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.
Mitochondrial proton leak is the largest single contributor to the standard metabolic rate (SMR) of a rat, accounting for about 20% of SMR. Yet the mechanisms by which proton leak occurs are incompletely understood. The available evidence suggests that both phospholipids and proteins in the mitochondrial inner membrane are important determinants of proton conductance. The uncoupling protein 1 homologues (e.g. UCP2, UCP3) may play a role in mediating proton leak, but it is unlikely they account for all of the observed proton conductance. Experimental data regarding the functions of these proteins include important ambiguities and contradictions which must be addressed before their function can be confirmed. The physiological role of the proton leak, and of the uncoupling protein 1 homologues, remains similarly unclear.
We assessed the ability of human uncoupling protein 2 (UCP2) to uncouple mitochondrial oxidative phosphorylation when expressed in yeast at physiological and supraphysiological levels. We used three different inducible UCP2 expression constructs to achieve mitochondrial UCP2 expression levels in yeast of 33, 283, and 4100 ng of UCP2/mg of mitochondrial protein. Yeast mitochondria expressing UCP2 at 33 or 283 ng/mg showed no increase in proton conductance, even in the presence of various putative effectors, including palmitate and all-trans-retinoic acid. Only when UCP2 expression in yeast mitochondria was increased to 4 g/mg, more than an order of magnitude greater than the highest known physiological concentration, was proton conductance increased. This increased proton conductance was not abolished by GDP. At this high level of UCP2 expression, an inhibition of substrate oxidation was observed, which cannot be readily explained by an uncoupling activity of UCP2. Quantitatively, even the uncoupling seen at 4 g/mg was insufficient to account for the basal proton conductance of mammalian mitochondria. These observations suggest that uncoupling of yeast mitochondria by UCP2 is an overexpression artifact leading to compromised mitochondrial integrity. Uncoupling protein 1 (UCP1)1 uncouples brown adipose tissue mitochondria, causing physiologically important, hormonally regulated, thermogenic proton cycling across the inner membrane. The functions of the UCP1 homologues, UCP2 and UCP3 (1-4), are currently uncertain (5-12). They have been demonstrated to uncouple mitochondrial oxidative phosphorylation in a number of experimental models, including proteoliposomes (13), yeast heterologous expression systems (1, 2, 14 -16), and transgenic mice (17). It is clear that, under some experimental conditions, heterologous or transgenic expression of these proteins can cause an increase in the proton conductance of the inner membrane (16, 18). However, it is less obvious whether these experimental observations of uncoupling are due to a native protein activity of the UCP1 homologues, or represent a more general disruption of mitochondrial function. None of the effects observed in genetically manipulated model systems has been repeated in natural systems where changes in the levels of UCP2 and/or UCP3 occur as a response to some environmental or physiological condition (19 -21).We have demonstrated that expression of UCP1 in yeast mitochondria can cause a nonspecific uncoupling that is not due to protein activity per se (22,23). This uncoupling artifact is present only at higher levels of UCP1 expression. At these levels, UCP1 expression in yeast also interferes with mitochondrial substrate oxidation. Similarly, Heidkaemper et al. (24) have concluded that both UCP1 and UCP3 can be expressed in an incompetent form that interferes with ATP production. They suggest that most of the UCP3 expressed in yeast mitochondria is nonfunctional. There is considerable evidence that, under some expression regimes, a substantial proportion o...
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