Antibody evidence for different conformational states of ADP, ATP translocator protein isolated from mitochondria.

Abstract

Consistent with the proposed reorientation mechanism for the ADP,ATP translocator protein of mitochondria, evidence was obtained for the existence of 2 distinct conformational states of the isolated translocator protein. Previous studies indicated that when the mitochondrial translocator protein is in the c-state (i.e., when its binding site faces the cytosol side) the protein binds primarily the ligand carboxyatractylate (CAT), and when the translocator protein is in the m-state (i.e., when its binding site faces the mitochondrial matrix) the translocator protein binds primarily bongkrekate. Direct evidence for this formulation has now come from the application of antibodies to the isolated translocator protein-ligand complex. Two antibodies was produced against the ADP,ATP translocator protein isolated from beef heart mitochondria. One antibody, which was produced against the protein isolated as the CAT-binding protein complex, was highly specific for that complex and did not react with the protein in the conformation state conferred by the bongkrekate ligand. This antibody did not cover the CAT-binding site, as evidenced by the exchange of unlabeled CAT with [35S]CAT bound to the translocator protein. The same antibody inhibited a transition of the protein from the c-state to the m-state, as evidenced by an inhibition of the displacement of [35S]CAT by bongkrekate (added jointly with ADP). The antibody apparently immobilized the translocator protein in the c-state. The 2nd antibody produced against the (somewhat less pure) ADP,ATP translocator protein, isolated as the bongkrekate-binding protein complex, did not react with the CAT-binding protein. Thus, the 2nd antibody appeared to be specific for the translocator protein in the m-state. Neither antibody inhibited mitochondrial ADP,ATP transport.