Cited by 94Mitochondrial Function and PathologyATP Synthase and ATPases ResearchAdipose Tissue and Metabolism

Artifactual uncoupling by uncoupling protein 3 in yeast mitochondria at the concentrations found in mouse and rat skeletal-muscle mitochondria

HARPER, James A., STUART, Jeff A., JEKABSONS, Mika B., ROUSSEL, Damien, BRINDLE, Kevin M., DICKINSON, Keith, JONES, Robert B., BRAND, Martin D.·2002·Biochemical Journal

Abstract

Western blots detected uncoupling protein 3 (UCP3) in skeletal-muscle mitochondria from wild-type but not UCP3 knock-out mice. Calibration with purified recombinant UCP3 showed that mouse and rat skeletal muscle contained 0.14μg of UCP3/mg of mitochondrial protein. This very low UCP3 content is 200–700-fold less than the concentration of UCP1 in brown-adipose-tissue mitochondria from warm-adapted hamster (24–84μg of UCP1/mg of mitochondrial protein). UCP3 was present in brown-adipose-tissue mitochondria from warm-adapted rats but was undetectable in rat heart mitochondria. We expressed human UCP3 in yeast mitochondria at levels similar to, double and 7-fold those found in rodent skeletal-muscle mitochondria. Yeast mitochondria containing UCP3 were more uncoupled than empty-vector controls, particularly at concentrations that were 7-fold physiological. However, uncoupling by UCP3 was not stimulated by the known activators palmitate and superoxide; neither were they inhibited by GDP, suggesting that the observed uncoupling was a property of non-native protein. As a control, UCP1 was expressed in yeast mitochondria at similar concentrations to that of UCP3 and at up to 50% of the physiological level of UCP1. Low levels of UCP1 gave palmitate-dependent and GDP-sensitive proton conductance but higher levels of UCP1 caused an additional GDP-insensitive uncoupling artifact. We conclude that the uncoupling of yeast mitochondria by high levels of UCP3 expression is entirely an artifact and provides no evidence for any native uncoupling activity of the protein.

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Crossref · 10.1042/bj3610049

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  • Massive overexpression of any mitochondrial membrane protein tends to disrupt native stoichiometry and mitochondrial membrane integrity, producing artifactual acceleration in proton conductance and increased energy expenditure.

    “Moreover, massive overexpression of any mitochondrial membrane protein tends to disrupt the native stoichiometry and integrity of the mitochondrial membrane, generating an artifactual acceleration in proton conductance and a concomitant increase in energy expenditure (Cadenas et al., 2002; Harper et al., 2002; Henique et al., 2010; Perdomo et al., 2004; Sebastian et al., 2007)”

    From Lipid-Induced Mitochondrial Stress and Insulin Action in Muscle (2012) · cites this paper as Harper et al., 2002

  • UCP-3 expression is 200-700 fold lower than UCP-1 in mouse skeletal muscle.

    “For instance, UCP-3 expression is expressed at a much lower level than UCP-1 (200–700 fold) in mouse skeletal muscle [21].”

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Authors
HARPER, J. A., STUART, J. A., JEKABSONS, M. B., ROUSSEL, D., BRINDLE, K. M., DICKINSON, K., JONES, R. B., & BRAND, M. D.
Affiliations
  • University of Cambridge (GB)
  • MRC Dunn Human Nutrition Unit, Hills Road, Cambridge CB2 2XY, U.K.
  • MRC Mitochondrial Biology Unit (GB)
  • BASF Pharma, Pennyfoot Street, Nottingham NG1 1GF, U.K.
Corresponding author
ROUSSEL, Damien
Venue
Biochemical Journal
Published
2001-12-17
Volume
361
Issue
1
Pages
49-56
ISSN
  • 0264-6021 (ISSN-L)
  • 1470-8728
OpenAlex
W4235973137
Cited by
87
References
0
Citations by year
2025: 1 · 2024: 2 · 2023: 1 · 2022: 2 · 2021: 2 · 2019: 2 · 2018: 2 · 2016: 3 · 2015: 2 · 2012: 2
FWCI
2.77
Type
journal article
Language
en
Open access
closed
Locations
Biochemical Journal (publishedVersion)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Mitochondrial Function and Pathology
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › ATP Synthase and ATPases Research
  • Health Sciences › Medicine › Physiology › Adipose Tissue and Metabolism
Keywords
UCP3, mitochondrial uncoupling, UCP1, skeletal muscle mitochondria, yeast mitochondria, recombinant protein expression, artifacts, palmitate, superoxide, gross domestic product, proton conductance, brown adipose tissue mitochondria

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