Fig. 6.
Cation-driven proton translocation by MdfA. (A and B) The ΔpH measurements in inverted membrane vesicles prepared from E. coli strain UTLΔmdfA::kan (A) or E. coli strain EP432 (B). Vesicles were diluted to 0.6 mg/ml membrane proteins in 1 mM Tris·HCl, pH 7/10 mM magnesium sulfate/1 μM ACMA. ATP·Tris (0.4 mM) was added to impose a ΔpH (internal acidic). In A and B Left, the first arrow indicates the time of addition of ATP, and the second arrow indicates addition of 100 mM sodium or potassium gluconate (as indicated). In B Right, a third arrow indicates the time of addition of 2.5 μM CCCP. Traces marked as control refer to vesicles prepared from cells not expressing MdfA. R112C stands for vesicles prepared from cells expressing the nonfunctional mutant R112C. WT refers to vesicles prepared from cells expressing wild-type MdfA. (C and D) ΔpH measurements in proteoliposomes using acridine orange as a fluorescent probe. A ΔpH (inside acidic) was imposed by means of an ammonium chloride chemical gradient across the membranes of liposomes devoid of protein (Control) or containing the nonfunctional mutant R112C, or wild-type MdfA (WT). The left arrow indicates time of addition of liposomes or proteoliposomes, the middle arrow indicates time of addition of 100 mM sodium gluconate (C) or 100 mM potassium gluconate (D), and the right arrow indicates time of addition of 20 μM CCCP.