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.
Highlights
Gradient increases exchange, it is striking that internal and external Li؉ with no gradient inhibits exchange
RSO vesicles from E. coli DW2 containing melibiose permease (MelB)-ST in the absence of Naϩ and Liϩ, i.e. in the presence of Hϩ, were excited at 290 nm (Fig. 4, top panel, Hϩ, black curve), yielding a typical Trp emission for membrane proteins with a max of 330 nm; D2G, which was added to the solution, was excited by the Trp emission, yielding a broad emission between 410 and 570 nm with a max of ϳ500 nm
Sugar Affinities—With RSO vesicles, diffFRET of D2G was measured in the presence of saturated Naϩ or Liϩ, and apparent binding constants for D2G were determined by the hyperbolic fitting (Fig. 6). appKd values for D2G in MelB of Salmonella typhimurium (MelB-ST) are 10.35 or 7.29 M in the presence of Naϩ or Liϩ, respectively, which are 3- or 5-fold higher than those of MelB of Escherichia coli (MelB-EC), respectively (Table 1)
Summary
Materials—[3H]Melibiose was custom-synthesized by PerkinElmer Life Sciences. 2Ј-(N-Dansyl)aminoalkyl-1-thio-D-galactopyranoside (D2G) was kindly provided by Drs H. Transport was initiated by adding 2 l of [3H]melibiose with a specific activity of 10 mCi/mmol at a final concentration of 0.4 mM in the absence or presence of either 20 mM NaCl or 20 mM LiCl and stopped at a given incubation time by diluting 60-fold with quenching buffer EC50 for Melibiose Displacement and appKd for Melibiose— RSO vesicles containing MelB in the absence or presence of 20 mM NaCl or LiCl were excited at 290 nm, and FRET signals of D2G, which was added at a concentration close to the appKd value, were traced at 500 nm. The increase of fluorescence intensity was plotted as a function of cation concentration, and K0.5 was determined by fitting with the hyperbola equation
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