The Homo sapiens Na+/H+ antiporter NHA2 (SLC9B2) transports Na+ or Li+ in exchange for protons across cell membranes, and its dysfunction results in various pathologies. The activity of HsNHA2 is specifically inhibited by the flavonoid phloretin. Using bioinformatic modeling, we predicted two amino acids (R177 and S178) as being important for the binding of phloretin to the HsNHA2 molecule. Functional expression of HsNHA2 in Saccharomyces cerevisiae and its site-directed mutagenesis revealed that while the R177T mutation resulted in an antiporter that was less sensitive to phloretin, the S178T mutation enhanced the inhibitory effect of phloretin on HsNHA2. Our data corroborate the transport properties of HsNHA2 and its interactions with an inhibitor and can be helpful for the development of new therapeutics targeting this antiporter and its pleiotropic physiological functions.

• Keywords
• Na+/H+ antiporter, human NHA2, phloretin inhibition, yeast,
• MeSH
• Phloretin * pharmacology   chemistry   metabolism   MeSH
• Humans MeSH
• Models, Molecular MeSH
• Mutagenesis, Site-Directed MeSH
• Sodium-Hydrogen Exchangers * genetics   antagonists & inhibitors   chemistry   metabolism   MeSH
• Saccharomyces cerevisiae genetics   metabolism   MeSH
• Amino Acid Sequence MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Phloretin * MeSH
• Sodium-Hydrogen Exchangers * MeSH

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.

• Keywords
• N-terminal auto-inhibition, Na+/H+ antiporter, human NHA2, phloretin, yeast,
• MeSH
• Humans MeSH
• Sodium-Hydrogen Exchangers * chemistry   genetics   MeSH
• Protons * MeSH
• Saccharomyces cerevisiae genetics   metabolism   MeSH
• Amino Acid Sequence MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Sodium-Hydrogen Exchangers * MeSH
• Protons * MeSH
• SLC9B2 protein, human MeSH Browser

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

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

Zygosaccharomyces rouxii is a fructophilic yeast that consumes fructose preferably to glucose. This behavior seems to be related to sugar uptake. In this study, we constructed Z. rouxii single-, double-, and triple-deletion mutants in the UL4 strain background (a ura3 strain derived from CBS 732(T)) by deleting the genes encoding the specific fructose facilitator Z. rouxii Ffz1 (ZrFfz1), the fructose/glucose facilitator ZrFfz2, and/or the fructose symporter ZrFsy1. We analyzed the effects on the growth phenotype, on kinetic parameters of fructose and glucose uptake, and on sugar consumption profiles. No growth phenotype was observed on fructose or glucose upon deletion of FFZ genes. Deletion of ZrFFZ1 drastically reduced fructose transport capacity, increased glucose transport capacity, and eliminated the fructophilic character, while deletion of ZrFFZ2 had almost no effect. The strain in which both FFZ genes were deleted presented even higher consumption of glucose than strain Zrffz1Δ, probably due to a reduced repressing effect of fructose. This study confirms the molecular basis of the Z. rouxii fructophilic character, demonstrating that ZrFfz1 is essential for Z. rouxii fructophilic behavior. The gene is a good candidate to improve the fructose fermentation performance of industrial Saccharomyces cerevisiae strains.

• MeSH
• Biological Transport genetics   MeSH
• Fermentation genetics   MeSH
• Fructose metabolism   MeSH
• Fungal Proteins genetics   metabolism   MeSH
• Gene Knockdown Techniques MeSH
• Glucose metabolism   MeSH
• Cell Proliferation genetics   MeSH
• Gene Expression Regulation, Fungal MeSH
• Saccharomyces cerevisiae genetics   metabolism   MeSH
• Zygosaccharomyces genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Fructose MeSH
• Fungal Proteins MeSH
• Glucose MeSH

Secreted aspartic proteinase Sapp1p of Candida parapsilosis represents one of the factors contributing to the pathogenicity of the fungus. The proteinase is synthesized as an inactive pre-pro-enzyme, but only processed Sapp1p is secreted into extracellular space. We constructed a plasmid containing the SAPP1 coding sequence under control of the ScGAL1 promoter and used it for proteinase expression in a Saccharomyces cerevisiae kex2Δ mutant. Because Sapp1p maturation depends on cleavage by Kex2p proteinase, the kex2Δ mutant secreted only the pro-form of Sapp1p. Characterization of this secreted proteinase form revealed that the Sapp1p signal peptide consists of 23 amino acids. Additionally, we prepared a plasmid with the SAPP1 coding sequence under control of its authentic CpSAPP1 promoter, which contains two GATAA motifs. While in C. parapsilosis SAPP1 expression is repressed by good low molecular weight nitrogen sources (e.g., ammonium ions), S. cerevisiae cells harboring this plasmid secreted a low concentration of active proteinase regardless of the type of nitrogen source used. Quantitative real-time PCR analysis of a set of genes related to nitrogen metabolism and uptake (GAT1, GLN3, STP2, GAP1, OPT1, and PTR2) obtained from S. cerevisiae cells transformed with either plasmid encoding SAPP1 under control of its own promoter or empty vector and cultivated in media containing various nitrogen sources also suggested that SAPP1 expression can be connected with the S. cerevisiae regulatory network. However, this regulation occurs in a different manner than in C. parapsilosis.

• MeSH
• Candida enzymology   genetics   MeSH
• Nitrogen metabolism   MeSH
• Endopeptidases metabolism   MeSH
• Real-Time Polymerase Chain Reaction MeSH
• Saccharomyces cerevisiae enzymology   genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Nitrogen MeSH
• Endopeptidases 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 transport activity and substrate specificity of two chimeras consisting of S. cerevisiae Nha1p's N-terminal regions (either first 125 or 184 AA) and the rest of the C. glabrata Cnh1p (up to the total protein length of 946 AA) were compared with those of the two native antiporters. Both chimeric transporters were functional upon expression in S. cerevisiae cells, their presence improved the ability of cells to grow in the presence of high external concentration of K(+), Na(+) or Rb(+) (as chlorides), but not in the presence of the smallest cation (Li(+)). Cation efflux confirmed the ability of chimeras to export cations and showed their significantly reduced transport capacity compared to the wild-type proteins. Despite the very high level of primary sequence identity (87 %) between the S. cerevisiae and C. glabrata plasma-membrane Na(+)/H(+) antiporters, various parts of these proteins are not exchangeable without affecting the antiporter's transport capacity.

• MeSH
• Candida glabrata drug effects   genetics   growth & development   metabolism   MeSH
• Potassium Chloride pharmacology   MeSH
• Sodium Chloride pharmacology   MeSH
• Fungal Proteins chemistry   genetics   metabolism   MeSH
• Molecular Sequence Data MeSH
• Sodium-Hydrogen Exchangers chemistry   genetics   metabolism   MeSH
• Cation Transport Proteins chemistry   genetics   metabolism   MeSH
• Recombinant Fusion Proteins chemistry   genetics   metabolism   MeSH
• Saccharomyces cerevisiae Proteins chemistry   genetics   metabolism   MeSH
• Saccharomyces cerevisiae drug effects   genetics   growth & development   metabolism   MeSH
• Amino Acid Sequence MeSH
• Sequence Analysis, DNA MeSH
• Sequence Alignment MeSH
• Salt Tolerance * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Potassium Chloride MeSH
• Sodium Chloride MeSH
• CNH1 protein, Candida albicans MeSH Browser
• Fungal Proteins MeSH
• Sodium-Hydrogen Exchangers MeSH
• NHA1 protein, S cerevisiae MeSH Browser
• Cation Transport Proteins MeSH
• Recombinant Fusion Proteins MeSH
• Saccharomyces cerevisiae Proteins 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

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.

• MeSH
• Metals, Alkali metabolism   MeSH
• Candida genetics   growth & development   metabolism   pathogenicity   MeSH
• Potassium metabolism   MeSH
• Microscopy, Fluorescence MeSH
• Fungal Proteins genetics   metabolism   MeSH
• Cations metabolism   MeSH
• Lithium metabolism   MeSH
• Membrane Proteins genetics   metabolism   MeSH
• Molecular Sequence Data MeSH
• Sodium-Hydrogen Exchangers genetics   metabolism   MeSH
• Cation Transport Proteins genetics   metabolism   MeSH
• Saccharomyces cerevisiae Proteins genetics   metabolism   MeSH
• Saccharomyces cerevisiae genetics   growth & development   metabolism   MeSH
• Protein Structure, Secondary MeSH
• Base Sequence MeSH
• Salts metabolism   MeSH
• Substrate Specificity MeSH
• Superoxide Dismutase-1 MeSH
• Superoxide Dismutase metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH
• Names of Substances
• Metals, Alkali MeSH
• CNH1 protein, Candida albicans MeSH Browser
• Potassium MeSH
• Fungal Proteins MeSH
• Cations MeSH
• Lithium MeSH
• Membrane Proteins MeSH
• Sodium-Hydrogen Exchangers MeSH
• NHA1 protein, S cerevisiae MeSH Browser
• Cation Transport Proteins MeSH
• Saccharomyces cerevisiae Proteins MeSH
• Salts MeSH
• Superoxide Dismutase-1 MeSH
• Superoxide Dismutase MeSH

Yarrowia lipolytica plasma-membrane Na+/H+ antiporter, encoded by the YlNHA2 gene, is a very efficient exporter of surplus sodium from the cytosol. Its heterologous expression in Saccharomyces cerevisiae wild-type laboratory strains increased their sodium tolerance more efficiently than the expression of ZrSod2-22 antiporter from the osmotolerant yeast Zygosaccharomvces rouxii.

• MeSH
• Antifungal Agents pharmacology   MeSH
• Gene Expression MeSH
• Fungal Proteins genetics   metabolism   MeSH
• Cloning, Molecular MeSH
• Sodium-Hydrogen Exchangers genetics   metabolism   MeSH
• Recombinant Proteins genetics   metabolism   MeSH
• Saccharomyces cerevisiae drug effects   genetics   growth & development   metabolism   MeSH
• Salts pharmacology   MeSH
• Yarrowia enzymology   genetics   MeSH
• Zygosaccharomyces enzymology   genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antifungal Agents MeSH
• Fungal Proteins MeSH
• Sodium-Hydrogen Exchangers MeSH
• Recombinant Proteins MeSH
• Salts MeSH