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+

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 Ni2+- 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.