The short C-terminal hydrophilic domain of NhaH Na+/H+ antiporter from Halobacillus dabanensis with roles in resistance to salt and in pH sensing

Abstract

The Na+/H+ antiporter plays key roles in maintaining low cytoplasmic Na+ level and pH homeostasis, while little is known about the Carboxyl-terminal hydrophilic tails of prokaryotic antiporters. In our previous study, the first Na+/H+ antiporter gene nhaH from moderate halophiles was cloned from Halobacillus dabanensis D-8 by functional complementation. A topological model suggested that only nine amino acid residues (395PLIKKLGMI403) existed in the hydrophilic C-terminal domain of NhaH. The C-terminal truncated mutant of NhaH was constructed by PCR strategy and designated as nhaHΔC. Salt tolerance experiment demonstrated that the deletion of hydrophilic C-terminal nine amino acid residues significantly inhibited the complementation ability of E. coli KNabc, in which three main Na+/H+ antiporters nhaA, nhaB and chaA were deleted. Everted membrane vesicles prepared from E. coli KNabc/nhaHΔC decreased both Na+/H+ and Li+/H+ exchange activities of NhaH, and also resulted in an acidic shift of its pH profile for Na+, indicating a critical role of the short C-terminal domain of NhaH antiporter in alkali cation binding/translocation and pH sensing.