Skip to main content
SpringerLink
Log in

Interplay between glutathione, Atx1 and copper. 1. Copper(I) glutathionate induced dimerization of Atx1

  • Original Paper
  • Published:
JBIC Journal of Biological Inorganic Chemistry Aims and scope Submit manuscript

Abstract

Copper is both an essential element as a catalytic cofactor and a toxic element because of its redox properties. Once in the cell, Cu(I) binds to glutathione (GSH) and various thiol-rich proteins that sequester and/or exchange copper with other intracellular components. Among them, the Cu(I) chaperone Atx1 is known to deliver Cu(I) to Ccc2, the Golgi Cu–ATPase, in yeast. However, the mechanism for Cu(I) incorporation into Atx1 has not yet been unraveled. We investigated here a possible role of GSH in Cu(I) binding to Atx1. Yeast Atx1 was expressed in Escherichia coli and purified to study its ability to bind Cu(I). We found that with an excess of GSH [at least two GSH/Cu(I)], Atx1 formed a Cu(I)-bridged dimer of high affinity for Cu(I), containing two Cu(I) and two GSH, whereas no dimer was observed in the absence of GSH. The stability constants (log β) of the Cu(I) complexes measured at pH 6 were 15–16 and 49–50 for CuAtx1 and Cu I2 (GS−)2(Atx1)2, respectively. Hence, these results suggest that in vivo the high GSH concentration favors Atx1 dimerization and that Cu I2 (GS−)2(Atx1)2 is the major conformation of Atx1 in the cytosol.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9

Similar content being viewed by others

Abbreviations

BCA:

Bicinchoninic acid

BCS:

Bathocuproine disulfonate

DTNB:

5,5′-Dithiobis(2-nitrobenzoate)

DTT:

Dithiothreitol

EPR:

Electron paramagnetic resonance

GSH:

Glutathione

Mops:

3-(N-Morpholino)propanesulfonic acid

PAGE:

Polyacrylamide gel electrophoresis

SDS:

Sodium dodecyl sulfate

TCEP:

Tris(2-carboxyethyl)phosphine hydrochloride

TTM:

Tetrathiomolybdate

References

  1. Jensen LT, Howard WR, Strain JJ, Winge DR, Culotta VC (1996) J Biol Chem 271:18514–18519

    Article  PubMed  CAS  Google Scholar 

  2. Calderone V, Dolderer B, Hartmann HJ, Echner H, Luchinat C, Del Bianco C, Mangani S, Weser U (2005) Proc Natl Acad Sci USA 102:51–56

    Article  PubMed  CAS  Google Scholar 

  3. Peña MM, Lee J, Thiele DJ (1999) J Nutr 129:1251–1260

    PubMed  Google Scholar 

  4. Balamurugan K, Schaffner W (2006) Biochim Biophys Acta 1763:737–746

    Article  PubMed  CAS  Google Scholar 

  5. Culotta VC, Yang M, O’Halloran TV (2006) Biochim Biophys Acta 1763:747–758

    Article  PubMed  CAS  Google Scholar 

  6. Cobine PA, Pierrel F, Winge DR (2006) Biochim Biophys Acta 1763:759–772

    Article  PubMed  CAS  Google Scholar 

  7. Singleton C, Le Brun NE (2007) Biometals 20:275–289

    Article  PubMed  CAS  Google Scholar 

  8. Portnoy ME, Schmidt PJ, Rogers RS, Culotta VC (2001) Mol Genet Genomics 265:873–882

    Article  PubMed  CAS  Google Scholar 

  9. Field LS, Luk E, Culotta VC (2002) J Bioenerg Biomembr 34:373–379

    Article  PubMed  CAS  Google Scholar 

  10. Xiao Z, Wedd AG (2002) Chem Commun 588–589

  11. Freedman JH, Ciriolo MR, Peisach J (1989) J Biol Chem 264:5598–5605

    PubMed  CAS  Google Scholar 

  12. Lin CM, Crawford BF, Kosman DJ (1993) J Gen Microbiol 139:1605–1615

    PubMed  CAS  Google Scholar 

  13. Ciriolo MR, Desideri A, Paci M, Rotilio G (1990) J Biol Chem 265:11030–11034

    PubMed  CAS  Google Scholar 

  14. Brouwer M, Brouwer-Hoexum T (1992) Biochemistry 31:4096–4102

    Article  PubMed  CAS  Google Scholar 

  15. Ferreira AM, Ciriolo MR, Marcocci L, Rotilio G (1993) Biochem J 292:673–676

    PubMed  CAS  Google Scholar 

  16. Mehra RK, Mulchandani P (1995) Biochem J 307:697–705

    PubMed  CAS  Google Scholar 

  17. Musci G, Di Marco S, Bellenchi GC, Calabrese L (1996) J Biol Chem 271:1972–1978

    Article  PubMed  CAS  Google Scholar 

  18. Meister A, Anderson ME (1983) Annu Rev Biochem 52:711–760

    Article  PubMed  CAS  Google Scholar 

  19. McDonald JW, Friesen GD, Rosenhein LD, Newton WE (1983) Inorg Chem Acta 72:205–210

    Article  CAS  Google Scholar 

  20. Pufahl RA, Singer CP, Peariso KL, Lin SJ, Schmidt PJ, Fahrni CJ, Culotta VC, Penner-Hahn JE, O’Halloran TV (1997) Science 278:853–856

    Article  PubMed  CAS  Google Scholar 

  21. Lin SJ, Pufahl RA, Dancis A, O’Halloran TV, Culotta VC (1997) J Biol Chem 272:9215–9220

    Article  PubMed  CAS  Google Scholar 

  22. Blair D, Diehl H (1961) Talanta 7:163–174

    Article  CAS  Google Scholar 

  23. Ellman GL (1959) Arch Biochem Biophys 82:70–77

    Article  PubMed  CAS  Google Scholar 

  24. Wright SK, Viola RE (1998) Anal Biochem 265:8–14

    Article  PubMed  CAS  Google Scholar 

  25. Krezel A, Lesniak W, Jezowska-Bojczuk M, Mlynarz P, Brasun J, Kozlowski H, Bal W (2001) J Inorg Biochem 84:77–88

    Article  CAS  Google Scholar 

  26. Kisker C, Schindelin H, Rees DC (1997) Annu Rev Biochem 66:233–267

    Article  PubMed  CAS  Google Scholar 

  27. Conklin MH, Hoffmann MR (1988) Environ Sci Technol 22:883–891

    Article  CAS  Google Scholar 

  28. Conklin MH, Hoffmann MR (1988) Environ Sci Technol 22:891–898

    Article  CAS  Google Scholar 

  29. Smith RM, Martell AE, Mothekaitis RJ (2001) NIST critically selected stability constants of metal complexes database. NIST Standard Reference Database 46

  30. Alderighi L, Gans P, Ienco A, Peters D, Sabatini A, Vacca A (1999) Coord Chem Rev 184:311–318

    Article  CAS  Google Scholar 

  31. Xiao Z, Loughlin F, George GN, Howlett GJ, Wedd AG (2004) J Am Chem Soc 126:3081–3090

    Article  PubMed  CAS  Google Scholar 

  32. Arnesano F, Banci L, Bertini I, Huffman DL, O’Halloran TV (2001) Biochemistry 40:1528–1539

    Article  PubMed  CAS  Google Scholar 

  33. Rosenzweig AC, Huffman DL, Hou MY, Wernimont AK, Pufahl RA, O’Halloran TV (1999) Struct Fold Des 7:605–617

    Article  CAS  Google Scholar 

  34. Kihlken MA, Leech AP, Le Brun NE (2002) Biochem J 368:729–739

    Article  PubMed  CAS  Google Scholar 

  35. Urvoas A, Moutiez M, Estienne C, Couprie J, Mintz E, Le Clainche L (2004) Eur J Biochem 271:993–1003

    Article  PubMed  CAS  Google Scholar 

  36. Arnesano F, Banci L, Bertini I, Cantini F, Ciofi-Baffoni S, Huffman DL, O’Halloran TV (2001) J Biol Chem 276:41365–41376

    Article  PubMed  CAS  Google Scholar 

  37. Wernimont AK, Huffman DL, Lamb AL, O’Halloran TV, Rosenzweig AC (2000) Nat Struct Biol 7:766–771

    Article  PubMed  CAS  Google Scholar 

  38. Ogra Y, Ohmichi M, Suzuki KT (1996) Toxicology 106:75–83

    Article  PubMed  CAS  Google Scholar 

  39. Österberg R, Ligaarden R, Persson D (1979) J Inorg Biochem 10:341–355

    Article  PubMed  Google Scholar 

  40. Corazza A, Harvey I, Sadler PJ (1996) Eur J Biochem 236:697–705

    Article  PubMed  CAS  Google Scholar 

  41. Pecci L, Montefoschi G, Musci G, Cavallini D (1997) Amino Acids 13:355–367

    Article  CAS  Google Scholar 

  42. Portnoy ME, Rosenzweig AC, Rae T, Huffman DL, O’Halloran TV, Culotta VC (1999) J Biol Chem 274:15041–15045

    Article  PubMed  CAS  Google Scholar 

  43. Walker JM, Huster D, Ralle M, Morgan CT, Blackburn NJ, Lutsenko S (2004) J Biol Chem 279:15376–15384

    Article  PubMed  CAS  Google Scholar 

  44. Morin I, Cuillel M, Lowe J, Crouzy S, Guillain F, Mintz E (2005) FEBS Lett 579:1117–1123

    Article  PubMed  CAS  Google Scholar 

  45. Banci L, Bertini I, Cantini F, Felli IC, Gonnelli L, Hadjiliadis N, Pierattelli R, Rosato A, Voulgaris P (2006) Nat Chem Biol 2:367–368

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

We thank Stéphane Ménage for performing the EPR measurements and Pierre Rousselot-Pailley for fruitful discussions. Funding of this work was provided in part by the Programme de Toxicologie Nucléaire Environnementale. The Institut des Métaux en Biologie de Grenoble provided access to EPR equipment. I.M. had a fellowship from the Programme de Toxicologie Nucléaire du CEA.

Author information

Author notes

  1. Olivier Jacquin

    Present address: Institut de Chimie B6c, Université de Liège, 4000, Liège, Belgium

  2. Isabelle Morin

    Present address: Institute for Ageing and Health, Henry Wellcome Laboratory for Biogerontology Research, University of Newcastle, Newcastle upon Tyne, NE4 6BE, UK

Authors and Affiliations

  1. Laboratoire de Chimie et Biologie des Métaux, CEA, DSV, iRTSV, 17 rue des Martyrs, Grenoble, 38054, France

    Roger Miras, Isabelle Morin, Olivier Jacquin, Martine Cuillel, Florent Guillain & Elisabeth Mintz

  2. CNRS, UMR 5249, Grenoble, 38054, France

    Roger Miras, Isabelle Morin, Olivier Jacquin, Martine Cuillel, Florent Guillain & Elisabeth Mintz

  3. Université Joseph Fourier, Grenoble, 38000, France

    Roger Miras, Isabelle Morin, Olivier Jacquin, Martine Cuillel, Florent Guillain & Elisabeth Mintz

Authors
  1. Roger Miras
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Isabelle Morin
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Olivier Jacquin
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Martine Cuillel
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Florent Guillain
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Elisabeth Mintz
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Elisabeth Mintz.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Miras, R., Morin, I., Jacquin, O. et al. Interplay between glutathione, Atx1 and copper. 1. Copper(I) glutathionate induced dimerization of Atx1. J Biol Inorg Chem 13, 195–205 (2008). https://doi.org/10.1007/s00775-007-0310-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00775-007-0310-2

Keywords

Search

Navigation