results suggest that though the two strains do not differ in the total potassium content, the regulation of intracellular potassium homeostasis is probably not the same in BY4741 and W303-1A cells. more...
- Published
- 2010
- Full Text
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40. Subinhibitory concentrations of fluconazole increase the intracellular sodium content in both fluconazole-resistant and -sensitive Candida albicans strains.
- Author
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Kolecka A, Krauke Y, Bujdakova H, and Sychrova H
- Subjects
- Candida albicans growth & development, Culture Media chemistry, Drug Resistance, Fungal, Humans, Osmotic Pressure, Sodium pharmacology, Temperature, Antifungal Agents pharmacology, Candida albicans chemistry, Candida albicans drug effects, Cytosol chemistry, Fluconazole pharmacology, Sodium analysis
- Abstract
Fluconazole-sensitive (SC 5314) and -resistant (clinical isolate 1173) Candida albicans strains were compared in terms of their osmotolerance and tolerance to toxic sodium cations. The two strains did not differ in their tolerance to high osmotic pressure in general but exhibited distinct sensitivities to sodium cations. Although the fluconazole-resistant 1173 strain contained, under all conditions tested, significantly lower intracellular amounts of Na+, it was much more sodium sensitive than the SC 5314 strain. The addition of subinhibitory concentrations of fluconazole to media supplemented with NaCl significantly influenced the growth of both strains and resulted in substantially elevated intracellular sodium concentrations compared with growth in medium containing NaCl but no fluconazole. more...
- Published
- 2009
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41. Membrane hyperpolarization drives cation influx and fungicidal activity of amiodarone.
- Author
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Maresova L, Muend S, Zhang YQ, Sychrova H, and Rao R
- Subjects
- Fluorescence, Humans, Immunoprecipitation, Ion Transport, Saccharomyces cerevisiae physiology, Amiodarone pharmacology, Membrane Proteins, Saccharomyces cerevisiae drug effects
- Abstract
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. more...
- Published
- 2009
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42. The salt tolerant yeast Zygosaccharomyces rouxii possesses two plasma-membrane Na+/H+-antiporters (ZrNha1p and ZrSod2-22p) playing different roles in cation homeostasis and cell physiology.
- Author
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Pribylova L, Papouskova K, and Sychrova H
- Subjects
- Amino Acid Sequence, Fungal Proteins genetics, Gene Expression, Molecular Sequence Data, Sequence Alignment, Sequence Deletion, Sodium-Hydrogen Exchangers genetics, Zygosaccharomyces genetics, Cations metabolism, Fungal Proteins metabolism, Homeostasis, Sodium Chloride metabolism, Sodium-Hydrogen Exchangers metabolism, Zygosaccharomyces physiology
- Abstract
Antiporters exporting Na(+) and K(+) in exchange for protons are conserved among yeast species. The only exception so far has been Zygosaccharomyces rouxii, an osmotolerant species closely related to Saccharomyces cerevisiae. Z. rouxii was described as possessing one plasma-membrane antiporter transporting only Na(+) (ZrSod2-22p in the CBS 732(T) type strain). We report the characterization of a second gene, ZrNHA1, encoding a new K(+)(Na(+))/H(+)-antiporter capable of both K(+) and Na(+) export. Synteny analyses suggested that ZrSOD2-22 originated by single duplication of the ZrNHA1 gene. Substrate specificities and transport properties of ZrNha1p and ZrSod2-22p were compared upon heterologous expression in S. cerevisiae, and then directly in Z. rouxii. Deletion mutants and phenotype analyses revealed that ZrSod2-22 antiporter is important for Na(+) detoxification, probably together with ZrEna1 ATPase; ZrNha1p is indispensable to maintain potassium homeostasis and ZrEna1p is not, in contrast to the situation in S. cerevisiae, involved in this function. more...
- Published
- 2008
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43. Functional expression of the voltage-gated neuronal mammalian potassium channel rat ether à go-go1 in yeast.
- Author
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Schwarzer S, Kolacna L, Lichtenberg-Fraté H, Sychrova H, and Ludwig J
- Subjects
- Animals, Barium Compounds pharmacology, Chlorides pharmacology, Ether-A-Go-Go Potassium Channels genetics, Hydrogen-Ion Concentration, Potassium metabolism, Potassium Channel Blockers pharmacology, Rats, Recombinant Proteins metabolism, Rubidium metabolism, Saccharomyces cerevisiae genetics, Ether-A-Go-Go Potassium Channels physiology, Saccharomyces cerevisiae metabolism
- Abstract
It has been shown previously that heterologous expression of inwardly rectifying potassium channels (K+-channels) from plants and mammals in K+-transport defective yeast mutants can restore the ability of growth in media with low [K+]. In this study, the functional expression of an outward rectifying mammalian K+-channel in yeast is presented for the first time. The outward-rectifying mammalian neuronal K+-channel rat ether à go-go channel 1 (rEAG1, Kv 10.1) was expressed in yeast (Saccharomyces cerevisiae) strains lacking the endogenous K+-uptake systems and/or alkali-metal-cation efflux systems. It was found that a truncated channel version, lacking almost the complete intracellular N-terminus (rEAG1 Delta 190) but not the full-length rEAG1, partially complemented the growth defect of K+-uptake mutant cells (trk1,2 Delta tok1 Delta) in media containing low K+ concentrations. The expression of rEAG1 Delta 190 in a strain lacking the cation efflux systems (nha1 Delta ena1-4 Delta) increased the sensitivity to high monovalent cation concentrations. Both phenotypes were observed, when rEAG1 Delta 190 was expressed in a trk1,2 Delta and nha1, ena1-4 Delta mutant strain. In the presence of K+-channel blockers (Cs+, Ba2+ and quinidine), the growth advantage of rEAG1 Delta 190 expressing trk1,2 tok1 Delta cells disappeared, indicating its dependence on functional rEAG1 channels. The results demonstrate that S. cerevisiae is a suitable expression system even for voltage-gated outward-rectifying mammalian K+-channels. more...
- Published
- 2008
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- View/download PDF
44. Tools for the genetic manipulation of Zygosaccharomyces rouxii.
- Author
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Pribylova L, de Montigny J, and Sychrova H
- Subjects
- Cloning, Molecular methods, Escherichia coli genetics, Genes, Reporter, Genetic Vectors, Green Fluorescent Proteins genetics, Metabolic Networks and Pathways genetics, Mutagenesis, Insertional methods, Plasmids, Sequence Deletion, Molecular Biology methods, Zygosaccharomyces genetics
- Abstract
A set of tools for the genetic manipulation of the osmotolerant yeast Zygosaccharomyces rouxii was developed. Auxotrophic mutants (ura3 leu2, ura3 ade2, ura3 leu2 ade2) derived from the CBS 732 type strain were prepared. Centromeric and episomal Z. rouxii/Escherichia coli shuttle plasmids with different marker genes (ScURA3, ZrLEU2, ZrADE2) and with multiple cloning sites were constructed, together with a plasmid enabling green fluorescent protein-tagging. A system for repeatable targeted gene deletion in Z. rouxii was established, involving first the integration of a PCR-generated loxP-kanMX-loxP cassette and second the removal of kanMX from the genome using a Z. rouxii plasmid harbouring cre recombinase. more...
- Published
- 2007
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45. The co-action of osmotic and high temperature stresses results in a growth improvement of Debaryomyces hansenii cells.
- Author
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Papouskova K and Sychrova H
- Subjects
- Food Contamination prevention & control, Humans, Kinetics, Potassium pharmacology, Saccharomyces cerevisiae drug effects, Saccharomyces cerevisiae growth & development, Saccharomycetales drug effects, Sodium pharmacology, Sorbitol pharmacology, Food Preservation methods, Osmotic Pressure, Saccharomycetales growth & development, Temperature
- Abstract
Debaryomyces hansenii is a salt tolerant yeast species, often isolated from sea water or found among other spoilage yeasts in several types of food. In this work, we examined the influence of temperature and increased osmotic pressure (two parameters also important in food industry) on D. hansenii growth. Several other authors showed that its growth at the normal yeast cultivation temperature (28 to 30 degrees C) is stimulated by the presence of sodium, in contrast to the growth of Saccharomyces cerevisiae, which is inhibited by the presence of sodium under the same experimental conditions. Here we show that the previously reported growth stimulation by sodium is temperature dependent in D. hansenii and can be observed under conditions that already amount to high temperature stress for D. hansenii. At a lower temperature (more convenient for D. hansenii cultivation), we found no significant improvement or even an inhibition of cell growth in the presence of Na(+). The growth of D. hansenii at high temperatures is also improved by the presence of potassium or sorbitol. Moreover, the temperature dependence of stimulatory effects of increased osmotic pressure in media does not seem to be unique for D. hansenii; similar relationships between the growth, cultivation temperature and presence of osmolytes we also observed for S. cerevisiae and Schizosaccharomyces pombe. more...
- Published
- 2007
- Full Text
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46. Osmoresistant yeast Zygosaccharomyces rouxii: the two most studied wild-type strains (ATCC 2623 and ATCC 42981) differ in osmotolerance and glycerol metabolism.
- Author
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Pribylova L, de Montigny J, and Sychrova H
- Subjects
- Chromosomes, Fungal genetics, Culture Media, Karyotyping, Osmotic Pressure, Salts, Zygosaccharomyces cytology, Glycerol metabolism, Zygosaccharomyces physiology
- Abstract
The yeast Zygosaccharomyces rouxii is known for its high tolerance to osmotic stress, which is thought to be caused by sets of specific genes. Relatively few Z. rouxii genes have been identified so far, all of them having homologues in Saccharomyces cerevisiae; none of them was Z. rouxii-specific. Most of the known Z. rouxii genes were isolated from two wild-type strains, ATCC 2623 and ATCC 42981. In this study, we compared these two strains with regard to some of their morphological, physiological and genomic properties. Important differences were found in their salt tolerance and assimilation of glycerol and karyotype; slight differences were also present in their cell morphology. The ATCC 42981 strain showed a higher resistance to salts, higher glycerol production and, unlike ATCC 2623, was able to assimilate glycerol. Under conditions of osmotic stress, the glycerol production in both Z. rouxii strains was much lower than in a S. cerevisiae S288c culture, which suggested the presence of a system that efficiently retains glycerol inside Z. rouxii cells. The karyotype analysis revealed that ATCC 42981 cells contain more chromosomes and have a bigger genome size than those of ATCC 2623. more...
- Published
- 2007
- Full Text
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47. Schizosaccharomyces pombe possesses two plasma membrane alkali metal cation/H antiporters differing in their substrate specificity.
- Author
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Papouskova K and Sychrova H
- Subjects
- Culture Media, Hydrogen-Ion Concentration, Potassium metabolism, Recombinant Proteins genetics, Recombinant Proteins metabolism, Saccharomyces cerevisiae drug effects, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae physiology, Schizosaccharomyces genetics, Schizosaccharomyces growth & development, Schizosaccharomyces physiology, Sodium metabolism, Sodium pharmacology, Substrate Specificity, Cations, Monovalent metabolism, Cell Membrane metabolism, Metals metabolism, Potassium pharmacology, Schizosaccharomyces metabolism, Sodium-Hydrogen Exchangers metabolism
- Abstract
The Schizosaccharomyces pombe plasma membrane Na(+)/H(+) antiporter, SpSod2p, has been shown to belong to the subfamily of yeast Na(+)/H(+) antiporters that only recognize Na(+) and Li(+) as substrates. Nevertheless, most of the studied plasma membrane alkali metal cation/H(+) antiporters from other yeasts have broader substrate specificities, exporting K(+) and Rb(+) as well. Such antiporters probably play two roles in the physiology of cells: the elimination of surplus toxic cations, and the regulation of stable intracellular K(+) content, pH and cell volume. The systematic sequencing of the Sch. pombe genome revealed the presence of an as-yet uncharacterized homolog of the Spsod2 gene (designated Spsod22). Spsod22 and Spsod2 were expressed in Saccharomyces cerevisiae cells lacking their own alkali metal cation efflux systems, and the transport properties of both Sch. pombe antiporters were compared to those of the Sac. cerevisiae Nha1 antiporter expressed under the same conditions. Here we show that SpSod22p has broad substrate specificity upon heterologous expression in Sac. cerevisiae cells and contributes to cell tolerance to high external levels of K(+). Thus, the Sch. pombe genome encodes two plasma membrane alkali metal cation/H(+) antiporters that play different roles in the physiology of the yeast. more...
- Published
- 2007
- Full Text
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48. Ring test assessment of the mKir2.1 growth based assay in Saccharomyces cerevisiae using parametric models and model-free fits.
- Author
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Hasenbrink G, Kolacna L, Ludwig J, Sychrova H, Kschischo M, and Lichtenberg-Fraté H
- Subjects
- Biomass, Densitometry, Dose-Response Relationship, Drug, Biological Assay, Drug Evaluation, Preclinical methods, Potassium Channels, Inwardly Rectifying antagonists & inhibitors, Potassium Channels, Inwardly Rectifying drug effects, Saccharomyces cerevisiae drug effects, Saccharomyces cerevisiae growth & development
- Abstract
Inward rectifying K+ (Kir) channels are a subfamily of the potassium channel superfamily. They mediate potassium influx into the cells, a process responding to the polarization state, a variety of intracellular messengers and specific auxiliary proteins, thereby they are involved in important physiological processes such as the pacemaker activity in the heart, insulin release, and potassium uptake in glial cells. The Saccharomyces cerevisiae mKir2.1 in vitro assay was subjected to a ring test assessment. Compound-associated mKir2.1 modulating effects were detected by growth determination of functionally complemented S. cerevisiae cells in a 96-well format within 15 h. Dose-response diagrams and EC50 value calculations were determined by parametric model and model-free fits using cubic spline interpolation. These characteristics were evaluated by statistical methods to determine reproducibility among working groups. Nonparametric bootstrap simulations of the variability of the data revealed that EC50 values of the mKir2.1 indicator strain were well-matched (81-92 microM), enabling unambiguous quantitative statements about inhibitory effects and no significant influence of the different laboratory conditions. Limitations of the assay include compounds/samples that are either insoluble under the conditions of the test or strongly cytotoxic to yeast. Thus, the described test is a sensitive and reliable tool that can be used in different laboratories and is applicable in drug discovery and development as simple and reliable prescreening procedure. more...
- Published
- 2007
- Full Text
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49. Characterisation of Zygosaccharomyces rouxii centromeres and construction of first Z. rouxii centromeric vectors.
- Author
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Pribylova L, Straub ML, Sychrova H, and de Montigny J
- Subjects
- Base Sequence, DNA Primers genetics, DNA, Fungal genetics, Genetic Vectors, Plasmids genetics, Saccharomyces cerevisiae genetics, Species Specificity, Transformation, Genetic, Centromere genetics, Zygosaccharomyces genetics
- Abstract
Zygosaccharomyces rouxii is a hemiascomycetous yeast known for its high osmotolerance, the basis of which still remains unknown. By exploring the Génolevures I database, four Z. rouxii fragments homologous to Saccharomyces cerevisiae centromeres were identified. Two of them were subjected to further analysis. Their function as centromeres in Z. rouxii was proved, and they were localized to Z. rouxii chromosomes II and VII, respectively. The species-specificity of centromeres was observed; plasmids with a Z. rouxii centromere were not recognized as centromeric in S. cerevisiae, and a S. cerevisiae centromere did not function as a centromere in Z. rouxii. Constructed plasmids bearing Z. rouxii centromeres serve as the first specific centromeric plasmids, and thus contribute to the so-far limited set of genetic tools needed to study the Z. rouxii specific features. more...
- Published
- 2007
- Full Text
- View/download PDF
50. Arabidopsis thaliana CHX17 gene complements the kha1 deletion phenotypes in Saccharomyces cerevisiae.
- Author
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Maresova L and Sychrova H
- Subjects
- Cinnamates pharmacology, DNA, Complementary, Gene Deletion, Genetic Complementation Test, Green Fluorescent Proteins genetics, Hydrogen-Ion Concentration, Hygromycin B analogs & derivatives, Hygromycin B pharmacology, Phenotype, Saccharomyces cerevisiae drug effects, Saccharomyces cerevisiae growth & development, Arabidopsis genetics, Arabidopsis Proteins genetics, Potassium-Hydrogen Antiporters genetics, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins genetics, Sodium-Hydrogen Exchangers genetics
- Abstract
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)., (Copyright 2006 John Wiley & Sons, Ltd.) more...
- Published
- 2006
- Full Text
- View/download PDF
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