title: Uncoupling of protein-3 induces an uncontrolled uncoupling of mitochondria after expression in muscle derived L6 cells.
abstract: The uncoupling proteins (UCPs) are thought to uncouple oxidative phosphorylation in the mitochondria and thus generate heat. One of the UCP isoforms, UCP3, is abundantly expressed in skeletal muscle, the major thermogenic tissue in humans. UCP3 has been overexpressed at high levels in yeast systems, where it leads to the uncoupling of cell respiration, suggesting that UCP3 may indeed be capable of dissipating the mitochondrial proton gradient. This effect, however, was recently shown to be a consequence of the high level of expression and incorrect folding of the protein and not to its intrinsic uncoupling activity. In the present study, we investigated the properties of UCP3 overexpressed in a relevant mammalian host system such as the rat myoblast L6 cell line. UCP3 was expressed in relatively low levels (< 1 microg x mg(-1) membrane protein) with the help of an adenovirus vector. Immunofluorescence microscopy of transduced L6 cells showed that UCP3 was expressed in more than 90% of the cells and that its staining pattern was characteristic for mitochondrial localization. The oxygen consumption of L6 cells under nonphosphorylating conditions increased concomitantly with the levels of UCP3 expression. However, uncoupling was associated with an inhibition of the maximal respiratory capacity of mitochondria and was not affected by purine nucleotides and free fatty acids. Moreover, recombinant UCP3 was resistant to Triton X-100 extraction under conditions that fully solubilize membrane bound proteins. Thus, UCP3 can be uniformly overexpressed in the mitochondria of a relevant muscle-derived cell line resulting in the expected increase of mitochondrial uncoupling. However, our data suggest that the protein is present in an incompetent conformation.
authors.affiliation: Metabolic and Cardiovascular Diseases, Novartis Pharmaceuticals Ltd, Basel, Switzerland.
authors.forename: Danilo
authors.initials: D
authors.lastname: Guerini
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Prati
authors.forename: Urvi
authors.initials: U
authors.lastname: Desai
authors.forename: Hans Peter
authors.initials: HP
authors.lastname: Nick
authors.forename: Rolf
authors.initials: R
authors.lastname: Flammer
authors.forename: Stephan
authors.initials: S
authors.lastname: Grüninger
authors.forename: Frederic
authors.initials: F
authors.lastname: Cumin
authors.forename: Machael
authors.initials: M
authors.lastname: Kaleko
authors.forename: Sheila
authors.initials: S
authors.lastname: Connelly
authors.forename: Michele
authors.initials: M
authors.lastname: Chiesi
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000502:physiology*; D002478:Cells, Cultured; D007473:Ion Channels; D008565:Membrane Proteins / Q000502:physiology; D026901:Membrane Transport Proteins; D008931:Mitochondria, Muscle / Q000378:metabolism*; D024101:Mitochondrial Proteins; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry; D051381:Rats; D000071256:Uncoupling Protein 1; D000071246:Uncoupling Protein 2; D000071247:Uncoupling Protein 3
chemical_list: D002352:Carrier Proteins; D007473:Ion Channels; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D024101:Mitochondrial Proteins; D011506:Proteins; C000604392:UCP3 protein, human; C000604394:Ucp3 protein, rat; D000071256:Uncoupling Protein 1; D000071246:Uncoupling Protein 2; D000071247:Uncoupling Protein 3