On the mechanism of mitochondrial uncoupling protein 1 function

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 UCP1 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.