Evidence for Mitochondrial Uptake of Glutathione by Dicarboxylate and 2-Oxoglutarate Carriers

The role of organic anion transporters in the mitochondrial uptake of glutathione (GSH) was investigated by assessing competition with substrates or inhibition with inhibitors of specific carriers and modulation of mitochondrial energetics. Potential artifacts in the transport methodology, including contamination of matrix space with extramitochondrial fluid, changes in matrix volume during incubations, efflux of transported GSH during sample processing, induction of the membrane permeability transition, contamination of the mitochondrial preparation with plasma membranes and GSH degradation, were corrected or eliminated. Substrates (i.e., malate, succinate) and an inhibitor (i.e., butylmalonate) of the dicarboxylate carrier, an inhibitor (i.e., phenylsuccinate) of the 2-oxoglutarate carrier, and glutamate produced significant inhibition of GSH uptake whereas substrates and inhibitors of the mono- and tricarboxylate carriers were generally without effect. Phosphoenolpyruvate, which is a substrate for the tricarboxylate carrier, inhibited GSH uptake, but this was due to induction of the membrane permeability transition and not to competition for uptake. Although glutamate inhibited GSH uptake, the converse did not occur. GSH uptake was pH-independent and aspartate had no effect, which suggest that the glutamate and glutamate-aspartate carriers are not involved in GSH uptake but that the glutamyl residue of GSH may be important in its transport. GSH uptake was dependent on phosphate and ATP generation. Hence, we conclude that both the dicarboxylate and 2-oxoglutarate carriers of the inner membrane can catalyze uptake of GSH into the matrix. The function of an additional, novel transporter cannot be excluded at present. This is the first study to define the function of mitochondrial anion carriers in GSH transport.