EVIDENCE THAT THE RAT HEPATIC MITOCHONDRIAL CARRIER IS DISTINCT FROM THE SINUSOIDAL AND CANALICULAR TRANSPORTERS FOR REDUCED GLUTATHIONE - EXPRESSION STUDIES IN XENOPUS-LAEVIS OOCYTES

Mitochondrial GSH derives hom a mitochondrial transport system (RmGshT ), which translocates cytosol GSH into the mitochondrial matrix. Mitoc hondria of oocytes, isolated 3-4 days after microinjection of total li ver mRNA, expressed a RmGshT compared with water-injected oocytes, The expressed RmGshT exhibited similar functional features as reported in isolated mitochondria of rat liver such as ATP stimulation, inhibitio n by glutamate, and insensitivity to inhibition by sulfobromophthalein -glutathione (BSP-GSH) and S-(2,4-dinitrophenyl)glutathione (DNP-GSH). The expressed RmGshT is localized in the inner mitochondrial membrane since expression is still observed in mitoplasts prepared from total liver mRNA-injected oocytes. Fractionation of poly(A)(+) RNA identifie d a single mRNA species of similar to 3-3.5 kilobases encoding for the RmGshT, which was stimulated by ATP and inhibited by glutamate but no t by BSP-GSH or DNP-GSH, Microinjection of this fraction did not lead to expression of plasma membrane GSH transport in intact oocytes, and conversely, oocytes microinjected with cRNA for rat liver sinusoidal G SH transporter (RsGshT) or rat liver canalicular GSH transporter (RcGs hT) did not express mitochondrial GSH transport activity. Thus, our re sults show the successful expression of the rat hepatic mitochondrial GSH carrier, which is different from RsGshT and RcGshT, and provide th e strategic basis for the cloning of this important carrier.