Molecular identification and functional characterization of a novel glutamate transporter in yeast and plant...

The genome of Saccharomyces cerevisiae encodes 35 members of the mitochondrial carrier family (MCF) and 58 MCF members are coded by the genome of Arabidopsis thaliana, most of which have been functionally characterized. Here two members of this family, Ymc2p from S. cerevisiae and BOU from Arabidopsis, have been thoroughly characterized. These proteins were overproduced in bacteria and reconstituted into liposomes. Their transport properties and kinetic parameters demonstrate that Ymc2p and BOU transport glutamate, and to a much lesser extent L-homocysteinesulfinate, but not other amino acids and many other tested metabolites. Transport catalyzed by both carriers was saturable, inhibited by mercuric chloride and dependent on the proton gradient across the proteoliposomal membrane. The growth phenotype of S. cerevisiae cells lacking the genes ymc2 and agc1, which encodes the only other S. cerevisiae carrier capable to transport glutamate besides aspartate, was fully complemented by expressing Ymc2p, Agc1p or BOU. Mitochondrial extracts derived from ymc2Δagc1Δ cells, reconstituted into liposomes, exhibited no glutamate transport at variance with wild-type, ymc2Δ and agc1Δ cells, showing that S. cerevisiae cells grown in the presence of acetate do not contain additional mitochondrial transporters for glutamate besides Ymc2p and Agc1p. Furthermore, mitochondria isolated from wild-type, ymc2Δ and agc1Δ strains, but not from the double mutant ymc2Δagc1Δ strain, swell in isosmotic ammonium glutamate showing that glutamate is transported by Ymc2p and Agc1p together with a H⁺. It is proposed that the function of Ymc2p and BOU is to transport glutamate across the mitochondrial inner membrane and thereby play a role in intermediary metabolism, C1 metabolism and mitochondrial protein synthesis.