UNIPORT OF MONOANIONIC L-MALATE IN MEMBRANE-VESICLES FROM LEUCONOSTOC-OENOS

L-malate transport was studied in membrane vesicles from Leuconostoc o enos MLE(-) (mutant lacking malolactic enzyme) which were fused with l iposomes 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 (Delta dpH), with th e inside alkaline, but was strongly inhibited by a membrane potential (Delta Psi) of normal polarity (inside negative). Imposition of a Delt a Psi, with the inside positive, by means of valinomycin-mediated pota ssium influx, resulted in a rapid accumulation of L-malate, indicating that L-malate was taken up in an anionic form. The results are consis tent with a uniport mechanism facilitating the uptake of monoanionic L -malate, the dominant species at the low pH of the experiments. Kineti c analysis of Delta pH-driven L-malate uptake in the pH range 3.0-5.8, yielded apparent affinity constants that varied less than twofold whe n calculated on the basis of the concentrations of monoanionic L-malat e, whereas the values differed 2-3 orders of magnitude for the other s pecies. At L-malate concentrations above 1 mM, a non-saturable transpo rt component became apparent which may reflect passive influx of L-mal ic acid. Substrate specificity studies indicated that citrate and L-ma late (and possibly D-lactate and L-lactate) compete for a single gener al carboxylate transport system. The carboxylate transport system cata lysed homologous L-malate and heterologous L-malate/citrate exchange w ith rates similar to the rate of L-malate efflux. Since metabolic ener gy is conserved during malolactic fermentation in L. oenos, the underl ying 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.