Reconstitution of novel mitochondrial uncoupling proteins UCP2 and UCP3

Reconstitution of novel mitochondrial uncoupling proteins, human UCP2 and UCP3, expressed in yeast, was performed to characterize fatty acid (FA)-induced H⁺ efflux in the resulted proteoliposomes. We now demonstrate for the first time that representatives of physiologically abundant long chain FAs, saturated or unsaturated, activate H⁺ translocation in UCP2- and UCP3-proteoliposomes. Efficiency of lauric, palmitic or linoleic acid was roughly the same, but oleic acid induced faster H⁺ uniport. We have confirmed that ATP and GTP inhibit such FA-induced H⁺ uniport mediated by UCP2 and UCP3. Coenzyme Q₁₀ did not further significantly activate the observed H⁺ efflux. In conclusion, careful instant reconstitution yields intact functional recombinant proteins, UCP2 and UCP3, the activity of which is comparable with UCP1.