The reconstituted mitochondrial adenine nucleotide translocator: Effects of lipid polymorphism

Abstract

This study investigates the role of polymorphic or nonbilayer lipids in the function of an integral membrane protein which is a key component of the mitochondrial energy transduction apparatus. The adenine nucleotide translocator (AdNT) has been isolated from rat heart mitochondria and reconstituted into ATP-containing liposomes composed of dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), and cardiolipin (CL). CL content was held constant at 11.1 mol%; the ratio of DOPC:DOPE was varied to manipulate R0, the intrinsic radius of curvature of the bilayer [S. M. Gruner (1985) Proc. Natl. Acad. Sci. USA 82, 3665-3669]. Translocator activity was determined fluorometrically, using a coupled enzyme system to measure ADP-induced efflux of ATP. Specific activity was calculated based on the number of functional translocators in each preparation, quantified using the tight-binding inhibitor carboxyatractylate (CAT). AdNT specific activity was a smooth function of Ro, with a maximum at a lipid composition similar to that of the inner mitochondrial membrane. Protein incorporation was constant at DOPC:DOPE ratios >1, but appeared to increase at ratios ≤1. The fraction of reconstituted AdNT incorporated in the native mitochondrial orientation, estimated from inhibition by 10 μM CAT, was independent of lipid composition and >85%. Leakage of encapsulated ATP increased at low R0 values both in the presence and absence of protein. © 1994 Academic Press. All rights reserved.