RESUMO
Western blots detected uncoupling protein 3 (UCP3) in skeletal-muscle mitochondria from wild-type but not UCP3 knock-out mice. Calibration with purified recombinant UCP3 showed that mouse and rat skeletal muscle contained 0.14 microg of UCP3/mg of mitochondrial protein. This very low UCP3 content is 200-700-fold less than the concentration of UCP1 in brown-adipose-tissue mitochondria from warm-adapted hamster (24-84 microg of UCP1/mg of mitochondrial protein). UCP3 was present in brown-adipose-tissue mitochondria from warm-adapted rats but was undetectable in rat heart mitochondria. We expressed human UCP3 in yeast mitochondria at levels similar to, double and 7-fold those found in rodent skeletal-muscle mitochondria. Yeast mitochondria containing UCP3 were more uncoupled than empty-vector controls, particularly at concentrations that were 7-fold physiological. However, uncoupling by UCP3 was not stimulated by the known activators palmitate and superoxide; neither were they inhibited by GDP, suggesting that the observed uncoupling was a property of non-native protein. As a control, UCP1 was expressed in yeast mitochondria at similar concentrations to that of UCP3 and at up to 50% of the physiological level of UCP1. Low levels of UCP1 gave palmitate-dependent and GDP-sensitive proton conductance but higher levels of UCP1 caused an additional GDP-insensitive uncoupling artifact. We conclude that the uncoupling of yeast mitochondria by high levels of UCP3 expression is entirely an artifact and provides no evidence for any native uncoupling activity of the protein.
Assuntos
Proteínas de Transporte/metabolismo , Mitocôndrias Musculares/metabolismo , Mitocôndrias/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Tecido Adiposo Marrom/metabolismo , Animais , Sequência de Bases , Proteínas de Transporte/genética , Cricetinae , DNA Complementar/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Técnicas In Vitro , Corpos de Inclusão/metabolismo , Canais Iônicos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Proteínas Mitocondriais , Músculo Esquelético/metabolismo , Prótons , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Superóxidos/farmacologia , Proteína Desacopladora 1 , Proteína Desacopladora 3RESUMO
Uncoupling protein 1 (UCP1) diverts energy from ATP synthesis to thermogenesis in the mitochondria of brown adipose tissue by catalysing a regulated leak of protons across the inner membrane. The functions of its homologues, UCP2 and UCP3, in other tissues are debated. UCP2 and UCP3 are present at much lower abundance than UCP1, and the uncoupling with which they are associated is not significantly thermogenic. Mild uncoupling would, however, decrease the mitochondrial production of reactive oxygen species, which are important mediators of oxidative damage. Here we show that superoxide increases mitochondrial proton conductance through effects on UCP1, UCP2 and UCP3. Superoxide-induced uncoupling requires fatty acids and is inhibited by purine nucleotides. It correlates with the tissue expression of UCPs, appears in mitochondria from yeast expressing UCP1, and is absent in skeletal muscle mitochondria from UCP3 knockout mice. Our findings indicate that the interaction of superoxide with UCPs may be a mechanism for decreasing the concentrations of reactive oxygen species inside mitochondria.