sodium proton exchanger
Dotaz
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European heart journal, ISSN 1520-765X vol. 1, suppl. K, July 1999
K49 s. : tab., il. ; 30 cm
- MeSH
- koronární nemoc farmakoterapie MeSH
- Na(+)-H(+) antiport antagonisté a inhibitory MeSH
- Publikační typ
- kongresy MeSH
- Konspekt
- Patologie. Klinická medicína
- NLK Obory
- kardiologie
- angiologie
American journal of cardiology, ISSN 0002-9149 vol. 83, 10A, May 1999
25G s. : il. ; 30 cm
- MeSH
- ischemická choroba srdeční MeSH
- Na(+)-H(+) antiport MeSH
- Publikační typ
- kongresy MeSH
- Konspekt
- Patologie. Klinická medicína
- NLK Obory
- kardiologie
- angiologie
Na(+)/H+exchangers form a broad family of transporters that mediate opposing fluxes of alkali metal cations and protons across cell membranes. They play multiple roles in different organisms (protection from toxic cations, regulation of cell volume or pH). Rat NHE2 exchanger was expressed in a Saccharomyces cerevisiae mutant strain lacking its own exporters of alkali metal cations. Though most of the overexpressed NHE2 remained entrapped in the secretory pathway, part of it reached the plasma membrane and mediated K+ efflux from the yeast. We demonstrate for the first time that a mammalian Na(+)/H+ exchanger transports alkali metal cations in yeast in the opposite direction than in mammalian cells, and that the substrate specificity of the rat NHE2 exchanger is limited only to potassium cations upon expression in yeast cells.
- MeSH
- draslík metabolismus MeSH
- finanční podpora výzkumu jako téma MeSH
- klonování DNA MeSH
- krysa rodu rattus MeSH
- Na(+)-H(+) antiport genetika metabolismus MeSH
- rekombinantní fúzní proteiny genetika metabolismus MeSH
- Saccharomyces cerevisiae cytologie genetika metabolismus MeSH
- substrátová specifita MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
The yeast Nha1p Na(+), K(+)/H(+) antiporter has a house-keeping role in pH and cation homeostasis. It is also needed to alleviate excess Na(+) or K(+) from the cytoplasm under high external concentrations of these cations. Erv14p, a putative cargo receptor for transmembrane proteins is required for trafficking of Nha1p from the endoplasmic reticulum to the plasma membrane. Sensitivity to high Na(+) concentrations of the erv14 mutant associated to the intracellular mislocalization of Nha1p-GFP, together with a lower Na(+) efflux, indicate the involvement of this mutual association to accomplish the survival of the yeast cell upon sodium stress. This observation is supported by the protein-protein interaction between Erv14p and Nha1p detected by the mating-based Split Ubiquitin System and co-immunoprecipitation assays. Our results indicate that even though Erv14p interacts with Nha1p through the TMD, the C-terminal is important not only for the efficient delivery of Nha1p to the plasma membrane but also for its dimerization to accomplish its role in yeast salt tolerance.
- MeSH
- biologický transport MeSH
- chlorid sodný metabolismus farmakologie MeSH
- draslík metabolismus farmakologie MeSH
- interakční proteinové domény a motivy MeSH
- kationty jednomocné MeSH
- membránové proteiny chemie genetika metabolismus MeSH
- multimerizace proteinu MeSH
- Na(+)-H(+) antiport chemie genetika metabolismus MeSH
- proteiny přenášející kationty chemie genetika metabolismus MeSH
- protony * MeSH
- regulace genové exprese u hub * MeSH
- rekombinantní fúzní proteiny chemie genetika metabolismus MeSH
- Saccharomyces cerevisiae - proteiny chemie genetika metabolismus MeSH
- Saccharomyces cerevisiae účinky léků genetika metabolismus MeSH
- sekundární struktura proteinů MeSH
- tolerance k soli MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: In yeast, 14-3-3 proteins bind to hundreds of phosphorylated proteins and play a role in the regulation of many processes including tolerance to NaCl. However, the mechanism of 14-3-3 involvement in the cell answer to salt or osmotic stresses is weakly understood. METHODS: We studied the role of the Saccharomyces cerevisiae 14-3-3 homologs Bmh1 and Bmh2 in the regulation of alkali-metal-cation homeostasis using the genetic-interaction approach. Obtained results were confirmed with the Bimolecular-Fluorescence-Complementation method. RESULTS: Deletion of BMH1, encoding the major 14-3-3 isoform, resulted in an increased sensitivity to Na+, Li+ and K+ and to cationic drugs but did not affect membrane potential. This bmh1Δ phenotype was complemented by overexpression of BMH2. Testing the genetic interaction between BMH genes and genes encoding plasma-membrane cation transporters revealed, that 14-3-3 proteins neither interact with the potassium uptake systems, nor with the potassium-specific channel nor with the Na+(K+)-ATPases. Instead, a genetic interaction was identified between BMH1 and NHA1 which encodes an Na+(K+)/H+ antiporter. In addition, a physical interaction between 14-3-3 proteins and the Nha1 antiporter was shown. This interaction does not depend on the phosphorylation of the Nha1 antiporter by Hog1 kinase. Our results uncovered a previously unknown interaction partner of yeast 14-3-3 proteins and provided evidence for the previously hypothesized involvement of Bmh proteins in yeast salt tolerance. GENERAL SIGNIFICANCE: Our results showed for the first time that the yeast 14-3-3 proteins and an alkali-metal-cation efflux system interact and that this interaction enhances the cell survival upon salt stress.
- MeSH
- alkalické kovy metabolismus MeSH
- fosforylace MeSH
- homeostáza fyziologie MeSH
- kationty metabolismus MeSH
- membránové potenciály MeSH
- Na(+)-H(+) antiport metabolismus MeSH
- proteiny 14-3-3 metabolismus MeSH
- proteiny přenášející kationty metabolismus MeSH
- protony MeSH
- průtoková cytometrie MeSH
- Saccharomyces cerevisiae - proteiny metabolismus MeSH
- Saccharomyces cerevisiae růst a vývoj metabolismus MeSH
- viabilita buněk MeSH
- western blotting MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem 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.
- MeSH
- lidé MeSH
- Na(+)-H(+) antiport * chemie genetika MeSH
- protony * MeSH
- Saccharomyces cerevisiae genetika metabolismus MeSH
- sekvence aminokyselin MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
