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
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.
- MeSH
- cytosol chemie MeSH
- draslík metabolismus MeSH
- exprese genu MeSH
- koncentrace vodíkových iontů MeSH
- lithium metabolismus toxicita MeSH
- proteiny přenášející kationty genetika metabolismus MeSH
- Saccharomyces cerevisiae účinky léků genetika metabolismus MeSH
- tolerance léku * MeSH
- Zygosaccharomyces účinky léků metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- MeSH
- biologický transport MeSH
- buněčná membrána metabolismus fyziologie MeSH
- draslík metabolismus farmakologie MeSH
- druhová specificita MeSH
- homeostáza MeSH
- hygromycin B farmakologie MeSH
- kationty MeSH
- lithium metabolismus farmakologie MeSH
- membránové potenciály MeSH
- mutace MeSH
- regulace genové exprese u hub MeSH
- Saccharomyces cerevisiae - proteiny genetika metabolismus MeSH
- Saccharomyces cerevisiae klasifikace genetika růst a vývoj fyziologie MeSH
- sodík metabolismus farmakologie MeSH
- tolerance k soli fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- duševní poruchy farmakoterapie MeSH
- lidé MeSH
- lithium * metabolismus terapeutické užití MeSH
- Check Tag
- lidé 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
- MeSH
- amiodaron metabolismus škodlivé účinky MeSH
- gonadotropiny hypofyzární metabolismus škodlivé účinky MeSH
- interferony metabolismus škodlivé účinky účinky léků MeSH
- interleukin-2 imunologie metabolismus škodlivé účinky MeSH
- kontrastní látky kontraindikace otrava MeSH
- lidé MeSH
- lithium metabolismus škodlivé účinky MeSH
- sloučeniny jodu metabolismus škodlivé účinky MeSH
- štítná žláza metabolismus sekrece účinky léků MeSH
- Check Tag
- lidé MeSH
- MeSH
- Alzheimerova nemoc farmakoterapie MeSH
- amyloidní plaky účinky léků MeSH
- apoptóza účinky záření MeSH
- bipolární porucha farmakoterapie MeSH
- exprese genu účinky léků MeSH
- finanční podpora výzkumu jako téma MeSH
- inositolfosfáty metabolismus MeSH
- kinasa 3 glykogensynthasy účinky léků MeSH
- lidé MeSH
- lithium farmakologie metabolismus terapeutické užití MeSH
- neurofibrilární klubka účinky léků MeSH
- přehledová literatura jako téma MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- MeSH
- Ca(2+)-Mg(2+)-ATPasa metabolismus MeSH
- hypothalamus metabolismus MeSH
- krysa rodu rattus MeSH
- lithium aplikace a dávkování metabolismus terapeutické užití MeSH
- modely nemocí na zvířatech MeSH
- mozková kůra metabolismus MeSH
- sodíko-draslíková ATPasa metabolismus MeSH
- thalamus metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- přehledy MeSH
- srovnávací studie MeSH
