In Saccharomyces cerevisiae, L-malic acid transport is not carrier mediated and is limited to slow, simple diffusion of the undissociated acid. Expression in S. cerevisiae of the MAE1 gene, encoding Schizosaccharomyces pombe malate permease, markedly increased L-malic acid uptake in this yeast. In this strain, at pH 3.5 (encountered in industrial processes), L-malic acid uptake involves Mae1p-mediated transport of the monoanionic form of the acid (apparent kinetic parameters: V max ؍ 8.7 nmol/mg/min; K m ؍ 1.6 mM) and some simple diffusion of the undissociated L-malic acid (K d ؍ 0.057 min ؊1 ). As total L-malic acid transport involved only low levels of diffusion, the Mae1p permease was further characterized in the recombinant strain. L-Malic acid transport was reversible and accumulative and depended on both the transmembrane gradient of the monoanionic acid form and the ⌬pH component of the proton motive force. Dicarboxylic acids with stearic occupation closely related to L-malic acid, such as maleic, oxaloacetic, malonic, succinic and fumaric acids, inhibited L-malic acid uptake, suggesting that these compounds use the same carrier. We found that increasing external pH directly inhibited malate uptake, resulting in a lower initial rate of uptake and a lower level of substrate accumulation. In S. pombe, proton movements, as shown by internal acidification, accompanied malate uptake, consistent with the proton/dicarboxylate mechanism previously proposed. Surprisingly, no proton fluxes were observed during Mae1p-mediated L-malic acid import in S. cerevisiae, and intracellular pH remained constant. This suggests that, in S. cerevisiae, either there is a proton counterflow or the Mae1p permease functions differently from a proton/dicarboxylate symport.Previous studies have provided evidence that L-malic acid is metabolized by Saccharomyces cerevisiae only in the presence of an assimilable carbon source (16,24,25). Exogenous L-malic acid (3 g/liter) is always consumed to a limited extent (10 to 20%), and the amount of degraded malate depends on the strain and culture conditions. This incomplete consumption of L-malic acid may be due to limited malate uptake and inefficiency of the enzyme systems involved in metabolism of the acid. Indeed, it has been reported that the transport of Lmalate is not carrier mediated in S. cerevisiae; the undissociated form of the acid slowly enters the cell by simple diffusion (28). During fermentation in the presence of malate, intracellular malate concentration in this yeast (close to 1 mM) is therefore lower than that in yeasts having a carrier protein for L-malate and able to metabolize L-malic acid completely (10 to 15 mM in Saccharomyces bailii) (20). The constitutive L-malic enzyme, thought to be responsible for the anaerobic metabolism of L-malate in S. cerevisiae, has a high K m for the substrate (50 mM) (16,17). Given the low levels of intracellular malate, the malic enzyme seems to function to a limited extent. Anaerobically grown S. cerevisiae cells also contain a ma...