Modulation of Expression of Rat Mitochondrial 2-Oxoglutarate Carrier in NRK-52E Cells Alters Mitochondrial Transport and Accumulation of Glutathione and Susceptibility to Chemically Induced Apoptosis

Abstract

We previously showed that two anion carriers of the mitochondrial inner membrane, the dicarboxylate carrier (DIC; Slc25a10) and oxoglutarate carrier (OGC; Slc25a11), transport glutathione (GSH) from cytoplasm into mitochondrial matrix. In the previous study, NRK-52E cells, derived from normal rat kidney proximal tubules, were transfected with the wild-type cDNA for the DIC expressed in rat kidney; DIC transfectants exhibited increased mitochondrial uptake and accumulation of GSH and were markedly protected from chemically induced apoptosis. In the present study, cDNAs for both wild-type (WT) and a double-cysteine mutant of rat OGC (rOGC and rOGC-C221,224S, respectively) were expressed in Escherichia coli, purified, and reconstituted into proteoliposomes to assess their function. Although both WT rOGC and rOGC-C221,224S exhibited transport properties for GSH and 2-oxoglutarate that were similar to those found in mitochondria of rat kidney proximal tubules, rates of transport and mitochondrial accumulation of substrates were reduced by >75% in rOGC-C221,224S compared with the WT carrier. NRK-52E cells were stably transfected with the cDNA for WT-rOGC and exhibited 10- to 20-fold higher GSH transport activity than nontransfected cells and were markedly protected from apoptosis induced by tert-butyl hydroperoxide (tBH) or S-(1,2-dichlorovinyl)-L-cysteine (DCVC). In contrast, cells stably transfected with the cDNA for rOGC-C221,224S were not protected from tBH- or DCVC-induced apoptosis. These results provide further evidence that genetic manipulation of mitochondrial GSH transporter expression alters mitochondrial and cellular GSH status, resulting in markedly altered susceptibility to chemically induced apoptosis.