Skip to main content
Biochemical Journal logoLink to Biochemical Journal
. 1993 Dec 15;296(Pt 3):693–700. doi: 10.1042/bj2960693

Cysteine residues are not essential for uncoupling protein function.

I Arechaga 1, S Raimbault 1, S Prieto 1, C Levi-Meyrueis 1, P Zaragoza 1, B Miroux 1, D Ricquier 1, F Bouillaud 1, E Rial 1
PMCID: PMC1137752  PMID: 8280067

Abstract

The uncoupling protein (UCP) of brown adipose tissue is a regulated proton carrier which allows uncoupling of mitochondrial respiration from ATP synthesis and, therefore, dissipation of metabolic energy as heat. In this article we demonstrate that, when UCP is expressed in Saccharomyces cerevisiae, it retains all its functional properties: proton and chloride transport, high-affinity binding of nucleotides and regulation of proton conductance by nucleotides and fatty acids. Site-directed mutagenesis demonstrates that sequential replacement by serine of cysteine residues in the UCP does not affect either its uncoupling activity or its regulation by nucleotides and fatty acids, and therefore establishes that none of the seven cysteine residues present in the wild-type UCP is critical for its activity. These data indicate that transport models involving essential thiol groups can be discounted and that chemical modification data require critical re-evaluation.

Full text

PDF
694

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bathgate B., Freebairn E. M., Greenland A. J., Reid G. A. Functional expression of the rat brown adipose tissue uncoupling protein in Saccharomyces cerevisiae. Mol Microbiol. 1992 Feb;6(3):363–370. doi: 10.1111/j.1365-2958.1992.tb01479.x. [DOI] [PubMed] [Google Scholar]
  2. Bouillaud F., Ricquier D., Thibault J., Weissenbach J. Molecular approach to thermogenesis in brown adipose tissue: cDNA cloning of the mitochondrial uncoupling protein. Proc Natl Acad Sci U S A. 1985 Jan;82(2):445–448. doi: 10.1073/pnas.82.2.445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bouillaud F., Weissenbach J., Ricquier D. Complete cDNA-derived amino acid sequence of rat brown fat uncoupling protein. J Biol Chem. 1986 Feb 5;261(4):1487–1490. [PubMed] [Google Scholar]
  4. Casteilla L., Blondel O., Klaus S., Raimbault S., Diolez P., Moreau F., Bouillaud F., Ricquier D. Stable expression of functional mitochondrial uncoupling protein in Chinese hamster ovary cells. Proc Natl Acad Sci U S A. 1990 Jul;87(13):5124–5128. doi: 10.1073/pnas.87.13.5124. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cullin C., Pompon D. Synthesis of functional mouse cytochromes P-450 P1 and chimeric P-450 P3-1 in the yeast Saccharomyces cerevisiae. Gene. 1988 May 30;65(2):203–217. doi: 10.1016/0378-1119(88)90457-x. [DOI] [PubMed] [Google Scholar]
  6. Dierks T., Salentin A., Krämer R. Pore-like and carrier-like properties of the mitochondrial aspartate/glutamate carrier after modification by SH-reagents: evidence for a performed channel as a structural requirement of carrier-mediated transport. Biochim Biophys Acta. 1990 Oct 19;1028(3):281–288. doi: 10.1016/0005-2736(90)90177-p. [DOI] [PubMed] [Google Scholar]
  7. Fernández M., Nicholls D. G., Rial E. The uncoupling protein from brown-adipose-tissue mitochondria. Chymotrypsin-induced structural and functional modifications. Eur J Biochem. 1987 May 4;164(3):675–680. doi: 10.1111/j.1432-1033.1987.tb11179.x. [DOI] [PubMed] [Google Scholar]
  8. Garlid K. D., Beavis A. D. Swelling and contraction of the mitochondrial matrix. II. Quantitative application of the light scattering technique to solute transport across the inner membrane. J Biol Chem. 1985 Nov 5;260(25):13434–13441. [PubMed] [Google Scholar]
  9. Guérin B., Labbe P., Somlo M. Preparation of yeast mitochondria (Saccharomyces cerevisiae) with good P/O and respiratory control ratios. Methods Enzymol. 1979;55:149–159. doi: 10.1016/0076-6879(79)55021-6. [DOI] [PubMed] [Google Scholar]
  10. Jezek P., Drahota Z. Sulfhydryl groups of the uncoupling protein of brown adipose tissue mitochondria. Distinction between sulfhydryl groups of the H+ channel and the nucleotide binding site. Eur J Biochem. 1989 Jul 15;183(1):89–95. doi: 10.1111/j.1432-1033.1989.tb14900.x. [DOI] [PubMed] [Google Scholar]
  11. Jezek P., Garlid K. D. New substrates and competitive inhibitors of the Cl- translocating pathway of the uncoupling protein of brown adipose tissue mitochondria. J Biol Chem. 1990 Nov 5;265(31):19303–19311. [PubMed] [Google Scholar]
  12. Jezek P. Sulfhydryl groups are involved in H+ translocation via the uncoupling protein of brown adipose tissue mitochondria. FEBS Lett. 1987 Jan 19;211(1):89–93. doi: 10.1016/0014-5793(87)81280-2. [DOI] [PubMed] [Google Scholar]
  13. Jones E. W. Tackling the protease problem in Saccharomyces cerevisiae. Methods Enzymol. 1991;194:428–453. doi: 10.1016/0076-6879(91)94034-a. [DOI] [PubMed] [Google Scholar]
  14. Klaus S., Casteilla L., Bouillaud F., Raimbault S., Ricquier D. Expression of the brown fat mitochondria uncoupling protein in Xenopus oocytes and important into mitochondrial membrane. Biochem Biophys Res Commun. 1990 Mar 16;167(2):784–789. doi: 10.1016/0006-291x(90)92094-g. [DOI] [PubMed] [Google Scholar]
  15. Klaus S., Casteilla L., Bouillaud F., Ricquier D. The uncoupling protein UCP: a membraneous mitochondrial ion carrier exclusively expressed in brown adipose tissue. Int J Biochem. 1991;23(9):791–801. doi: 10.1016/0020-711x(91)90062-r. [DOI] [PubMed] [Google Scholar]
  16. Klingenberg M., Winkler E. The reconstituted isolated uncoupling protein is a membrane potential driven H+ translocator. EMBO J. 1985 Dec 1;4(12):3087–3092. doi: 10.1002/j.1460-2075.1985.tb04049.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lin C. S., Klingenberg M. Characteristics of the isolated purine nucleotide binding protein from brown fat mitochondria. Biochemistry. 1982 Jun 8;21(12):2950–2956. doi: 10.1021/bi00541a023. [DOI] [PubMed] [Google Scholar]
  18. Menick D. R., Lee J. A., Brooker R. J., Wilson T. H., Kaback H. R. Role of cysteine residues in the lac permease of Escherichia coli. Biochemistry. 1987 Feb 24;26(4):1132–1136. doi: 10.1021/bi00378a022. [DOI] [PubMed] [Google Scholar]
  19. Milner R. E., Wilson S., Arch J. R., Trayhurn P. Acute effects of a beta-adrenoceptor agonist (BRL 26830A) on rat brown-adipose-tissue mitochondria. Increased GDP binding and GDP-sensitive proton conductance without changes in the concentration of uncoupling protein. Biochem J. 1988 Feb 1;249(3):759–763. doi: 10.1042/bj2490759. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Murdza-Inglis D. L., Patel H. V., Freeman K. B., Jezek P., Orosz D. E., Garlid K. D. Functional reconstitution of rat uncoupling protein following its high level expression in yeast. J Biol Chem. 1991 Jun 25;266(18):11871–11875. [PubMed] [Google Scholar]
  21. Nelson D. R., Lawson J. E., Klingenberg M., Douglas M. G. Site-directed mutagenesis of the yeast mitochondrial ADP/ATP translocator. Six arginines and one lysine are essential. J Mol Biol. 1993 Apr 20;230(4):1159–1170. doi: 10.1006/jmbi.1993.1233. [DOI] [PubMed] [Google Scholar]
  22. Nicholls D. G. Hamster brown-adipose-tissue mitochondria. Purine nucleotide control of the ion conductance of the inner membrane, the nature of the nucleotide binding site. Eur J Biochem. 1976 Feb 16;62(2):223–228. doi: 10.1111/j.1432-1033.1976.tb10151.x. [DOI] [PubMed] [Google Scholar]
  23. Nicholls D. G., Lindberg O. Brown-adipose-tissue mitochondria. The influence of albumin and nucleotides on passive ion permeabilities. Eur J Biochem. 1973 Sep 3;37(3):523–530. doi: 10.1111/j.1432-1033.1973.tb03014.x. [DOI] [PubMed] [Google Scholar]
  24. Nicholls D. G., Locke R. M. Thermogenic mechanisms in brown fat. Physiol Rev. 1984 Jan;64(1):1–64. doi: 10.1152/physrev.1984.64.1.1. [DOI] [PubMed] [Google Scholar]
  25. Palmieri F., Bisaccia F., Iacobazzi V., Indiveri C., Zara V. Mitochondrial substrate carriers. Biochim Biophys Acta. 1992 Jul 17;1101(2):223–227. [PubMed] [Google Scholar]
  26. Peachey T., French R. R., York D. A. Regulation of GDP binding and uncoupling-protein concentration in brown-adipose-tissue mitochondria. The effects of cold-acclimation, warm-reacclimation and noradrenaline. Biochem J. 1988 Jan 15;249(2):451–457. doi: 10.1042/bj2490451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Phelps A., Wohlrab H. Mitochondrial phosphate transport. The Saccharomyces cerevisiae (threonine 43 to cysteine) mutant protein explicitly identifies transport with genomic sequence. J Biol Chem. 1991 Oct 25;266(30):19882–19885. [PubMed] [Google Scholar]
  28. Prieto S., Bouillaud F., Ricquier D., Rial E. Activation by ATP of a proton-conducting pathway in yeast mitochondria. Eur J Biochem. 1992 Sep 1;208(2):487–491. doi: 10.1111/j.1432-1033.1992.tb17212.x. [DOI] [PubMed] [Google Scholar]
  29. Rial E., Aréchaga I., Sainz-de-la-Maza E., Nicholls D. G. Effect of hydrophobic sulphydryl reagents on the uncoupling protein and inner-membrane anion channel of brown-adipose-tissue mitochondria. Eur J Biochem. 1989 Jun 1;182(1):187–193. doi: 10.1111/j.1432-1033.1989.tb14816.x. [DOI] [PubMed] [Google Scholar]
  30. Rial E., Nicholls D. G. Chemical modification of the brown-fat-mitochondrial uncoupling protein with tetranitromethane and N-ethylmaleimide. A cysteine residue is implicated in the nucleotide regulation of anion permeability. Eur J Biochem. 1986 Dec 15;161(3):689–694. doi: 10.1111/j.1432-1033.1986.tb10494.x. [DOI] [PubMed] [Google Scholar]
  31. Rial E., Nicholls D. G. The mitochondrial uncoupling protein from guinea-pig brown adipose tissue. Synchronous increase in structural and functional parameters during cold-adaptation. Biochem J. 1984 Sep 15;222(3):685–693. doi: 10.1042/bj2220685. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Rial E., Poustie A., Nicholls D. G. Brown-adipose-tissue mitochondria: the regulation of the 32000-Mr uncoupling protein by fatty acids and purine nucleotides. Eur J Biochem. 1983 Dec 1;137(1-2):197–203. doi: 10.1111/j.1432-1033.1983.tb07815.x. [DOI] [PubMed] [Google Scholar]
  33. Robillard G. T., Konings W. N. A hypothesis for the role of dithiol-disulfide interchange in solute transport and energy-transducing processes. Eur J Biochem. 1982 Oct;127(3):597–604. doi: 10.1111/j.1432-1033.1982.tb06914.x. [DOI] [PubMed] [Google Scholar]
  34. Sims P. J., Waggoner A. S., Wang C. H., Hoffman J. F. Studies on the mechanism by which cyanine dyes measure membrane potential in red blood cells and phosphatidylcholine vesicles. Biochemistry. 1974 Jul 30;13(16):3315–3330. doi: 10.1021/bi00713a022. [DOI] [PubMed] [Google Scholar]
  35. Spector A. A., Fletcher J. E., Ashbrook J. D. Analysis of long-chain free fatty acid binding to bovine serum albumin by determination of stepwise equilibrium constants. Biochemistry. 1971 Aug 17;10(17):3229–3232. doi: 10.1021/bi00793a011. [DOI] [PubMed] [Google Scholar]
  36. Trayhurn P., Ashwell M., Jennings G., Richard D., Stirling D. M. Effect of warm or cold exposure on GDP binding and uncoupling protein in rat brown fat. Am J Physiol. 1987 Feb;252(2 Pt 1):E237–E243. doi: 10.1152/ajpendo.1987.252.2.E237. [DOI] [PubMed] [Google Scholar]
  37. Trayhurn P., Milner R. E. A commentary on the interpretation of in vitro biochemical measures of brown adipose tissue thermogenesis. Can J Physiol Pharmacol. 1989 Aug;67(8):811–819. doi: 10.1139/y89-128. [DOI] [PubMed] [Google Scholar]
  38. Wright A. P., Bruns M., Hartley B. S. Extraction and rapid inactivation of proteins from Saccharomyces cerevisiae by trichloroacetic acid precipitation. Yeast. 1989 Jan-Feb;5(1):51–53. doi: 10.1002/yea.320050107. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES