THE RECONSTITUTED MITOCHONDRIAL ADENINE-NUCLEOTIDE TRANSLOCATOR - EFFECTS OF LIPID POLYMORPHISM

This study investigates the role of polymorphic or nonbilayer lipids i n the function of an integral membrane protein which is a key componen t of the mitochondrial energy transduction apparatus. The adenine nucl eotide translocator (AdNT) has been isolated from rat heart mitochondr ia and reconstituted into ATP-containing liposomes composed of dioleoy lphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), and cardiolipin (CL). CL content was held constant at 11.1 mol%; the r atio of DOPC:DOPE was varied to manipulate R(0), the intrinsic radius of curvature of the bilayer [S. M. Gruner (1985) Proc. Natl. Acad. Sci . USA 82, 3665-3669]. Translocator activity was determined fluorometri cally, using a coupled enzyme system to measure ADP-induced efflux of ATP. Specific activity was calculated based on the number of functiona l translocators in each preparation, quantified using the tight-bindin g inhibitor carboxyatractylate (CAT). AdNT specific activity was a smo oth function of Ro, with a maximum at a lipid composition similar to t hat of the inner mitochondrial membrane. Protein incorporation was con stant at DOPC:DOPE ratios >1, but appeared to increase at ratios less than or equal to 1. The fraction of reconstituted AdNT incorporated in the native mitochondrial orientation, estimated from inhibition by 10 mu M CAT, was independent of lipid composition and >85%. Leakage of e ncapsulated ATP increased at low R(0) values both in the presence and absence of protein. (C) 1994 Academic Press, Inc.