title: Monomers of the NhaA Na+/H+ antiporter of Escherichia coli are fully functional yet dimers are beneficial under extreme stress conditions at alkaline pH in the presence of Na+ or Li+.
abstract: NhaA, the Na(+)/H(+) antiporter of Escherichia coli, exists in the native membrane as a homodimer of which two monomers have been suggested to be attached by a beta-hairpin at the periplasmic side of the membrane. Constructing a mutant deleted of the beta-hairpin, NhaA/Delta(Pro(45)-Asn(58)), revealed that in contrast to the dimeric mobility of native NhaA, the mutant has the mobility of a monomer in a blue native gel. Intermolecular cross-linking that monitors dimers showed that the mutant exists only as monomers in the native membrane, proteoliposomes, and when purified in beta-dodecyl maltoside micelles. Furthermore, pull-down experiments revealed that, whereas as expected for a dimer, hemagglutinin-tagged wild-type NhaA co-purified with His-tagged NhaA on a Ni(2+)-NTA affinity column, a similar version of the mutant did not. Remarkably, under routine stress conditions (0.1 m LiCl, pH 7 or 0.6 m NaCl, pH 8.3), the monomeric form of NhaA is fully functional. It conferred salt resistance to NhaA- and NhaB-deleted cells, and whether in isolated membrane vesicles or reconstituted into proteoliposomes exhibited Na(+)/H(+) antiporter activity and pH regulation very similar to wild-type dimers. Remarkably, under extreme stress conditions (0.1 m LiCl or 0.7 m NaCl at pH 8.5), the dimeric native NhaA was much more efficient than the monomeric mutant in conferring extreme stress resistance.
authors.affiliation: Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, 91904 Jerusalem, Israel.
authors.forename: Abraham
authors.initials: A
authors.lastname: Rimon
authors.affiliation: Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, 91904 Jerusalem, Israel.
authors.forename: Tzvi
authors.initials: T
authors.lastname: Tzubery
authors.affiliation: Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, 91904 Jerusalem, Israel. Electronic address: etana@vms.huji.ac.il.
authors.forename: Etana
authors.initials: E
authors.lastname: Padan
mesh_terms: D019281:Dimerization; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000502:physiology*; D006863:Hydrogen-Ion Concentration; D008094:Lithium / Q000494:pharmacology*; D012964:Sodium / Q000494:pharmacology*; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry* / Q000502:physiology*
chemical_list: D029968:Escherichia coli Proteins; C102839:NhaA protein, E coli; D017923:Sodium-Hydrogen Exchangers; D008094:Lithium; D012964:Sodium