Oligomerization of NhaA, the Na+/H+ antiporter of Escherichia coli in the membrane and its functional and structural consequences

Recently, a two-dimensional crystal structure of NhaA, the Na+/H+ antiporte r of Escherichia coli has been obtained [Williams, K. A., Kaufer, U. G., Pa dan, E., Schuldiner, S. and Kuhlbrandt, W. (1999) EMBO J., 18, 3558-3563]. In these crystals NhaA exists as a dimer. Using biochemical and genetic app roaches here we show that NhaA exists in the native membrane as a homooligo mer. Functional complementation between the polypeptides of NhaA was demons trated by coexpression of pairs of conditional lethal (at high pH in the pr esence of Naf) mutant alleles of nhaA in EP432, a strain lacking antiporter s. Physical interaction in the membrane was shown between the His-tagged Nh aA polypeptide which is readily affinity purified from DM-solubilized membr anes with a Ni2+-NTA column and another which is not; only when coexpressed did both copurify on the column. The organization of the oligomer in the m embrane was studied in situ by site-directed cross-linking experiments. Cys teine residues were introduced-one per NhaA-into certain loops of Cys-less NhaA, so that only intermolecular cross-linking could take place, Different linker-size cross-linkers were applied to the membranes, and the amount of the cross-linked protein was analyzed by mobility shift on SDS-PAGE. The r esults are consistent with homooligomeric NhaA and the location of residue 254 in the interface between monomers. Intermolecular cross-linking of V254 C caused an acidic shift in the pH profile of NhaA.