3 ASPARTIC RESIDUES IN MEMBRANE-SPANNING REGIONS OF NA+ H+ ANTIPORTERFROM VIBRIO-ALGINOLYTICUS PLAY A ROLE IN THE ACTIVITY OF THE CARRIER/

The Na+/H+ antiporter gene from Vibrio alginolyticus restores the grow th of an nhaA-defective strain of Escherichia coli, NM81, in a high Na Cl medium (Nakamura, T., Komano, Y., Itaya, E., Tsukamoto, K., Tsuchiy a, T. and Unemoto, T. (1994) Biochim. Biophys. Acta 1190, 465-468). Th is gene, named nhaAv, allowed the nhaA-defective coli strains, NM81(De lta haA) and RS1 (Delta nhaA, chnA(-)), to extrude Na+ at alkaline pH. The extrusion of Na+ occurred against its chemical gradient in the pr esence of membrane-permeable amine. Thus, the nhaAv gene product is fu nctional as an electrogenic Na+/H+ antiporter in E. coli cells. The Nh aAv protein has only four acidic amino acid residues in the putative m embrane-spanning regions, that is, Asp-57, Asp-125. Asp-155 and Asp-15 6, and these Asp residues are conserved in NhaA from E. coli. Asp-lll, which is predicted to be in a loop region between the transmembrane s egments is also conserved in NhaA. Thus, each conserved Asp residue wa s replaced with asparagine by a site-directed mutagenesis. E. coli NM8 1 cells containing a plasmid harboring the nhaAv gene mutated at Asp-1 25, -155 or -156 could neither grow in a high NaCl medium nor extrude Naf at alkaline pH against its chemical gradient. These results show t hat Asp-125, -155 and -156, but not Asp-57 and -111, play a role in th e activity of the Na+/H+ antiporter, NhaAv.