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

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.(undefined

Isolation and characterization of <i>Schizosaccharomyces pombe</i> mutants with defective NAD-dependent malic enzyme

To obtain NAD-dependent malic enzyme mutants of Schizosaccharomyces pombe, a colony color indicator screening system was developed. Mutants defective for malic acid utilization (mau mutants) are yellow, while wild-type colonies are blue on the defined bromcresol green based indicator medium. NAD-dependent malic enzyme mutants were distinguished from other mau mutants by subsequent, starch gel electrophoresis, spectrophotometry, complementation tests, and intermediate pool analysis with cell-free extracts. </jats:p

Malate transport in Schizosaccharomyces pombe.

The transport of malate was studied in a Schizosaccharomyces pombe wild-type strain and in mutant strains unable to utilize malic acid. Two groups of such mutants, i.e., malic enzyme-deficient and malate transport-defective mutants, were differentiated by a 14C-labeled L-malate transport assay and by starch gel electrophoresis followed by activity staining for malic enzyme (malate dehydrogenase [oxaloacetate decarboxylating] [NAD+]; 1.1.1.38) and malate dehydrogenase (1.1.1.37). Transport of malate in S. pombe was constitutive and strongly inhibited by inhibitors of oxidative phosphorylation and of the formulation of proton gradients. Transport was a saturable function of the malate concentration. The apparent Km and Vmax values for transport by the parent were 3.7 mM and 40 nmol/min per mg of protein, respectively, while those of the malic enzyme-deficient mutant were 5.7 mM and 33 nmol/min per mg of protein, respectively. Malate transport was pH and temperature dependent. The specificity of transport was studied with various substrates, including mono- and dicarboxylic acids, and the possibility of a common transport system for dicarboxylic acids is discussed

The mae1 gene of Schizosaccharomyces pombe encodes a permease for malate and other C4 dicarboxylic acids

The mae1 gene of the yeast Schizosaccharomyces pombe was identified on the basis of its ability to complement a mutant defective in the transport of malic acid. Analysis of the DNA sequence revealed an open reading frame of 1314 base pairs, encoding a polypeptide of 438 amino acids with a predicted molecular weight of 49 kDa. A hydropathy profile of the predicted amino acid sequence revealed a protein with ten membrane-spanning or associated domains and hydrophilic N- and C- termini. The predicted secondary structure of the protein is similar to models proposed for other integral membrane proteins from both prokaryotes and eukaryotes. The S. pombe mae1 gene encodes a single mRNA of 1.5 kb. The mae1 gene is expressed constitutively and is not subject to catabolite repression as was previously reported for the malate permease systems of Candida utilis and Hansenula anomala. The mae1 gene was mapped 2842 bp 5' to the MFm1 gene on chromosome I. Transport assays revealed that the mae1 gene encodes a permease involved in the uptake of L-malate, succinate and malonic acid. The sequence of the S. pombe mae1 gene is available in GenBank under Accession Number U21002.Articl