Characterization ofSchizosaccharomyces pombeMalate Permease by Expression inSaccharomyces cerevisiae

Abstract

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...

Key Process Conditions for Production of C 4 Dicarboxylic Acids in Bioreactor Batch Cultures of an Engineered Saccharomyces cerevisiae Strain

“…This observation might be explained by the equilibrium thermodynamics of product export (8,43). Export of malate and succinate in S. cerevisiae RWB525 has been shown to be strongly dependent on expression of the heterologous malate transporter SpMae1 (51), which seems to catalyze electroneutral proton-coupled symport of the monoanion species of these dicarboxylates (9,36). Export via this transport mechanism would become progressively more difficult as the extracellular pH decreases (Fig.…”

Antisense Inhibition of the Iron-Sulphur Subunit of Succinate Dehydrogenase Enhances Photosynthesis and Growth in Tomato via an Organic Acid–Mediated Effect on Stomatal Aperture

“…Given that malate has often been described as a component of mechanisms that sense high concentrations of CO 2 (Vavasseur and Raghavendra, 2005;Geiger et al, 2009), we next analyzed whether the transgenic lines in addition exhibited differential expression of the currently known (and putative) malate transporters or if this response was mediated merely at the substrate level. Three malate transporters have been cloned that are responsible for cytosol to vacuole and cytosol to apoplast exchange (Emmerlich et al, 2003;Lee et al, 2008;Meyer et al, 2010b), and, by analogy to microbial systems, it had been thought likely that the SLOW ANION CHANNEL-ASSOCIATED1 (SLAC1) also transports malate (Camarasa et al, 2001;Negi et al, 2008). Recent evidence demonstrated by functional expression in Xenopus laevis oocytes that guard cell-expressed Arabidopsis SLAC1 encodes a weak voltage-dependent, anionselective plasma membrane channel rather than a malate transporter (Geiger et al, 2009).…”

SLAC1 is required for plant guard cell S-type anion channel function in stomatal signalling

“…3), which contain a C4-dicarboxylate transporter/malic acid transport protein domain (InterPro: IPR004695) defined from the Escherichia coli TehA and Schizosaccharomyces pombe Mae1 proteins. Mae1 is involved in malate uptake 16 . TehA and Mae1 lack the long hydrophilic tail present in the N terminus of SLAC1, but show a weak, 15-20% amino-acid identity over the transmembrane region with SLAC1 ( Supplementary Fig.…”