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

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Abstract
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.