title: A hydrophobic filter confers the cation selectivity of Zygosaccharomyces rouxii plasma-membrane Na+/H+ antiporter.
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 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.
authors.affiliation: Department of Membrane Transport, Institute of Physiology, Academy of Sciences of the Czech Republic, v.v.i., Videnska 1083, Prague 4, Czech Republic 142 20. Electronic address: olga.zimmermannova@fgu.cas.cz.
authors.forename: Olga
authors.initials: O
authors.lastname: Kinclova-Zimmermannova
authors.affiliation: Drug Resistance Mechanism and Modulation Group, Molecular and Structural Basis of Infectious Systems, National Centre for Scientific Research and Lyon I University Laboratory No. 5086, Institute of Biology and Chemistry of Proteins, Lyon 69 367, France. Electronic address: pierre.falson@ibcp.fr.
authors.forename: Pierre
authors.initials: P
authors.lastname: Falson
authors.affiliation: Department of Membrane Transport, Institute of Physiology, Academy of Sciences of the Czech Republic, v.v.i., Videnska 1083, Prague 4, Czech Republic 142 20. Electronic address: denis.cmunt@fgu.cas.cz.
authors.forename: Denis
authors.initials: D
authors.lastname: Cmunt
authors.affiliation: Department of Membrane Transport, Institute of Physiology, Academy of Sciences of the Czech Republic, v.v.i., Videnska 1083, Prague 4, Czech Republic 142 20. Electronic address: hana.sychrova@fgu.cas.cz.
authors.forename: Hana
authors.initials: H
authors.lastname: Sychrova
mesh_terms: D000595:Amino Acid Sequence; D002412:Cations / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D008094:Lithium / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017354:Point Mutation; D011188:Potassium / Q000378:metabolism; D011487:Protein Conformation; D012964:Sodium / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D020068:Zygosaccharomyces / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D002412:Cations; D005656:Fungal Proteins; D017923:Sodium-Hydrogen Exchangers; D008094:Lithium; D012964:Sodium; D011188:Potassium
keywords: plasma membrane; potassium transport; sodium proton exchanger; substrate specificity; yeast