On the Mechanism of Mitochondrial Uncoupling Protein 1 Function

Eamon P Breen,Sebastien G Gouin,Andrew F Murphy,Lee R Haines,Angela M Jackson,Terry W Pearson,Paul V Murphy,Richard K Porter

doi:10.1074/jbc.m511575200

Abstract

Native uncoupling protein 1 (UCP 1) was purified from rat mitochondria by hydroxyapatite chromatography and identified by peptide mass mapping and tandem mass spectrometry. Native and expressed UCP 1 were reconstituted into liposomes, and proton flux through UCP 1 was shown to be fatty acid-dependent and GDP-sensitive. To investigate the mechanism of action of UCP 1, we determined whether hydrophilic modification of the omega-carbon of palmitate effected its transport function. We show that proton flux was greater through native UCP 1-containing proteoliposomes when facilitated by less hydrophilically modified palmitate (palmitate > omega-methoxypalmitate > omega-hydroxypalmitate with little or no proton flux due to glucose-O-omega-palmitate or undecanesulfonate). We show that non-proton-dependent charge transfer was greater when facilitated by less hydrophilically modified palmitate (palmitate/undecanesulfonate > omega-methoxypalmitate > omega-hydroxypalmitate, with no non-proton-dependent charge transfer flux due to glucose-O-omega-palmitate). We show that the GDP-inhibitable oxygen consumption rate in brown adipose tissue mitochondria was reversed by palmitate (as expected) but not by glucose-O-omega-palmitate. Our data are consistent with the model that UCP 1 flips long-chain fatty acid anions and contradict the "cofactor" model of UCP 1 function.

Highlights

Despite our knowledge about the physiological function of uncoupling protein 1 (UCP 1) in BAT, the mechanism of its uncoupling action is still unclear [7,8,9]
The first proposes that UCP 1 acts as a proton conduit across the mitochondrial inner membrane and, importantly, that fatty acids act as cofactors/activators providing an additional carboxyl moiety at a key intramembrane site, enhancing the rate of proton movement [8, 10, 11]
Hydoxyapatite chromatography procedures previously used for purification of UCP 1 from hamster BAT [23] were adapted for rat BAT, and identification of the purified protein was determined using mass spectrometry

Summary

Introduction

Despite our knowledge about the physiological function of UCP 1 in BAT, the mechanism of its uncoupling action is still unclear [7,8,9]. Sample Preparation for Mass Spectrometry—A 30- to 33-kDa colloidal Coomassie Brilliant Blue G-250-stained protein band was excised from a one-dimensional SDS-PAGE gel (Fig. 1A) and was transferred to a 1.5-ml Eppendorf microcentrifuge tube (previously autoclaved and rinsed with 50% high-performance liquid chromatography grade methanol to remove any contaminants) containing 100 ␮l of 20% (w/v) ammonium sulfate.

Results

Conclusion