title: On the mechanism of mitochondrial uncoupling protein 1 function.
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.
authors.affiliation: School of Biochemistry and Immunology, Trinity College Dublin, College Green, Dublin 2, Ireland.
authors.forename: Eamon P
authors.initials: EP
authors.lastname: Breen
authors.forename: Sebastien G
authors.initials: SG
authors.lastname: Gouin
authors.forename: Andrew F
authors.initials: AF
authors.lastname: Murphy
authors.forename: Lee R
authors.initials: LR
authors.lastname: Haines
authors.forename: Angela M
authors.initials: AM
authors.lastname: Jackson
authors.forename: Terry W
authors.initials: TW
authors.lastname: Pearson
authors.forename: Paul V
authors.initials: PV
authors.lastname: Murphy
authors.forename: Richard K
authors.initials: RK
authors.lastname: Porter
mesh_terms: D002001:Adipose Tissue, Brown / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000838:Anions; D002352:Carrier Proteins / Q000737:chemistry / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005227:Fatty Acids / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000737:chemistry; D007473:Ion Channels; D007477:Ions; D008081:Liposomes / Q000378:metabolism; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000737:chemistry / Q000502:physiology*; D008928:Mitochondria / Q000378:metabolism; D024101:Mitochondrial Proteins; D008969:Molecular Sequence Data; D010101:Oxygen Consumption; D019308:Palmitic Acid / Q000737:chemistry; D010449:Peptide Mapping; D010766:Phosphorylation; D011510:Proteolipids / Q000378:metabolism; D011522:Protons; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D014475:Uncoupling Agents / Q000737:chemistry; D000071256:Uncoupling Protein 1
chemical_list: D000838:Anions; D002352:Carrier Proteins; D005227:Fatty Acids; D007473:Ion Channels; D007477:Ions; D008081:Liposomes; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D011510:Proteolipids; D011522:Protons; D011994:Recombinant Proteins; C000604373:Ucp1 protein, rat; D014475:Uncoupling Agents; D000071256:Uncoupling Protein 1; C020485:proteoliposomes; D006153:Guanosine Diphosphate; D019308:Palmitic Acid