Reconstitution of the uncoupling protein of brown adipose tissue mitochondria. Demonstration of GDP-sensitive halide anion uniport.
The fluorescent anion indicator 6-methoxy-N-(3-sulfopropyl)quinolinium was trapped in proteoliposomes reconstituted with purified 32-kDa uncoupling protein and used to detect GDP-sensitive uniports of Cl-, Br-, and I-. Transport of these halide anions was rapid and potential-dependent. F- and nitrate were found to inhibit Cl- uptake competitively, suggesting that these anions are also substrates for transport. This preparation also exhibited H+(OH-) transport, showing that the reconstituted uncoupling protein possesses both halide and H+ transport functions, as is observed in intact brown adipose tissue mitochondria. Cl- transport was inhibited to the residual level observed in liposomes without protein when GDP was present on both sides of the membrane. Cl- transport was inhibited by about 50% when GDP was present only on one side of the membrane. We infer that uncoupling protein reconstitutes into proteoliposomes with a 1:1 ratio of sidedness orientation. The Km values for Cl- uniport were 100 and 65 mM, respectively, in GDP-loaded and non-GDP-loaded vesicles. Participation of the inner membrane anion channel in the observed transport is rendered unlikely by the fact that this carrier is insensitive to GDP. A variety of additional experiments probing for inner membrane anion channel yielded uniformly negative results, confirming the absence of contamination by this protein. Our results therefore demonstrate that the uncoupling protein mediates anion translocation, a function previously reported as lacking in the reconstituted system.
Jezek
P
P
Department of Pharmacology, Medical College of Ohio, Toledo 43699.
Orosz
D E
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Garlid
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D002001:Adipose Tissue, Brown / Q000378:metabolism*; D000818:Animals; D000838:Anions; D001965:Bromides / Q000378:metabolism*; D002352:Carrier Proteins; D002712:Chlorides / Q000378:metabolism*; D006224:Cricetinae; D005456:Fluorescent Dyes; D006153:Guanosine Diphosphate / Q000494:pharmacology*; D007454:Iodides / Q000378:metabolism*; D007473:Ion Channels; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008647:Mesocricetus; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins; D008970:Molecular Weight; D011510:Proteolipids / Q000378:metabolism; D011806:Quinolinium Compounds; D000071256:Uncoupling Protein 1
D000838:Anions; D001965:Bromides; D002352:Carrier Proteins; D002712:Chlorides; D005456:Fluorescent Dyes; D007454:Iodides; D007473:Ion Channels; D008081:Liposomes; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D011510:Proteolipids; D011806:Quinolinium Compounds; D000071256:Uncoupling Protein 1; C020485:proteoliposomes; D006153:Guanosine Diphosphate; C052335:6-methoxy-N-(3-sulfopropyl)quinolinium