Fig. 5: L-glutamate transport driven by the PMF from the L-malate decarboxylation pathway.

a Cartoon of the co-reconstituted L-malate decarboxylation pathway and GltP in liposomes. The coupled transport of 3H⁺ and 1 L-glutamate by GltP is driven by the PMF from the L-malate decarboxylation pathway. b SDS-polyacrylamide gel of purified GltP in DDM (Lane 1), reconstituted at LPR 100 (w/w) (Lane 2), and co-reconstituted with MleP at LPR 100 (w/w) (Lane 3) in E. coli polar lipids:egg PC 3:1 (mol ratio) liposomes. MleS was encapsulated in the vesicles of Lane 2 and 3. (Uncropped gel in Supplementary Fig. 1b). c Glutamate transport upon addition of 10 mM L-malate in MleP LPR 250 - GltP LPR 250 proteoliposomes containing L-malate decarboxylation components. In curves blue and black, proteoliposomes were pre-incubated for 5 min with external 20 µM Na-L-¹⁴C-glutamate before addition of 10 mM of L-malate (blue) or succinate (black) (Glu→Mal and Glu→Succ). In the orange curve, 10 mM L-malate was added at t = 0, and, after 30 min of incubation, the uptake was started by addition of 20 µM Na-L-¹⁴C-glutamate (Mal→Glu). d Dissipation of ΔΨ and ΔpH by 1 µM valinomycin and 1 µM nigericin, respectively, at t = 3 h. Data correspond to an individual experiment with a single preparation of proteoliposomes. e Cartoon of the L-glutamate transport (in symport with H⁺) driven by ΔΨ and ΔpH, which are formed by valinomycin-mediated K⁺ diffusion (ΔΨ) and acetate (AcO⁻)/acetic acid (AcOH) diffusion (ΔpH) potentials in GltP liposomes. f Comparison of the L-glutamate transport driven by the PMF from the L-malate decarboxylation pathway (blue) with that from K⁺ and acetate diffusion potentials in MleP-GltP proteoliposomes (n = 1). All the experiments were performed at 30 °C and pH 7. Data for the blue curves in (c, d, and f). are presented as the mean of L-glutamate uptake (nmol of internalized L-glutamate per mg of GltP) ± SD from independent replicates (n = 5) with different preparations of proteoliposomes. a, e were created with Biorender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.