Biochemical characterization of the C4-dicarboxylate transporter DctA from Bacillus subtilis

Abstract

Bacterial secondary transporters of the DctA family mediate ion-coupled uptake of C(4)-dicarboxylates. Here, we have expressed the DctA homologue from Bacillus subtilis in the Gram-positive bacterium Lactococcus lactis. Transport of dicarboxylates in vitro in isolated membrane vesicles was assayed. We determined the substrate specificity, the type of cotransported ions, the electrogenic nature of transport, and the pH and temperature dependence patterns. DctA was found to catalyze proton-coupled symport of the four C(4)-dicarboxylates from the Krebs cycle (succinate, fumurate, malate, and oxaloacetate) but not of other mono- and dicarboxylates. Because (i) succinate-proton symport was electrogenic (stimulated by an internal negative membrane potential) and (ii) the divalent anionic form of succinate was recognized by DctA, at least three protons must be cotransported with succinate. The results were interpreted in the light of the crystal structure of the homologous aspartate transporter Glt(Ph) from Pyrococcus horikoshii.

FIG. 1.

Purification of DctA_Bs. (A) Protein samples from different steps throughout the purification were run on an SDS-12.5% polyacrylamide gel, which was stained with Coomassie brilliant blue. Lanes: 1, membrane vesicles (10 μg of protein); 2, DDM-soluble fraction; 3, insoluble fraction; 4, flowthrough from the nickel-Sepharose column; 5, wash fraction from the Ni-Sepharose column; 6 to 8, three elution fractions from the nickel-Sepharose column; and 9, peak fraction (at an elution volume of ∼11 ml) from the size exclusion chromatography step. The arrow indicates DctA_Bs. M, markers; Mw, molecular mass. (B) The peak elution fraction from the nickel-Sepharose column (lane 7) was subjected to size exclusion chromatography with a Superdex-200 column. The chromatogram is shown.

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.

FIG. 3.

Succinate transport by DctA_Bs in membrane vesicles in the presence of putative transport inhibitors. Transport of [¹⁴C]succinate (3.1 μM) in the presence of , , and ΔΨ (−118 mV) was measured as described in the legend to Fig. 2A. The external buffer was supplemented with a 50-fold excess of the indicated compounds. Initial transport rates were determined as described in the legend to Fig. 2B and normalized with respect to the rate without inhibitors [100%, corresponding to 37.5 pmol (mg protein·s)⁻¹]. Measurements were performed in quadruplicate, and standard deviations are shown.

FIG. 4.

Succinate counterflow by DctA_Bs. Membrane vesicles were loaded with 50 mM potassium phosphate (pH 7.0) supplemented with 1 mM succinate (closed circles), fumarate (open circles), malate (closed triangles), oxaloacetate (open triangles), maleate (open squares), citrate (closed squares), or orotate (closed diamonds) and diluted 200-fold in external buffer (50 mM potassium phosphate, pH 7.0) supplemented with 3.1 μM [¹⁴C]succinate. As a negative control, membrane vesicles that were not loaded with any substrate (open diamonds) or vesicles loaded with 1 mM succinate (crosses) in which ThiT was expressed were used. Measurements were done in triplicate; error bars indicate standard deviations.

FIG. 5.

pH dependence of succinate transport. Membrane vesicles were loaded with a mixture of 50 mM K-MES and 50 mM K-HEPES adjusted to a pH of 6, 7, or 8, and the loaded vesicles were diluted in isosmotic Na-MES-Na-HEPES buffer adjusted to a pH of 5, 6, or 7. The external buffer contained 3.1 μM [¹⁴C]succinate and 0.5 μM valinomycin.

FIG. 6.

pH dependence of succinate transport during counterflow. Membrane vesicles were loaded with 50 mM potassium phosphate, supplemented with 1 mM unlabeled succinate, at the indicated pHs. Membrane vesicles were diluted 100-fold in the same external buffer but supplemented with 3.1 μM [¹⁴C]succinate instead of the unlabeled succinate. Error bars indicate the standard deviations, taken from three measurements.

FIG. 7.

Succinate transport in the presence of membrane potentials of different magnitudes. Membrane vesicles were loaded with 100 mM Na-HEPES buffer (pH 7.5) supplemented with 10 mM KCl. Membrane vesicles were diluted in external buffer consisting of Na-MES, pH 5.5, and 3.1 μM [¹⁴C]succinate supplemented with 0 mM KCl (closed circles), 2 mM KCl (open circles), 10 mM KCl (closed triangles), or 50 mM KCl (open triangles) in the presence of 0.5 μM valinomycin. The concentration of Na-MES was adjusted to ensure that the external and internal solutions were isosmotic. As a control, succinate transport in membrane vesicles in which ThiT was expressed was measured with 0 mM KCl in the external buffer (closed squares). The values of the K⁺ equilibrium potentials are indicated.