Purification and Proteomic Analysis of Chloroplasts and their Sub-Organellar Compartments

  • Daniel Salvi
  • Norbert Rolland
  • Jacques Joyard
  • Myriam Ferro
Part of the Methods in Molecular Biology™ book series (MIMB, volume 432)


Sub-cellular proteomics has proven to be a powerful approach to link the information contained in sequenced genomes from eukaryotic cells to the functional knowledge provided by studies of cell compartments. Chloroplasts are plant-specific organelles and are the site of photosynthesis and also of many other essential metabolic pathways, like syntheses of amino acids, vitamins, and pigments. They contain several sub-organellar compartments: the envelope (the two-membrane system surrounding the organelle), the stroma (the internal soluble phase), and the thylakoid membranes (the internal membrane system). There is a link between these compartments and the functions of their constitutive proteins. One way to bring into view the sub-proteomes of the chloroplast is to develop proteomic analyses based (1) on the use of highly purified sub-fractions of the chloroplast and (2) on mass spectrometry (MS)-based analyses for protein identification. To illustrate such strategies, this chapter describes the methods for purification of chloroplasts from Arabidopsis leaves and for the specific recovery of highly pure sub-organellar fractions of envelope, stroma, and thylakoids. Subsequently, methods are described to analyze by MS the proteins recovered from these fractions.

Key words

chloroplast chloroplast envelope stroma thylakoids mass spectrometry proteome 


  1. 1.
    Ferro, M., Salvi, D., Rivi‘ere-Rolland, H., Vermat, T., Seigneurin-Berny, D., Grunwald, D., et al. (2002) Integral membrane proteins of the chloroplast envelope: identification and subcellular localization of new transporters. Proc. Natl. Acad. Sci. U.S.A. 99, 11487–11492.CrossRefPubMedGoogle Scholar
  2. 2.
    Ferro, M., Salvi, D., Brugi‘ere, S., Miras, S., Kowalski, S., Louwagie, M., et al. (2003) Proteomics of the chloroplast envelope membranes from Arabidopsis thaliana. Mol. Cell. Proteomics 2, 325–345.Google Scholar
  3. 3.
    Ferro, M., Seigneurin-Berny, D., Rolland, N., Chapel, A., Salvi, D., Garin, J., et al. (2002) Organic solvent extraction as a versatile procedure to identify hydrophobic chloroplast membrane proteins. Electrophoresis 21, 3517–3526.CrossRefGoogle Scholar
  4. 4.
    Seigneurin-Berny, D., Rolland, N., Garin, J., and Joyard, J. (1999) Differential extraction of hydrophobic proteins from chloroplast envelope membranes: a subcellular-specific proteomic approach to identify rare intrinsic membrane proteins. Plant J. 19, 217–228.CrossRefPubMedGoogle Scholar
  5. 5.
    Douce, R. and Joyard, J. (1982) Purification of the chloroplast envelope. In Methods in Chloroplast Molecular Biology (Edelman, M., Hallick, R., and Chua, N. H., eds.), Elsevier/North-Holland, Amsterdam, pp. 139–256.Google Scholar
  6. 6.
    Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254.CrossRefPubMedGoogle Scholar
  7. 7.
    Peltier, J. B, Emanuelsson, O., Kalume, D. E., Ytterberg, J., Friso, G., Rudella, A., et al. (2002) Central functions of the lumenal and peripheral thylakoid proteome of Arabidopsis determined by experimentation and genome-wide prediction. Plant Cell 14, 211–236.CrossRefPubMedGoogle Scholar
  8. 8.
    Peltier, J. B., Friso, G., Kalume, D. E., Roepstorff, P., Nilsson, F., Adamska, I., et al. (2000) Proteomics of the chloroplast: systematic identification and targeting analysis of lumenal and peripheral thylakoid proteins. Plant Cell 12, 319–341.CrossRefPubMedGoogle Scholar
  9. 9.
    Schubert, M., Petersson, U. A., Haas, B. J., Funk, C., Schroder, W. P., and Kieselbach, T. (2002) Proteome map of the chloroplast lumen of Arabidopsis thaliana. J. Biol. Chem. 277, 8354–8365.CrossRefPubMedGoogle Scholar
  10. 10.
    Seigneurin-Berny, D., Gravot, A., Auroy, P., Mazard, C., Kraut, A., Finazzi, G., et al. (2006) HMA1, a new Cu-ATPase of the chloroplast envelope, is essential for growth under adverse light conditions. J. Biol. Chem. 281, 2882–2892.CrossRefPubMedGoogle Scholar
  11. 11.
    Santoni, V., Molloy, M., and Rabilloud, T. (2000a) Membrane proteins and proteomics: un amour impossible? Electrophoresis 21, 1054–1070.CrossRefGoogle Scholar
  12. 12.
    Santoni, V., Kieffer, S., Desclaux, D., Masson, F., and Rabilloud, T. (2000b) Membrane proteomics: use of additive main effects with multiplicative interaction model to classify plasma membrane proteins according to their solubility and electrophoretic properties. Electrophoresis 21, 3329–3344.CrossRefGoogle Scholar
  13. 13.
    Brugi‘ere, S., Kowalski, S., Ferro, M., Seigneurin-Berny, D., Miras, S., Salvi, D., et al. (2004) The hydrophobic proteome of mitochondrial membranes from Arabidopsis cell suspensions. Phytochemistry 23, 1693–1707.CrossRefGoogle Scholar
  14. 14.
    Marmagne, A., Rouet, M. A., Ferro, M., Rolland, N., Alcon, C., Joyard, J., et al. (2004) Identification of new intrinsic proteins in Arabidopsis plasma membrane proteome. Mol. Cell. Proteomics 3, 675–691.Google Scholar
  15. 15.
    Adessi, C., Miége, C., Albrieux, C., and Rabilloud, T. (1997) Two-dimensional electrophoresis of membrane proteins: a current challenge for immobilized pH gradients. Electrophoresis 18, 127–135.CrossRefPubMedGoogle Scholar
  16. 16.
    Friso, G., Giacomelli, L., Ytterberg, A. J., Peltier, J. B., Rudella, A., Sun, Q., et al. (2004) In-depth analysis of the thylakoid membrane proteome of Arabidopsis thaliana chloroplasts: new proteins, new functions, and a plastid proteome database. Plant Cell 16, 478–499.CrossRefPubMedGoogle Scholar
  17. 17.
    Chua, N. H. (1980) Electrophoretic analysis of chloroplast proteins. Methods Enzymol. 69, 434–436.CrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Daniel Salvi
    • 1
    • 2
    • 3
  • Norbert Rolland
    • 1
    • 2
    • 3
  • Jacques Joyard
    • 1
    • 2
    • 3
  • Myriam Ferro
    • 4
    • 5
    • 6
  1. 1.Laboratoire de Physiologie Cellulaire VégétaleGrenoble
  2. 2.Université Joseph Fourier/CNRS UMR-5168/INRA/CEA-GrenobleGrenoble
  3. 3.iRTSVGrenoble
  4. 4.CEA, DSV, iRTSV, Laboratoire de l’Etude de la Dynamique des ProtéomesGrenoble
  5. 5.Université Joseph FourierGrenoble
  6. 6.INSERMGrenoble

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