Polymorphic lipids in the function of the mitochondrial adenine nucleotide translocator

Abstract

Lipids are amphiphilic molecules that are major components of biological membranes. In an aqueous environment, they typically form ordered, bilayer structures. However, some naturally occurring lipids, when isolated and hydrated under physiologic conditions, will preferentially form inverted, non-bilayer structures, e.g., the hexagonal H{dollar}\sb{lcub}\rm II{rcub}{dollar} phase which consists of lipid cylinders with the polar head groups facing in lining an aqueous core. When incorporated into a bilayer, such polymorphic lipids impart considerable intramembrane stress. This dissertation describes research carried out to test the hypothesis that polymorphic lipids play a key role in modulating the behavior of integral membrane proteins. Biochemically active membranes such as the energy-transducing membranes of mitochondria and chloroplasts are highly enriched in polymorphic lipids. The adenine nucleotide translocator (AdNT), an integral protein of the mitochondrial inner membrane (i.m.) which catalyzes the exchange of ADP for ATP across the i.m., has been isolated and reconstituted into liposomes. The polymorphic lipid content of the reconstituted system was systematically varied, and the effects of this variation on translocator function were determined. The mitochondrial AdNT was successfully reconstituted using a modification of existing protocols. The reconstituted system was characterized and found to have properties consistent with those previously reported for both in situ and reconstituted AdNT. A method was developed for the determination of translocator specific activity in the reconstituted system based on tight-binding inhibitor theory. Reconstituted AdNT activity was found to be a smooth function of polymorphic lipid content, with a maximum at a lipid composition similar to that of the i.m. Liposome leakiness increased with increasing polymorphic lipid content. No significant effect of polymorphic lipid content on either extent of AdNT incorporation or AdNT orientation was detected.