There are three different sodium transport systems (Ena1-4p, Nha1p, Nhx1p) in Saccharomyces cerevisiae. The effect of their absence on the tolerance to alkali-metal cations and on the membrane potential was studied. All three sodium transporters were found to participate in the maintenance of Na+, Li+, K+ and Cs+ homeostasis. Measurements of the distribution of a fluorescent potentiometric probe (diS-C3(3) assay) in cell suspensions showed that the lack of all three transporters depolarizes the plasma membrane. The overexpression of the Na+,K+/H+ antiporter Nha1 resulted in the hyperpolarization of the plasma membrane and consequently increased the sensitivity to Cs+, Tl+ and hygromycin B. This is the first evidence that the activity of a Na+,K+/H+ antiporter could play a role in the homeostatic regulation of the plasma membrane potential in yeast cells.

• MeSH
• draslík metabolismus   MeSH
• financování organizované MeSH
• koncentrace vodíkových iontů MeSH
• membránové potenciály MeSH
• membránové proteiny fyziologie   MeSH
• Na(+)-H(+) antiport fyziologie   MeSH
• proteiny přenášející kationty fyziologie   MeSH
• Saccharomyces cerevisiae - proteiny fyziologie   MeSH
• Saccharomyces cerevisiae fyziologie   růst a vývoj   MeSH
• sodík metabolismus   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
• alkalické kovy metabolismus   MeSH
• Candida MeSH
• draslík metabolismus   MeSH
• financování organizované MeSH
• fluorescenční mikroskopie MeSH
• fungální proteiny genetika   metabolismus   metabolismus   MeSH
• kationty metabolismus   MeSH
• lithium metabolismus   MeSH
• membránové proteiny genetika   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• Na(+)-H(+) antiport genetika   metabolismus   MeSH
• proteiny přenášející kationty genetika   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   růst a vývoj   MeSH
• sekundární struktura proteinů MeSH
• sekvence nukleotidů MeSH
• soli metabolismus   MeSH
• substrátová specifita MeSH
• superoxiddismutasa metabolismus   MeSH
• Publikační typ
• srovnávací studie MeSH

The physiological role of Candida albicans Cnh1, a member of the Na+/H+ antiporter family, was characterized. Though CaCnh1p had broad substrate specificity and mediated efflux of at least four alkali metal cations upon heterologous expression in Saccharomyces cerevisiae, its presence in C. albicans cells was important especially for potassium homeostasis. In C. albicans, CaCnh1p tagged with GFP was localized in the plasma membrane of cells growing as both yeasts and hyphae. Deletion of CNH1 alleles did not affect tolerance to NaCl, LiCl or CsCl, but resulted in increased sensitivity to high external concentrations of KCl and RbCl. The potassium and rubidium tolerance of a cnh1 homozygous mutant was fully restored by reintegration of CNH1 into the genome. The higher sensitivity of the cnh1/cnh1 mutant to external KCl was caused by a lower K+ efflux from these cells. Together, the functional characterization of the CaCnh1 antiporter in C. albicans revealed that this antiporter plays a significant role in C. albicans physiology. It ensures potassium and rubidium tolerance and participates in the regulation of intracellular potassium content of C. albicans cells.

• MeSH
• antibakteriální látky farmakologie   MeSH
• buněčná membrána chemie   MeSH
• Candida albicans metabolismus   MeSH
• cesium farmakologie   MeSH
• chlorid draselný farmakologie   MeSH
• chlorid lithný farmakologie   MeSH
• chlorid sodný farmakologie   MeSH
• chloridy farmakologie   MeSH
• delece genu MeSH
• draslík metabolismus   MeSH
• exprese genu MeSH
• financování organizované MeSH
• fungální léková rezistence MeSH
• fungální proteiny analýza   genetika   metabolismus   MeSH
• homeostáza MeSH
• hyfy chemie   MeSH
• klonování DNA MeSH
• kvasinky chemie   MeSH
• Na(+)-H(+) antiport analýza   genetika   metabolismus   MeSH
• rubidium farmakologie   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• substrátová specifita MeSH
• testy genetické komplementace MeSH

• MeSH
• agregace erytrocytů MeSH
• cystická fibróza MeSH
• krevní proteiny MeSH
• lidé MeSH
• membránové proteiny MeSH
• techniky in vitro MeSH
• Check Tag
• lidé MeSH

• MeSH
• buněčná membrána MeSH
• hemolýza MeSH
• viry ultrastruktura   MeSH

• MeSH
• buněčná membrána MeSH
• membránové potenciály MeSH
• svaly fyziologie   MeSH
• vápník MeSH

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.

• MeSH
• bodová mutace MeSH
• draslík metabolismus   MeSH
• fungální proteiny chemie   genetika   metabolismus   MeSH
• hydrofobní a hydrofilní interakce MeSH
• kationty metabolismus   MeSH
• konformace proteinů MeSH
• lithium metabolismus   MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• Na(+)-H(+) antiport chemie   genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sodík metabolismus   MeSH
• substrátová specifita MeSH
• Zygosaccharomyces chemie   genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• biologie buňky MeSH
• buněčná membrána fyziologie   MeSH
• fluidita membrány genetika   MeSH
• lipidy fyziologie   MeSH
• membránové lipidy fyziologie   MeSH
• tekutiny a sekrety tělesné fyziologie   MeSH
• Publikační typ
• kongresy MeSH
• sborníky MeSH

• Konspekt
• Buněčná biologie. Cytologie
• NLK Obory
• cytologie, klinická cytologie
• biochemie
• biologie

• MeSH
• antigeny virové MeSH
• buněčná membrána MeSH
• experimenty na zvířatech MeSH
• imunologické techniky MeSH
• lymfatické uzliny cytologie   imunologie   MeSH
• onkogenní viry imunologie   MeSH
• radioimunoanalýza MeSH
• skot MeSH
• Check Tag
• skot MeSH

The family of Nha antiporters mediating the efflux of alkali metal cations in exchange for protons across the plasma membrane is conserved in all yeast species. Yarrowia lipolytica is a dimorphic yeast, phylogenetically very distant from the model yeast Saccharomyces cerevisiae. A search in its sequenced genome revealed two genes (designated as YlNHA1 and YlNHA2) with homology to the S. cerevisiae NHA1 gene, which encodes a plasma membrane alkali metal cation/H+ antiporter. Upon heterologous expression of both YlNHA genes in S. cerevisiae, we showed that Y. lipolytica antiporters differ not only in length and sequence, but also in their affinity for individual substrates. While the YlNha1 protein mainly increased cell tolerance to potassium, YlNha2p displayed a remarkable transport capacity for sodium. Thus, Y. lipolytica is the first example of a yeast species with two plasma membrane alkali metal cation/H+ antiporters differing in their putative functions in cell physiology; cell detoxification vs. the maintenance of stable intracellular pH, potassium content and cell volume.

• MeSH
• antiportéry metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• časové faktory MeSH
• draslík metabolismus   MeSH
• exprese genu MeSH
• financování organizované MeSH
• fungální proteiny metabolismus   MeSH
• rekombinantní fúzní proteiny metabolismus   MeSH
• Saccharomyces cerevisiae cytologie   MeSH
• sekvenční analýza proteinů MeSH
• sodík metabolismus   MeSH
• soli farmakologie   MeSH
• transport proteinů MeSH
• Yarrowia fyziologie   metabolismus   účinky léků   MeSH