A mitochondrial uncoupling artifact can be caused by expression of uncoupling protein 1 in yeast

Uncoupling protein 1 (UCP1) from mouse was expressed in yeast and the speci fic (GDP-inhibitable) and artifactual (GDP-insensitive) effects on mitochon drial uncoupling were assessed. UCP 1 provides a GDP-inhibitable model syst em to help interpret the uncoupling effects of high expression in yeast of other members of the mitochondrial carrier protein family, such as the UCP1 homologues UCP2 and UCP3. Yeast expressing UCP1 at modest levels (approx. 1 mug/mg of mitochondrial protein) showed no growth defect, normal rates of chemically uncoupled respiration and an increased non-phosphorylating prot on conductance that was completely GDP-sensitive. The catalytic-centre acti vity of UCP1 in these yeast mitochondria was similar to that in mammalian b rown-adipose-tissue mitochondria. However, yeast expressing UCP1 at higher levels (approx. 11 mug/mg of mitochondrial protein) showed a growth defect. Their mitochondria had depressed chemically uncoupled respiration rates an d an increased proton conductance that was partly GDP-insensitive. Thus, al though UCP1 shows native behaviour at modest levels of expression in yeast, higher levels (or rates) of expression can lead to an uncoupling that is n ot a physiological property of the native protein and is therefore artifact ual. This observation might be important in the interpretation of results f rom experiments in which the functions of UCP1 homologues are verified by t heir ability to uncouple yeast mitochondria.