Transport of malic acid in the yeast Schizosaccharomyces pombe: evidence for a proton-dicarboxylate symport

Abstract

The transport system for malic acid present in Schizosaccharomyces pombe cells, growing in batch culture on several corbon sources, has been studied. It was found that the diarboxylic acid carrier of S. pombe is a proton-dicarboxylate symporter that allows transport and accumulation as a function of pH with the following kinetic parameters at pH 5·0: Vmax = 0·01 nmol of total malic acids 1 mg (dry weight) of cells, 1and Km = 0·1mM total malic acid uptake (pH 5·0) was accompanied by disappearance of extracellular protons, the uptake rates of which followed Michaelis-Menten kinetics as a function of the acid concentration. The Km values, calculated as the concentrations either of anions or of undissociated acid, at various extracellular pH values, pointed to the monoanionic form as the transported species. Furthermore, accumulated free acid suffered rapid efflux after the addition of the portonophore carbonyl cyanid m-chlorophenyl hydrazone. These results suggested that the transport system was a dicarboxylateproton symporter. Growth of cells in a medium with glucose (up to 14%, w/v) and malic acid (1·5%, w/v) also resulted in proton-dicarboxylate activity, suggesting that the system, besides being constitutive, was still active at high glucose concentrations. The following dicarboxylic acids acted as competitive inhibitors of malic acid transport at pH 5·0: D- malic acid, succinic acid, fumaric acid oxaloacetic acid, -Ketoglutaric acid, maleic acid, maleic and malonic acid. In addition all of these dicarboxylic acids induced proton movements that followed MichaelisMenten kinetics. It was concluded that the malic negatively charged form (probably the monoanionic form) was transported by a proton-symport mechanism and that the carrier appeared to be a common dicarboxylat transport sysmem. The undissociated acid entered the cells slowly by simple diffusion.