Mechanism of melibiose/cation symport of the melibiose permease of Salmonella typhimurium

Abstract

The MelB permease of Salmonella typhimurium (MelB-ST) catalyzes the coupled symport of melibiose and Na(+), Li(+), or H(+). In right-side-out membrane vesicles, melibiose efflux is inhibited by an inwardly directed gradient of Na(+) or Li(+) and stimulated by equimolar concentrations of internal and external Na(+) or Li(+). Melibiose exchange is faster than efflux in the presence of H(+) or Na(+) and stimulated by an inwardly directed Na(+) gradient. Thus, sugar is released from MelB-ST externally prior to the release of cation in agreement with current models proposed for MelB of Escherichia coli (MelB-EC) and LacY. Although Li(+) stimulates efflux, and an outwardly directed Li(+) gradient increases exchange, it is striking that internal and external Li(+) with no gradient inhibits exchange. Furthermore, Trp → dansyl FRET measurements with a fluorescent sugar (2'-(N-dansyl)aminoalkyl-1-thio-β-D-galactopyranoside) demonstrate that MelB-ST, in the presence of Na(+) or Li(+), exhibits (app)K(d) values of ∼1 mM for melibiose. Na(+) and Li(+) compete for a common binding pocket with activation constants for FRET of ∼1 mM, whereas Rb(+) or Cs(+) exhibits little or no effect. Taken together, the findings indicate that MelB-ST utilizes H(+) in addition to Na(+) and Li(+). FRET studies also show symmetrical emission maximum at ∼500 nm with MelB-ST in the presence of 2'-(N-dansyl)aminoalkyl-1-thio-β-D-galactopyranoside and Na(+), Li(+), or H(+), which implies a relatively homogeneous distribution of conformers of MelB-ST ternary complexes in the membrane.

FIGURE 1.

[³H]Melibiose transport. A and B, downhill transport in DW2 intact cells with overexpressed MelB-ST (A) or MelB-EC (B). Calculated intracellular [³H]melibiose concentrations were plotted as a function of time. C, uptake with RSO vesicles containing MelB-ST. Square, circle, and triangle represent data that were obtained in the presence of H⁺, Na⁺, and Li⁺, respectively. Na⁺ or Li⁺ (20 mm) was added by premixing with [³H]melibiose. Error bars indicate S.E.

FIGURE 2.

Effect of ΔΨ on [³H]melibiose uptake. Active transport with RSO vesicles (pH 7.5) containing MelB-ST was carried out in the absence and presence of 20 mm Na⁺ (circle) or Li⁺ (triangle). At the 2-min time point of transport time course, 50 μm valinomycin (Vm, dashed lines), 1 μm nigericin (Ng, dotted lines), or both (dashed and dotted lines) were added into RSO vesicles.

FIGURE 3.

[³H]Melibiose efflux and exchange. Intracellular [³H]melibiose was expressed as a percentage of the zero time point in the presence of external 200 mm Na⁺ or Li⁺. Open (Efflux) and filled (exchange) symbols represent equimolar cation concentrations across the membrane. The dashed line represents external 200 mm Na⁺ or Li⁺. A, de-energized RSO vesicles were pre-equilibrated with 20 mm [³H]melibiose without Na⁺ and Li⁺. Right-filled hexagons and stars represent the presence of external Na⁺ and Li⁺, respectively. The dotted line represents the presence of 20 mm external cations. RSO vesicles were also treated with 3.6 mm MIANS for 5 h at 23 °C and diluted into 100 mm potassium P_i, pH 7.5, in the absence of melibiose (crossed square). B and C, de-energized RSO vesicles were pre-equilibrated with 20 mm [³H]melibiose and 20 mm Na⁺ (B) or Li⁺ (C). The left-filled symbol represents the dilution buffer without Na⁺ or Li⁺ (20/0 mm). The right-filled symbol represents the dilution buffer containing 200 mm Na⁺ or Li⁺ (20/200 mm).

FIGURE 4.

FRET. RSO vesicles prepared from DW2 cells without or with MelB at a protein concentration of 0.5 mg/ml were excited at 290 nm. Emissions were plotted between 410 and 570 nm. Sugar and cation were added into RSO vesicles sequentially according to the following order: black lines, Trp emission; red lines, D²G emission (10 μm); green lines, Na⁺, Li⁺, Rb⁺, or Cs⁺ (20 mm); blue lines, Na⁺ or Li⁺ (20 mm), Rb⁺ or Cs⁺ (100 mm); cyan and magenta lines, melibiose (130 mm). In MelB-ST with Rb⁺, green and cyan curves were removed for clarity. Data were measured with Hitachi F-7000 fluorescence spectrophotometer. A.U., absorbance units.

FIGURE 5.

Deduced FRET spectra. A and C, _diffFRET emission before and after the addition of excess melibiose was calculated from Fig. 4. Curves filled with light or dark gray represent the deduced _diffFRET spectra in the presence of Na⁺ or H⁺, and the open blue curves represent _diffFRET spectrum in the presence of Li⁺. B and D, Na⁺- and Li⁺-dependent FRET were calculated by subtracting the H⁺-coupled _diffFRET and plotting in filled and open curves, respectively. A.U., absorbance units.

FIGURE 6.

^appK_d for D²G. Titration of D²G was measured by FRET in the presence of H⁺ (square), Na⁺ (circle), or Li⁺ (triangle) with Hitachi F-7000 fluorescence spectrophotometer. _diffFRET representing the bound D²G was integrated between 460 and 540 nm for MelB-ST and 440 and 530 nm for MelB-EC, and the hyperbolic function was applied to fit data (Table 1). Poor D²G FRET signal in the absence of Na⁺ and Li⁺ precluded accurate measurements in MelB-ST. A.U., absorbance units.

FIGURE 7.

EC₅₀ for melibiose displacement of D²G binding. With SLM-8100/DMX fluorescence spectrophotometer, displacements of D²G by melibiose at increasing concentrations were measured on a time trace with λ_Ex 290 nm and λ_Em 500 nm and expressed as percentages of a total bound D²G. EC₅₀ in the absence (square) or presence of Na⁺ (circle) or Li⁺ (triangle) was determined by hyperbolic fitting, and ^appK_d for melibiose was calculated by the equation: ^appK_d = EC₅₀/{1 + [D²G]/K_d for D²G} (Table 1).

FIGURE 8.

Cation activation constants for D²G FRET. On a time trace with λ_Ex 290 nm and λ_Em 500 nm, Na⁺ (circle) or Li⁺ (triangle) at increasing concentrations was added after 10 μm D²G was added. The stimulation in FRET signal was plotted as the concentration of Na⁺ or Li⁺ presented in solution, and K_0.5^Na and K_0.5^Li were determined by hyperbolic fitting (Table 1). A.U., absorbance units.

FIGURE 9.

Cation competition. On a time trace at λ_Ex 290 nm and λ_Em 500 nm with SLM-8100/DMX fluorescence spectrophotometer, RSO containing MelB-ST was incubated with 5.6 mm Li⁺ (5-fold K_0.5) after the addition of 10 μm D²G and followed by a saturated concentration of either Li⁺ (40 mm, green curve) or Na⁺ (44 mm, red curve) as the arrows indicate. As a control, Na⁺ (44 mm) was directly added without Li⁺ (blue curve). The FRET signals are displaced with 130 mm melibiose. A.U., absorbance units.