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,th...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 Halo-bacillus 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 nhaH△C.Salt tolerance experiment demonstrated that the deletion of hydrophilic C-terminal nine amino acid resi-dues 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/nhaH△C 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.展开更多
基金the National High Technology Research and Development Program of China (Grant No. 2003AA241150)International Cooperation Program for Science and Technology (Grant No.2006DFA31060)
文摘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 Halo-bacillus 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 nhaH△C.Salt tolerance experiment demonstrated that the deletion of hydrophilic C-terminal nine amino acid resi-dues 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/nhaH△C 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.
