Purification Techniques of Subcellular Compartments for Analytical and Preparative Purposes

  • Laurence Aubry
  • Gérard Klein
Part of the Methods in Molecular Biology™ book series (MIMB, volume 346)


In the following protocols, broken cells are the starting material of all downstream purifications of functional organelles or intact subcellular membranes. The choice of the breakage method has direct and deep repercussions on the quality of subsequent steps. Breaking vegetative amoebae by shear stress with a steel ball cell cracker preserves the integrity of subcellular organelles and in particular that of lysosomes, the rupture of which is very deleterious to further purifications.

In this chapter, we propose purification schemes for plasma membrane, nuclei, mitochondria, and endocytic compartments. Plasma membranes are purified without any cell coating by partition between aqueous polymer phases. Nuclei and mitochondria are purified by differential centrifugations in adequate buffer conditions. Endosomes are magnetically isolated after feeding the cells with colloidal iron dextran and phagosomes by flotation on a sucrose gradient after feeding amoebae with latex beads.

As analytical approaches, we propose procedures to label the plasma membrane and the endo-lysosomal compartments by biotinylation and to separate early and late compartments on a Percoll gradient.

Key Words

Plasma membrane nuclei mitochondria endocytic compartment gradient purification Dictyostelium 


  1. 1.
    Balch, W. E. and Rothman, J. E. (1985) Characterization of protein transport between successive compartments of the Golgi apparatus: asymmetric properties of donor and acceptor activities in a cell-free system. Arch. Biochem. Biophys. 240, 413–425.PubMedCrossRefGoogle Scholar
  2. 2.
    Laurent, O., Bruckert, F., Adessi, C., and Satre, M. (1998) In vitro reconstituted Dictyostelium discoideum early endosome fusion is regulated by Rab7-but proceeds in the absence of ATP-Mg2+ from the bulk solution. J. Biol. Chem. 273, 793–739.PubMedCrossRefGoogle Scholar
  3. 3.
    Goodloe-Holland, C. M. and Luna, E. J. (1987) Purification and characterization of Dictyostelium discoideum plasma membranes. Methods Cell Biol. 28, 103–128.PubMedCrossRefGoogle Scholar
  4. 4.
    Brunette, D. M. and Till, J. E. (1971) A rapid method for the isolation of L-cell surface membranes using an aqueous two-phase polymer system. J. Memb. Biol. 5, 215–224.CrossRefGoogle Scholar
  5. 5.
    Nellen, W., Datta, S., Reymond, C., et al. (1987) Molecular biology in Dictyostelium: tools and applications. Methods Cell Biol. 28, 67–100.PubMedCrossRefGoogle Scholar
  6. 6.
    Charlesworth, M. C. and Parish, R. W. (1975) The isolation of nuclei and basic nucleoproteins from the cellular slime mold Dictyostelium discoideum. Eur. J. Biochem. 54, 307–316.PubMedCrossRefGoogle Scholar
  7. 7.
    Bof, M., Brandolin, G., Satre, M., and Klein, G. (1999) The mitochondrial adenine nucleotide translocator from Dictyostelium discoideum. Functional characterization and DNA sequencing. Eur. J. Biochem. 259, 795–800.PubMedCrossRefGoogle Scholar
  8. 8.
    Rodriguez-Paris, J. M., Nolta, K. V., and Steck, T. L. (1993) Characterization of lysosomes isolated from Dictyostelium discoideum by magnetic fractionation. J. Biol. Chem. 268, 9110–9116.PubMedGoogle Scholar
  9. 9.
    Adessi, C., Chapel, A., Vincon, M., Rabilloud, T., Klein, G., Satre, M., and Garin, J. (1995) Identification of major proteins associated with Dictyostelium discoideum endocytic vesicles. J. Cell Sci. 108, 3331–3337.PubMedGoogle Scholar
  10. 10.
    Aubry, L., Klein, G., Martiel, J. L., and Satre, M. (1993) Kinetics of endosomal pH evolution in Dictyostelium discoideum amoebae. Study by fluorescence spectroscopy. J. Cell Sci. 105, 861–866.PubMedGoogle Scholar
  11. 11.
    Brenot, F., Aubry, L., Martin, J. B., Satre, M., and Klein, G. (1992) Kinetics of endosomal acidification in Dictyostelium discoideum amoebae. 31P-NMR evidence for a very acidic early endosomal compartment. Biochimie 74, 883–895.PubMedCrossRefGoogle Scholar
  12. 12.
    Beinert, H. (1978) Micromethods for the quantitative determination of iron and copper in biological material. Meth. Enzymol. 54, 435–445.PubMedCrossRefGoogle Scholar
  13. 13.
    Desjardins, M., Huber, L. A., Parton, R. G., and Griffiths, G. (1994) Biogenesis of phagolysosomes proceeds through a sequential series of interactions with the endocytic apparatus. J. Cell Biol. 124, 677–688.PubMedCrossRefGoogle Scholar
  14. 14.
    Gotthardt, D., Warnatz, H. J., Henschel, O., Bruckert, F., Schleicher, M., and Soldati, T. (2002) High-resolution dissection of phagosome maturation reveals distinct membrane trafficking phases. Mol. Biol. Cell 13, 3508–3820.PubMedCrossRefGoogle Scholar
  15. 15.
    Chia, C. P. and Luna, E. J. (1989) Phagocytosis in Dictyostelium discoideum is inhibited by antibodies directed primarily against common carbohydrate epitopes of a major cell-surface plasma membrane glycoprotein. Exp. Cell Res. 181, 11–26.PubMedCrossRefGoogle Scholar
  16. 16.
    Aubry, L., Mattei, S., Blot, B., Sadoul, R., Satre, M., and Klein, G. (2002) Biochemical characterization of two analogues of the apoptosis-linked gene 2-protein in Dictyostelium discoideum and interaction with a physiological partner in mammals, murine Alix. J. Biol. Chem. 277, 21,947–21,954.PubMedCrossRefGoogle Scholar
  17. 17.
    Bof, M., Brenot, F., Gonzalez, C., Klein, G., Martin, J. B., and Satre, M. (1992) Dictyostelium discoideum mutants resistant to the toxic action of methylene diphosphonate are defective in endocytosis. J. Cell Sci. 101, 139–144.Google Scholar
  18. 18.
    Steck, T. L. and Lavasa, M. (1994) A general method for plasma membrane isolation by colloidal gold density shift. Anal. Biochem. 223, 47–50.PubMedCrossRefGoogle Scholar
  19. 19.
    Aubry, L., Klein, G., Martiel, J. L., and Satre, M. (1997) Fluid-phase endocytosis in the amoebae of the cellular slime mold Dictyostelium discoideum: mathematical modelling of kinetics and pH evolution. J. Theor. Biol. 184, 89–98.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2006

Authors and Affiliations

  • Laurence Aubry
    • 1
  • Gérard Klein
    • 1
  1. 1.Laboratoire de Biochimie et Biophysique des Systèmes Intégrés (UMR5092), Département Réponse et Dynamique Cellulaires, CNRS-CEA-UJFCEA-GrenobleGrenoble Cedex 09France

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