Multidrug transport protein norM from vibrio cholerae simultaneously couples to sodium- and proton-motive force

Abstract

Membrane transporters belonging to the multidrug and toxic compound extrusion family mediate the efflux of unrelated pharmaceuticals from the interior of the cell in organisms ranging from bacteria to human. These proteins are thought to fall into two classes that couple substrate efflux to the influx of either Na(+) or H(+). We studied the energetics of drug extrusion by NorM from Vibrio cholerae in proteoliposomes in which purified NorM protein was functionally reconstituted in an inside-out orientation. We establish that NorM simultaneously couples to the sodium-motive force and proton-motive force, and biochemically identify protein regions and residues that play important roles in Na(+) or H(+) binding. As the positions of protons are not available in current medium and high-resolution crystal structures of multidrug and toxic compound extrusion transporters, our findings add a previously unrecognized parameter to mechanistic models based of these structures.

Keywords: Bioenergetics; Enzyme Mechanisms; Membrane Proteins; Membrane Transport; Multidrug Transporters.

FIGURE 1.

Ethidium binding and transport by NorM-VC in lactococcal membranes. A, Western analysis of total membrane protein in membrane vesicles (5 μg/lane) using anti-His₅ antibody shows that wild type (WT) and mutant proteins are expressed at similar levels in the cytoplasmic membrane of L. lactis. B, EMA photolabeling of NorM-VC in the presence or absence of Na⁺. Lactococcal membrane vesicles (25 mg) containing NorM-VC (WT) or lacking NorM protein (CTR) were incubated with 10 μm EMA in 50 mm Tris-Cl buffer (pH 7.4) containing 10 mm Na₂SO₄ or K₂SO₄, and 5 mm MgSO₄ at 37 °C for 15 min. Membrane vesicles were exposed to UV irradiation and solubilized total membrane proteins (10 μg) were loaded on a 12% SDS-PAGE gel. Coomassie staining (left panel, top) confirmed equal loading NorM-VC (WT) and lack of the protein in control lanes (CTR). Band intensity of ethidium fluorescence emission of NorM-VC signal (left panel, bottom) was quantified using ImageJ (right panel). **, p < 0.05, significantly different. C, ATP-depleted cells were preloaded with 2 μm ethidium until a saturation level was reached. Active ethidium efflux was initiated by addition of 25 mm glucose as a source of metabolic energy, and 1 mm Na₂SO₄ (or K₂SO₄ in control experiments) at the arrow. D, rate of active ethidium efflux (■) (right y axis) as a function of the Na⁺ concentration in the buffer. For measurements of facilitated ethidium efflux (●) (left y axis), ATP-depleted ethidium-loaded cells were diluted 40-fold in buffer after which the efflux rate was determined as a function of the external Na⁺ concentration in the absence of glucose. The rate of ethidium efflux in control cells lacking NorM-VC protein was subtracted from the rate observed in NorM-VC expressing cells. E–G, ionophores nigericin (N) or valinomycin (V) or both (0.5 μm each) were added to ethidium-loaded cells 3 min prior to the addition of glucose and 1 mm Na⁺.

FIGURE 2.

Ethidium transport by NorM-VC in DNA-loaded proteoliposomes. A, availability of the NH₂-terminal His₁₀ tag in WT NorM to cleavage by Proteinase K (+PK) at the external side of proteoliposomes compared with control without the protease (−PK). To make the His tag in the liposomal lumen accessible, 1% Triton X-100 was added to the proteoliposomes before proteolysis (+PK/Tx100). Subsequently, the remaining His tag was detected on an immune blot (2.5 μg of protein per lane) probed with anti-His₅ antibody. B–D, ethidium transport in proteoliposomes with imposed chemical Na⁺ gradient (ΔpNa, interior high), sodium-motive force (SMF) (= membrane potential (Δψ)-ZΔpNa, interior positive and high, in which Z ≅ 58 mV at 20 °C), chemical proton gradient (ΔpH, interior acid), proton-motive force (PMF = Δψ-ZΔpH, interior positive and acid), or SMF plus PMF (= Δψ-ZΔpNa-ZΔpH). Experiments with imposed PMF and ΔpH in C were performed in the presence of Na⁺ ([Na⁺]_in = [Na⁺]_out = 1 mm), whereas those in D were performed in the complete absence of Na⁺ in plastic containers. The data represent observations in at least three separate experiments using independent batches of purified proteins, cells, and proteoliposomes. Error bars represent the mean ± S.E.

FIGURE 3.

Structure model of outward-facing NorM-VC. Stereoview (PDB 3MKT) showing TM1–6 in gray and TM7–12 in green. Asp-36 (red) and neighboring Asn-174 and Asn-178 residues (light blue) are shown in a blue circle. Glu-255 (purple) and Asp-371 (orange) are shown in a purple circle with neighboring aromatic residues in yellow. Top, extracellular side; bottom, intracellular side.

FIGURE 4.

Role of Asp-36 and neighboring residues in Na⁺ coupling. A and B, in contrast to observations for WT, active ethidium efflux in cells expressing the D36N (A) or N174A (B) mutant was not stimulated by the addition of 1 mm Na⁺. C, rate of facilitated ethidium efflux as a function of Na⁺ concentration in ATP-depleted cells containing the N174A mutant demonstrates reduced affinity of the mutant for Na⁺ compared with WT (compare Fig. 1D). D and E, ethidium transport in DNA-loaded proteoliposomes containing purified WT NorM-VC (D) or N174A protein (E), in which the SMF was imposed artificially. The standard SMF was imposed at [Na⁺]_in/[Na⁺]_out = 100/1 mm, whereas “SMF (low Na⁺)” was imposed at [Na⁺]_in/[Na⁺]_out = 10/0.1 mm. Traces are based on observations in four separate experiments using independent batches of cells and proteoliposomes. Error bars represent the mean ± S.E.

FIGURE 5.

Role of Glu-255, Asp-371, and neighboring residues in Na⁺ coupling. A, B, D, E, and H, active ethidium efflux activity in cells expressing E255Q (A), D371N (B), Y367A (D), F395A (E), or F288A (H) mutant was performed as described in the legend to Fig. 1C. C, F, and G, rates of facilitated ethidium efflux in ATP-depleted cells containing D371N (C), F395A (F), or F288A (G) mutant were measured as a function of the Na⁺ concentration as described for Fig. 1D. Error bars represent the mean ± S.E. of four independent observations.

FIGURE 6.

Studies on H⁺ coupling. A, effect of saturating amounts of ethidium (Et) on the pH of a low-buffered solution containing purified WT NorM-VC, carboxyl to amide mutants or no protein (CTR). In the histogram, only the decrease in pH/min for WT was significantly elevated compared with CTR (p < 0.005). B, ethidium transport in proteoliposomes containing purified D371N NorM-VC with imposed standard SMF ± PMF (as described in Fig. 2B), or Δψ (interior positive) or PMF (interior positive and acid) in the presence of Na⁺ ([Na⁺]_in = [Na⁺]_out = 1 mm as described in the legend to Fig. 2C). C, steady-state level in B was determined in four independent experiments. Error bars represent the mean ± S.E. **, p < 0.05, significantly different. The difference between ethidium uptake in the presence of SMF versus SMF + PMF was not significant for the D371A mutant.