Physiological levels of mammalian uncoupling protein 2 do not uncouple yeast mitochondria

assessed the ability of human uncoupling protein 2 (UCP2) to uncouple mitoc hondrial oxidative phosphorylation when expressed in yeast at physiological and supraphysiological levels. We used three different inducible UCP2 expr ession constructs to achieve mitochondrial UCP2 expression levels in yeast of 33, 283, and 4100 ng of UCP2/mg of mitochondrial protein. Yeast mitochon dria expressing UCP2 at 33 or 283 ng/mg showed no increase in proton conduc tance, even in the presence of various putative effecters, including palmit ate and all-trans-retinoic acid. Only when UCP2 expression in yeast mitocho ndria was increased to 4 mug/mg, more than an order of magnitude greater th an the highest known physiological concentration, was proton conductance in creased. This increased proton conductance was not abolished by GDP, At thi s high level of UCP2 expression, an inhibition of substrate oxidation was o bserved, which cannot be readily explained by an uncoupling activity of UCP 2. Quantitatively, even the uncoupling seen at 4 mug/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 ov erexpression artifact leading to compromised mitochondrial integrity.