title: Characteristics of the turnover of uncoupling protein 3 by the ubiquitin proteasome system in isolated mitochondria.
abstract: Uncoupling protein 3 (UCP3) is implicated in mild uncoupling and the regulation of mitochondrial ROS production. We previously showed that UCP3 turns over rapidly in C2C12 myoblasts, with a half-life of 0.5-4h, and that turnover can be reconstituted in vitro. We show here that rapid degradation of UCP3 in vitro in isolated brown adipose tissue mitochondria required the 26S proteasome, ubiquitin, ATP, succinate to generate a high membrane potential, and a pH of 7.4 or less. Ubiquitin containing lysine-48 was both necessary and sufficient to support UCP3 degradation, implying a requirement for polyubiquitylation at this residue. The 20S proteasome did not support degradation. UCP3 degradation was prevented by simultaneously blocking matrix ATP generation and import, showing that ATP in the mitochondrial matrix was required. Degradation did not appear to require a transmembrane pH gradient, but was very sensitive to membrane potential: degradation was halved when membrane potential decreased 10-20mV from its resting value, and was not significant below about 120mV. We propose that matrix ATP and a high membrane potential are needed for UCP3 to be polyubiquitylated through lysine-48 of ubiquitin and exported to the cytosolic 26S proteasome, where it is de-ubiquitylated and degraded.
authors.affiliation: Buck Institute for Research and Aging, Novato, CA 94945, USA. smookerj@buckinstitutte.org
authors.forename: Shona A
authors.initials: SA
authors.lastname: Mookerjee
authors.forename: Martin D
authors.initials: MD
authors.lastname: Brand
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D005260:Female; D007473:Ion Channels / Q000378:metabolism*; D053078:Membrane Potential, Mitochondrial; D051379:Mice; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000378:metabolism*; D032446:Myoblasts / Q000166:cytology / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D025801:Ubiquitin / Q000378:metabolism*; D000071247:Uncoupling Protein 3
chemical_list: D007473:Ion Channels; D024101:Mitochondrial Proteins; D025801:Ubiquitin; C000604393:Ucp3 protein, mouse; C000604394:Ucp3 protein, rat; D000071247:Uncoupling Protein 3; D000255:Adenosine Triphosphate; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease