Skip to main content
SpringerLink
Log in

Chlamydomonas reinhardtii thioredoxins: structure of the genes coding for the chloroplastic m and cytosolic h isoforms; expression in Escherichia coli of the recombinant proteins, purification and biochemical properties

  • Research Article
  • Published:
Plant Molecular Biology Aims and scope Submit manuscript

Abstract

Based on known amino acid sequences, probes have been generated by PCR and used for the subsequent isolation of cDNAs and genes coding for two thioredoxins (m and h) of Chlamydomonas reinhardtii. Thioredoxin m, a chloroplastic protein, is encoded as a preprotein of 140 amino acids (15 101 Da) containing a transit peptide of 34 amino acids with a very high content of Ala and Arg residues. The sequence for thioredoxin h codes for a 113 amino acid protein with a molecular mass of 11817 Da and no signal sequence. The thioredoxin m gene contains a single intron and seems to be more archaic in structure than the thioredoxin h gene, which is split into 4 exons. The cDNA sequences encoding C. reinhardtii thioredoxins m and h have been integrated into the pET-3d expression vector, which permits efficient production of proteins in Escherichia coli cells. A high expression level of recombinant thioredoxins was obtained (up to 50 mg/l culture). This has allowed us to study the biochemical/biophysical properties of the two recombinant proteins. Interestingly, while the m-type thioredoxin was found to have characteristics very close to the ones of prokaryotic thioredoxins, the h-type thioredoxin was quite different with respect to its kinetic behaviour and, most strikingly, its heat denaturation properties.

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

Similar content being viewed by others

Abbreviations

DTT:

dithiothreitol

FBPase:

Fructose 1,6-biphosphate phosphatase

FTR:

ferredoxin-thioredoxin reductase

IPTG:

isopropyl thiogalactoside

NADP-MDH:

NADPH-dependent malate dehydrogenase

NMR:

nuclear magnetic resonance

NTR:

NADPH-dependent thioredoxin reductase

References

  1. Holmgren A: Thioredoxin. Annu Rev Biochem 54: 237–271 (1985).

    Google Scholar 

  2. Katti S, Le Master D, Eklund H: Crystal structure of thioredoxin from Escherichia coli at 1.68 Å resolution. J Mol Biol 212: 167–184 (1990).

    Google Scholar 

  3. Holmgren A: Thioredoxin catalyzes the reduction of insulin disulfides by dithiothreitol and dihydrolipoamide. J Biol Chem 254: 9627–9632 (1979).

    Google Scholar 

  4. Buchanan BB: Role of light in the regulation of chloro-plast enzymes. Annu Rev Plant Physiol 31: 341–374 (1980).

    Google Scholar 

  5. Buchanan BB: Regulation of CO2 assimilation in oxygenic photosynthesis. The ferredoxin-thioredoxin system. Arch Biochem Biophys 288: 1–8 (1991).

    Google Scholar 

  6. Droux M, Miginiac-Maslow M, Jacquot JP, Crawford NA, Kosower NS, Buchanan BB: Ferredoxin-thioredoxin reductase: a catalytically active dithiol group links photo-reduced ferredoxin to thioredoxin functional in photosynthetic enzyme regulation. Arch Biochem Biophys 256: 372–380 (1987).

    Google Scholar 

  7. Florencio FJ, Yee BC, Johnson TC, Buchanan BB: An NADP/thioredoxin system in leaves: purification and characterization of NADP thioredoxin reductase and thioredoxin h from spinach. Arch Biochem Biophys 266: 496–507 (1988).

    Google Scholar 

  8. Kobrehel K, Yee BC, Buchanan BB: Role of the NADP/thioredoxin system in the reduction of α-amylase and trypsin inhibitor proteins. J Biol Chem 266: 16135–16140 (1991).

    Google Scholar 

  9. Bodenstein-Lang J, Buch A, Follmann H: Animal and plant mitochondria contain specific thioredoxin. FEBS Lett 258: 22–26 (1989).

    Google Scholar 

  10. Harris E: The Chlamydomonas Sourcebook: A Comprehensive Guide to Biology and Laboratory Use. Academic Press, San Diego (1989).

    Google Scholar 

  11. Kindle KL, Richards KL, Stern DB: Engineering the chloroplast genome: techniques and capabilities for chloroplast transformation in Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 88: 1721–1725 (1991).

    Google Scholar 

  12. Sodeinde OA, Kindle KL: Homologous recombination in the nuclear genome of Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 90: 9199–9203 (1993).

    Google Scholar 

  13. Huppe H, Picaud A, Buchanan BB, Miginiac-Maslow M: Identification of an NADP/thioredoxin system in Chlamydomonas reinhardtii. Plant 186: 115–121 (1991).

    Google Scholar 

  14. Huppe HC, de Lamotte-Guéry F, Jacquot JP, Buchanan BB: The ferredoxin-thioredoxin system of a green alga, Chlamydomonas reinhardtii. Identification and characterization of thioredoxins and ferredoxin-thioredoxin reductase components. Planta 180: 341–351 (1990).

    Google Scholar 

  15. Schmitter JM, Jacquot JP, de Lamotte-Guéry, Beauvallet C, Dutka S, Gadal P, Decottignies P: Purification, properties and complete amino acid sequence of ferredoxin from a green alga, Chlamydomonas reinhardtii. Eur J Biochem 172: 405–412 (1988).

    Google Scholar 

  16. Decottignies P, Schmitter JM, Jacquot JP, Dutka S, Picaud A, Gadal P: Purification, characterization and complete amino acid sequence of a thioredoxin from a green alga Chlamydomonas reinhardtii. Arch Biochem Biophys 280: 112–121 (1990).

    Google Scholar 

  17. Decottignies P, Schmitter JM, Dutka S, Jacquot JP, Miginiac-Maslow M: Characterization and primary structure of a second thioredoxin from the green alga, Chlamydomonas reinhardtii. Eur J Biochem 198: 505–512 (1991).

    Google Scholar 

  18. Rogers WJ, Hodges M, Decottignies P, Schmitter JM, Gadal P, Jacquot JP: Isolation of a cDNA fragment coding for Chlamydomonas reinhardtii ferredoxin and expression of the recombinant protein in Escherichia coli cells. FEBS Lett 310: 240–245 (1992).

    Google Scholar 

  19. Jacquot JP, Stein M, Hodges M, Miginiac Maslow M: PCR cloning of a nucleotidic sequence coding for the mature part of Chlamydomonas reinhardtii thioredoxin Ch2. Nucl Acids Res 20: 617 (1992).

    Google Scholar 

  20. Lancelin JM, Stein M, Jacquot JP: Secondary structure and protein folding of recombinant chloroplastic thioredoxin Ch2 from the green alga Chlamydomonas reinhardtii as determined by 1H NMR. J Biochem 114: 421–431 (1993).

    Google Scholar 

  21. Dyson HJ, Holmgren A, Wright PE: Assignment of the proton NMR spectrum of reduced and oxidized thioredoxin: sequence specific assignment and global fold. Biochemistry 28: 7074–7087 (1989).

    Google Scholar 

  22. Dyson HJ, Guppert GP, Case DA, Holmgren A, Wright P: Three dimensional solution structure of the reduced form of Escherichia coli thioredoxin determined by nuclear magnetic resonance spectroscopy. Biochemistry 29: 4129–4136 (1990).

    Google Scholar 

  23. Forman-Kay JD, Clore GM, Driscoll PC, Wingfield PT, Gronenborn AM: High resolution three dimensional structure of reduced recombinant human thioredoxin in solution. Biochemistry 30: 2685–2698 (1991).

    Google Scholar 

  24. Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).

    Google Scholar 

  25. Campell WH, Gowri G: Codon usage in higher plants, green algae and cyanobacteria. Plant Physiol 92: 1–11 (1990).

    Google Scholar 

  26. Ausubel FM et al. (eds) Current Protocols in Molecular Biology, vol. 1, chapter 6. John Wiley, New York (1987).

    Google Scholar 

  27. Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW: Use of bacteriophage T7 RNA polymerase to direct selective high level expression of cloned genes. Meth Enzymol 185: 60–89 (1990).

    Google Scholar 

  28. de Lamotte-Guéry F, Miginiac-Maslow M, Decottignies P, Stein M, Minard P, Jacquot JP: Mutation of a negatively charged amino acid in thioredoxin modifies its reactivity with chloroplastic anzymes. Eur J Biochem 196: 287–294 (1991).

    Google Scholar 

  29. Jacquot JP, Issakidis E, Decottignies P, Lemaire M, Miginiac-Maslow M: Analysis and manipulation of target enzymes for thioredoxin control. Meth Enzymol 252: 240–252 (1995).

    Google Scholar 

  30. Jacquot JP, Rivera-Madrid R, Marinho P, Kollarova M, Le Maréchal P, Miginiac-Maslow M, Meyer Y: Arabidopsis thaliana NADPH thioredoxin reductase. J Mol Biol 235: 1357–1363 (1994).

    Google Scholar 

  31. Jacquot JP, Keryer E, Issakidis E, Decottignies P, Miginiac-Maslow M, Schmitter JM, Crétin C: Properties of recombinant NADP-malate dehydrogenases from Sorghum vulgare leaves expressed in Escherichia coli cells. Eur J Biochem 199: 47–51 (1991).

    Google Scholar 

  32. Issakidis E, Saarinen M, Decottignies P, Jacquot JP, Crétin C, Gadal P, Miginiac-Maslow M: Identification and characterization of the second regulatory disulfide bridge of recombinant Sorghum leaf NADP-malate dehydrogenase. J Biol Chem 269: 3511–3517 (1994).

    Google Scholar 

  33. Schäger H, von Jagow G: Tricine-sodium dodecyl sulfate polyacrylamide gel electrophoresis for the separation of proteins in the 1 to 100 kDa range. Anal Biochem 166: 368–379 (1987).

    Google Scholar 

  34. Wetterauer B, Véron M, Miginiac-Maslow M, Decottignies P, Jacquot JP: Biochemical characterization of thioredoxin I from Dictyostelium discoideum. Eur J Biochem 209: 643–649 (1992).

    Google Scholar 

  35. von Heijne G, Steppuhn J, Herrmann RG: Domain structure of mitochondrial and chloroplast targeting peptides. Eur J Biochem 180: 535–545 (1989).

    Google Scholar 

  36. Wedel N, Clausmeyer S, Herrmann RG, Gardet-Salvi L, Schürmann P: Nucleotide sequence of cDNAs encoding the entire precursor polypeptide for thioredoxin m from spinach chloroplasts. Plant Mol Biol 18: 527–533 (1992).

    Google Scholar 

  37. Lancelin JM, Bally I, Arlaud G, Blackledge M, Gans P, Stein M, Jacquot JP: NMR structures of ferredoxin chloroplastic transit peptide from Chlamydomonas reinhardtii promoted by trifluoroethanol in aqueous solution. FEBS Lett 343: 261–266 (1994).

    Google Scholar 

  38. Tonissen KF, Wells RE: Isolation and characterization of human thioredoxin encoding genes. Gene 102: 221–228 (1991).

    Google Scholar 

  39. Kaghad M, Dessarps F, Jacquemin-Sablon H, Caput D, Fradelizi D, Wollman EE: Genomic cloning of human thioredoxin encoding gene: mapping of the transcription start point and analysis of the promoter. Gene 140: 273–278 (1994).

    Google Scholar 

  40. Brugidou C, Marty I, Chartier Y, Meyer Y: The Nicotiana tabacum encodes two cytoplasmic genes, which are differently expressed. Mol Gen Genet 238: 285–293 (1993).

    Google Scholar 

  41. Breathnach R, Chambon P: Organization and expression of eukaryotic split genes coding for proteins. Annu Rev Biochem 50: 349–383 (1981).

    Google Scholar 

  42. Patthy L: Introns and exons. Curr Opin Struct Biol 4: 383–392 (1994).

    Google Scholar 

  43. Reith M, Munholland J: A high resolution map of the chloroplast genome of the red alga Porphyra purpurea. Plant Cell 5: 465–475 (1993).

    Google Scholar 

  44. Reynolds AE, Chesnick JB, Woolford J, Cattolico RA: Chloroplast encoded thioredoxin genes in the red algae Porphyra yezoensis and Griffithsia pacifica: evolutionary implications. Plant Mol Biol 25: 13–21 (1994).

    Google Scholar 

  45. Hirel PH, Schmitter JM, Dessen P, Fayat G, Blanquet S: Extent of N terminal excision from Escherichia coli cells in governed by the side chain length of the penultimate amino acid. Proc Natl Acad Sci USA: 86: 8247–8251 (1989).

    Google Scholar 

  46. Wollman E, d'Auriol L, Rimsky L, Shaw A, Jacquot JP, Wingfield P, Graber P, Dessarps F, Robin P, Galibert F, Bertoglio J, Fradelizi D: Cloning and expression of a cDNA for human thioredoxin. J Biol Chem 263: 15506–15512 (1988).

    Google Scholar 

  47. Creighton TE (ed) Protein Structure; A Practical Approach. IRL Press, Oxford (1989).

    Google Scholar 

  48. Mittard V, Morelle N, Brutscher B, Simorre JP, Marion D, Stein M, Jacquot JP, Lirsac PN, Lancelin JM: 1H, 13C, 15N-NMR resonance assignments of oxidized thioredoxin h from the eukaryotic green alga Chlamydomonas reinhardtii using new methods based on three-dimensional triple-resonance NMR spectroscopy and computer-assisted backbone assignment. Eur J Biochem 229: 473–485 (1995).

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Laboratoire de Physiologie Végétale Moléculaire, URA 1128 CNRS, Université de Paris-Sud, Bâtiment 630, 91405, Orsay Cedex, France

    Mariana Stein, Jean-Pierre Jacquot, Emmanuelle Jeannette, Paulette Decottignies, Michael Hodges & Myroslawa Miginiac-Maslow

  2. Institut de Biologie Structurale, CEA-CNRS 41, Avenue des Martyrs 38027, Grenoble Cedex, France

    Jean-Marc Lancelin & Virginie Mittard

  3. Laboratoire de Biochimie Ecole Polytechnique, 91128, Palaiseau Cedex, France

    Jean-Marie Schmitter

Authors
  1. Mariana Stein
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jean-Pierre Jacquot
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Emmanuelle Jeannette
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Paulette Decottignies
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Michael Hodges
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jean-Marc Lancelin
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Virginie Mittard
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jean-Marie Schmitter
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Myroslawa Miginiac-Maslow
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

Dedicated to the memory of Claude Crétin

Rights and permissions

Reprints and permissions

About this article

Cite this article

Stein, M., Jacquot, JP., Jeannette, E. et al. Chlamydomonas reinhardtii thioredoxins: structure of the genes coding for the chloroplastic m and cytosolic h isoforms; expression in Escherichia coli of the recombinant proteins, purification and biochemical properties. Plant Mol Biol 28, 487–503 (1995). https://doi.org/10.1007/BF00020396

Download citation

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00020396

Key words

Search

Navigation