On the Mechanism of Fatty Acid-induced Proton Transport by Mitochondrial Uncoupling Protein

Keith D Garlid,David E Orosz,Martin Modrianský,Stefano Vassanelli,Petr Jezek

doi:10.1074/jbc.271.5.2615

Abstract

Uncoupling protein mediates electrophoretic transport of protons and anions across the inner membrane of brown adipose tissue mitochondria. The mechanism and site of proton transport, the mechanism by which fatty acids activate proton transport, and the relationship between fatty acids and anion transport are unknown. We used fluorescent probes to measure H+ and anion transport in vesicles reconstituted with purified uncoupling protein and carried out a comparative study of the effects of laurate and its close analogue, undecanesulfonate. Undecanesulfonate was transported by uncoupling protein with a Km value similar to that observed for laurate as it activated H+ transport. Both laurate and undecanesulfonate inhibited Cl- with competitive kinetics. Undecanesulfonate inhibited laurate-induced H+ transport with competitive kinetics. Undecanesulfonate and laurate differed in two important respects. (i) Laurate caused uncoupling protein-mediated H+ transport, whereas undecanesulfonate did not. (ii) Lauric acid was rapidly transported across the bilayer by nonionic diffusion, whereas undecanesulfonic was not. We infer that the role of uncoupling protein in H+ transport is to transport fatty acid anions and that fatty acids induce H+ transport because they can diffuse electroneutrally across the membrane. According to this hypothesis, uncoupling protein is a pure anion porter and does not transport protons; rather it is designed to enable fatty acids to behave as cycling protonophores.

Highlights

Bioenergetic Aspects of the Fatty Acid Protonophore Hypothesis—In mitochondria containing 10% UCP protein, the Vmax for laurate (Table I) translates to about 2000 nmol/(mg of mitochondrial protein1⁄7min), roughly equal to the maximum rate of proton ejection by the redox chain. This is not unexpected, because mitochondrial transporters are normally synthesized in quantities more than sufficient to carry out their tasks
When the need for thermogenesis is great, mammals synthesize more UCP, to the extent that UCP levels reach 15% of mitochondrial protein (24, 25)
Transported anions and FAs interact with a common site on UCP which we have called the FA docking site (6, 8)

Summary

EXPERIMENTAL PROCEDURES

Purification and Reconstitution of UCP—Brown adipose tissue mitochondria were isolated from Syrian hamsters, and UCP was purified and reconstituted into proteoliposomes using protocols described previously (12). Measurement of UCP-mediated Hϩ flux was obtained from changes in SPQ fluorescence due to quenching by the anion of TES buffer (12, 13). Internal medium contained 84.4 mM TEA2SO4, 0.6 mM TEA-EGTA, and 28.8 mM TEA-TES, pH 7.2. (iii) Laurate caused increases in the rates of valinomycin-induced Hϩ or ClϪ transport in liposomes. Lysis, detected when internal probe was exposed to external medium, was apparent with high doses of longer chain FAs or alkylsulfonates. This effect was amplified by increasing valinomycin concentration, suggesting ion pair transport of laurate with the valinomycin-Kϩ complex (16). Materials for protein purification and liposome formation were obtained from sources described in Jezek et al (5)

RESULTS

TABLE I Kinetic parameters for anionic substrates of uncoupling protein

DISCUSSION