Expression of uncoupling proteins in a mammalian cell culture system (HEK293) and assessment of their protein function.

Immortalised cultured cells are powerful tools to assess the function of ectopically expressed proteins. However, it must be ensured that the protein of interest is functional in the host system and display native behaviour. In particular, mitochondrial uncoupling proteins (UCPs) displayed (non-native) artefactual uncoupling when expressed in yeast or can possess functions upon reconstitution in proteoliposomes that cannot be reproduced in isolated mitochondria. In the light of newly discovered UCP1 orthologues and paralogues (UCP2, UCP3, plant UCP), comparative functional studies require a system with identical mitochondrial, cellular, and genetic backgrounds. In this chapter, the protocols for the ectopic expression of mouse UCP1 in the human embryonic kidney (HEK293) cell line are introduced. In isolated cell culture mitochondria, the proton leak can be measured and modulators of UCP1 activity can be tested. As mouse UCP1 in this system shows native behaviour, this may be a suitable system to directly compare the functional relationships between different UCPs.