Artefactual uncoupling following UCP3 expression in yeast mitochondria

Abstract

Much of the evidence supporting a mitochondrial uncoupling function for the homologues of UCPl (e.g. UCP2, UCP3) comes from experiments in which the levels of protein expression have been genetically manipulated. Here we present evidence, from yeast genetically modified to express these proteins, that these experimental manipulations can lead to an uncoupling of oxidative phosphorylation that does not represent a native activity of the protein. We used UCP1, whose native uncoupling activity can be fully inhibited by GDP, to demonstrate that artefactual uncoupling occurs at higher levels of protein expression. UCP3 similarly uncouples when expressed at these high levels. When we expressed UCP3 in yeast mitochondria at levels similar to, or 2-fold greater than, those found in mouse skeletal muscle (73 ? 53 ng / mg mitochondria1 protein) no uncoupling was observed. UCP3 only uncoupled at levels of expression that were 3-fold or more than in the mouse. This uncoupling was insensitive to GDP. The stimulation of uncoupling by palmitate in UCP1-expressing mitochondria was not duplicated in UCPZ or UCP3 expressors.42 Discriminatory antibody raised to UCP-3 used to detect protein in a number of systems The human UCP-3 (hUCP-3) and human UCP-2 (hUCP-2) proteins were overexpressed in the E. coli strain, BL21 pLysS using the expression vector PET 21d. mg amounts of U C P protein were expressed and purified in inclusion bodies to a high level (70%) of purity. Inclusion bodies were isolated by previously published protocol (Echtay et al,, 1999). Antibodies raised in rabbit to a hydrophilic peptide from rat UCP-3 (rUCP-3) protein (residues 149-163) were tested in this system. Western immunoblotting analysis, using the anti-UCP-3 peptide, detected E. coli expressed hUCP-3 but not hUCP-3 protein.Uncoupling protein 3 (UCP-3) is preferentially expressed in skeletal muscle (I).It has been shown in mice that low-intensity exercise bouts of 2 hourdday for 2 weeks produce increases in UCP-3 mRNA levels 3 hours post-exercise which decay to resting levels 24hours post-exercise (2). The aim of this study was to investigate the effects of endurance swimming training on UCP-3 protein expression in mouse skeletal muscle.Control animals performed no exercise while trained animals performed bouts of weighted exercise endurance training for up to 2 hours duration, 5 daydweek, for 17 weeks. 24hours following the training program animals were killed by C 0 2 asphyxiation,and the hind limb muscles soleus, extensor digitorum longus (EDL), plantaris and gastrocnemius were dissected free and intact.Muscle homogenates were prepared and analysed using western blot analysis for total UCP-3 protein content,corrected for mitochondrial number according to cytochrome-c content.Our data show an increase in UCP-3 protein expression in EDL and plantaris from trained mice compared with the control mice.As yet no detectable changes are seen in soleus and gastrocnemius.These results suggest that UCP-3 does play some as of yet undetermined role during endurance ex...