FIG. 2.

Succinate transport by DctA_Bs in membrane vesicles. (A) Membrane vesicles containing DctA_Bs (circles) or ThiT (negative control; triangles) were loaded with 100 mM K-HEPES (pH 7.5). The vesicles were diluted 100-fold in an isosmotic Na-MES buffer, pH 5.5 (consisting of ∼135 mM MES [morpholineethanesulfonic acid] adjusted to pH 5.5 with NaOH), containing 3.1 μM [¹⁴C]succinate in the presence (closed symbols) or absence (open symbols) of 0.5 μM valinomycin. In this way, chemical gradients for protons and sodium ions (and ) were created. In the presence of valinomycin, an additional K⁺ diffusion potential, ΔΨ (Nernst potential, −118 mV), was created. (B) Initial [¹⁴C]succinate transport rates in the presence of various gradients. Columns correspond to the presence of gradients as follows: 1, , , and ΔΨ; 2, and ΔΨ; 3, and ; 4, ; 5, and ΔΨ; and 6, negative control. Initial transport rates were calculated from data for the 15-s time point (see panel A). Data were normalized, and the highest measured rate [47.4 pmol (mg protein·s)⁻¹] was set at 100%. Proton and sodium ion gradients (and ) in the presence or absence of ΔΨ were created as described in the legend to panel A. To create in the absence of , external methylglucamine-MES buffer (pH 5.5) was used instead of Na-MES buffer. Again, the addition of valinomycin was used to create a K⁺ diffusion membrane potential (ΔΨ) where indicated. To create and ΔΨ in the absence of , Na-HEPES (pH 7.5) was used as the external buffer and valinomycin was present. ThiT-harboring vesicles were used as a control in the presence of all three gradients. All data are averages of three independent measurements; error bars indicate standard deviations.