FIGURE 4.

Na⁺/H⁺ antiporter activities in the inverted membrane vesicles of E. coli expressing wild type NhaS3 or NhaS3 variants. A, antiporter activities of the NhaS3 variants. The locations of the negatively charged residues (Asp or Glu) that were replaced with Ala or Asn/Gln are indicated with arrows (top). The Roman numerals designate the numbers of the putative transmembrane spanning domains predicted by the TopPredII algorithm and counted from the N terminus of NhaS3. Na⁺/H⁺ antiporter activity was determined in inverted membrane vesicles by measuring the percentage of fluorescence dequenching observed after addition of 5 mm NaCl (bottom). B, immunological detection of NhaS3 variants containing mutations at Asp²¹⁷, Asp²¹⁸, and Glu⁴⁰² that showed no transport activities in A. The membrane proteins isolated from the strains expressing the nhaS3 variants were separated by SDS-polyacrylamide gel electrophoresis (12% acrylamide) and blotted, and the blots were probed using anti-NhaS3 antibodies. WT, wild type; EV, empty vector.