ESSENTIAL ASPARTIC-ACID RESIDUES, ASP-133, ASP-163 AND ASP-164, IN THE TRANSMEMBRANE HELICES OF A NA+ H+ ANTIPORTER (NHAA) FROM ESCHERICHIA-COLI/

The importance of negatively charged residues in transmembrane helices of many cation-coupled transporters has been widely demonstrated, Fou r Asp residues were located in the putative transmembrane helices of t he Escherichia coli Na+/H+ antiporter, NhaA, We replaced each of these Asp residues by Asn in plasmid encoded nhaA and expressed these const ructs in an E, coli mutant defective in both nhaA and nhaB, Substituti on of Asp-65 or Asp-282 (in the extramembrane region) had no effect on supporting the host mutant growth in the high NaCl- or LiCl-containin g medium, and these two mutants had normal Na+/H+ and Li+/H+ antiporte r activities, In contrast, substitution of Asp-133, Asp-163 or Asp-164 was detrimental to survival of the host mutant and impaired both Na+/ H+ and Li+/H+ antiporter activities, These three Asp residues, conserv ed in the nhaA homologs from different species and which are located c losely in the 3rd and 4th putative transmembrane helices, appear to pl ay important roles in cation binding and transport.