A Hydrophobic Filter Confers the Cation Selectivity of Zygosaccharomyces rouxii Plasma-Membrane Na+/H+ Antiporter. Issue 8 (24th April 2015)

• Record Type: Journal Article
• Authors: Kinclova-Zimmermannova, Olga; Falson, Pierre; Cmunt, Denis; Sychrova, Hana
• Is Part Of: Journal of molecular biology. Volume 427:Issue 8(2015:Apr. 15)
• Page Start: 1681
• Page End: 1694
• Publication Date: 2015-04-24
• Subjects: 3D three-dimensional -- TMS transmembrane segments -- CPA cation/proton antiporter; yeast -- plasma membrane -- sodium proton exchanger -- substrate specificity -- potassium transport; Molecular biology -- Periodicals; Biology -- Periodicals; Biochemistry -- Periodicals; Bacteriology -- Periodicals; Molecular Biology -- Periodicals; Biochemistry -- Periodicals; Biologie moléculaire -- Périodiques; Biologie -- Périodiques; Biochimie -- Périodiques; Moleculaire biologie; Biochemistry; Biology; Molecular biology; Periodicals; 572.805
• DOI: 10.1016/j.jmb.2015.02.012
• Languages: English
• ISSNs: 0022-2836

Abstract

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 Zr Sod2-22 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 Zr Sod2-22 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. Graphical abstract: Highlights: Yeast Na + /H + antiporters differ in cation selectivity for Li +, Na + and/or K + . Three-dimensional model reveals a hydrophobic filter in the cation translocation pathway. T141, A179 and V375 (transmembrane segments 4, 5 and 11, respectively) form a cationic filter in Z. rouxii Sod2-22. Cation selectivity is altered by repositioning of a –CH3 within the filter. Region conferring alkali-metal cation specificity of Na + /H + antiporters is found.