Uniport of Monoanionic L‐malate in Membrane Vesicles from Leuconostoc Oenos

L‐malate transport was studied in membrane vesicles from Leuconostoc oenos MLE(–) (mutant lacking malolactic enzyme) which were fused with liposomes containing beef heart cytochrome c oxidase as a proton‐motive‐force‐generating system. In these hybrid membranes, accumulation of L‐malate was observed in response to a pH gradient (pH), with the inside alkaline, but was strongly inhibited by a membrane potential (ψ) of normal polarity (inside negative). Imposition of a ψ, with the inside positive, by means of valinomycin‐mediated potassium influx, resulted in a rapid accumulation of L‐malate, indicating that L‐malate was taken up in an anionic form. The results are consistent with a uniport mechanism facilitating the uptake of monoanionic L‐malate, the dominant species at the low pH of the experiments. Kinetic analysis of pH‐driven L‐malate uptake in the pH range 3.0–5.8, yielded apparent affinity constants that varied less than twofold when calculated on the basis of the concentrations of monoanionic L‐malate, whereas the values differed 2–3 orders of magnitude for the other species. At L‐malate concentrations above 1 mM, a nonsaturable transport component became apparent which may reflect passive influx of L‐malic acid. Substrate specificity studies indicated that citrate and L‐malate (and possibly D‐lactate and L‐lactate) compete for a single general carboxylate transport system. The carboxylate transport system catalysed homologous L‐malate and heterologous L‐malate/citrate exchange with rates similar to the rate of L‐malate efflux. Since metabolic energy is conserved during malolactic fermentation in L. oenos, the underlying mechanism most likely involves electrogenic monoanionic L‐malate uptake, in combination with H⁺ consumption in the cytoplasm, followed by diffusion outwards of lactic acid plus carbon dioxide.