Functional reconstitution of rat uncoupling protein following its high level expression in yeast.
Small mammals, including human infants, rely on nonshivering thermogenesis for a substantial portion of their body heat during exposure to cold. This thermogenesis is mediated in large part by the uncoupling protein, which is found exclusively within the inner membrane of brown adipose tissue mitochondria. The sole function of uncoupling protein is to provide a regulated transport pathway for electrophoretic back-flux of H+ ions into the mitochondrial matrix, thereby dissipating the protonmotive force and producing heat. Thus, uncoupling protein is unique with respect to both its physiological role and its tissue expression. We have now achieved high level expression of rat uncoupling protein in yeast, with import into yeast mitochondria at levels, 70-100 micrograms/mg of mitochondrial protein, similar to those observed in brown adipose tissue mitochondria from cold-adapted rats. When the expressed protein was purified and reconstituted into liposomes, the proteoliposomes exhibited GDP-sensitive proton and chloride uniports that were inhibited by GDP with Ki values similar to those obtained with native protein. Moreover, the molecular activities of the expressed protein with respect to Cl- and H+ transport were indistinguishable from those of native protein. The availability of unlimited amounts of functional, expressed uncoupling protein will now permit application of site-directed mutagenesis to the many intriguing aspects of uncoupling protein structure and function.
Murdza-Inglis
D L
DL
Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada.
Patel
H V
HV
Freeman
K B
KB
Jezek
P
P
Orosz
D E
DE
Garlid
K D
KD
D002001:Adipose Tissue, Brown / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D015153:Blotting, Western; D002352:Carrier Proteins; D002712:Chlorides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D006153:Guanosine Diphosphate / Q000494:pharmacology; D006859:Hydrogen / Q000378:metabolism; D007473:Ion Channels; D008565:Membrane Proteins / Q000235:genetics*; D024101:Mitochondrial Proteins; D008969:Molecular Sequence Data; D019301:Oleic Acid; D009829:Oleic Acids / Q000494:pharmacology; D010085:Oxidative Phosphorylation; D010100:Oxygen / Q000378:metabolism; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D000071256:Uncoupling Protein 1
D002352:Carrier Proteins; D002712:Chlorides; D007473:Ion Channels; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D009829:Oleic Acids; D000071256:Uncoupling Protein 1; D006153:Guanosine Diphosphate; D019301:Oleic Acid; D006859:Hydrogen; D010100:Oxygen