Figure 6

Cation-driven proton translocation by MdtM. Cation-driven proton translocation by MdtM at alkaline pH was measured by the fluorescence dequenching of acridine orange upon addition of Na⁺ gluconate (A) or K⁺ gluconate (B) to inverted vesicles derived from antiporter-deficient E. coli TO114 cells that overexpressed recombinant wild-type MdtM (black traces) or the dysfunctional MdtM D22A mutant (grey traces). Respiration-dependent generation of ΔpH (acid inside) was established by addition of lactate as indicated and once the fluorescence quench of acridine orange reached a steady state, Na⁺ gluconate or K⁺ gluconate was added to a final concentration of 100 mM. Addition of 100 μM CCCP at the time indicated was used to completely dissipate ΔpH. The traces are representative of experiments performed in triplicate on at least two separate preparations of inverted vesicles. The fluorescence intensity of each measurement is represented as a percentage of the initial acridine orange fluorescence signal prior to addition of lactate.