The Isolation of Clathrin-Coated Vesicles and Purification of Their Protein Components

  • Antony P. Jackson
Part of the Methods in Molecular Biology book series (MIMB, volume 19)


Coated vesicles are implicated in a number of receptor-mediated transport events within eukaryotic cells. In particular, they are required for the export of newly synthesized lysosomal enzymes from the trans-Golgi network, and at the plasma membrane they are responsible for receptor-mediated endocytosis of selected ligands (1). Coated vesicles are the best characterized of all transport organelles, owing in large measure to the ease with which they can be purified.


Fraction Collector Isolation Buffer Coated Vesicle Dounce Homogenizer Sodium Thiocyanate 
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  1. 1.
    Brodsky, F. M. (1988) Living with clathrin: its role in intracellular membrane traffic. Science 242, 1396–1402.PubMedCrossRefGoogle Scholar
  2. 2.
    Zaremba, S. and Keen, J. H. (1983) Assembly polypeptides from coated vesicles mediate reassembly of unique clathrin coats. J. Cell Biol. 97, 1339–1347.PubMedCrossRefGoogle Scholar
  3. 3.
    Pearse, B. M. F. (1989) Characterization of coated-vesicle adaptors: their reassembly with clathrin and with recycling receptors. Meth. Cell Biol. 31,229–243.CrossRefGoogle Scholar
  4. 4.
    Pearse, B. M. F. and Robinson, M. S. (1984) Purification and properties of 100-kd proteins from coated vesicles and their reconstitution with clathrin. EMBOJ. 3, 1951–1957.Google Scholar
  5. 5.
    Manfredi, J. J. and Bazari, W. L. (1987) Purification and characterization of two distinct complexes of assembly polypeptides from calf brain coated vesicles that differ in their polypeptide composition and kinase activities. J. Biol. Chem. 262, 12,182–12,188.PubMedGoogle Scholar
  6. 6.
    Ahle, S., Mann, A., Eichelsbacher, U., and Ungewickell, E. (1988) Structural relationships between clathrin assembly proteins from the Golgi and the plasma membrane. EMBO J. 7, 919–929.PubMedGoogle Scholar
  7. 7.
    Robinson, M. S. (1989) Cloning of cDNAs encoding two related 100-kD coated vesicle proteins (α-adaptins). J. Cell Bio. 108, 833–842.CrossRefGoogle Scholar
  8. 8.
    Kirchhausen, T., Nathanson, K. L., Matsui, W., Vaisber, A., Chow, E. P., Burne, C., Keen J. H., and Davis, A. E. (1989) Structural and functional division into two domains of the large (100 to 115-kDa) chains of the clathrin-associated protein complex AP-2. Proc. Natl. Acad. Sci. USA 86, 2612–2616.PubMedCrossRefGoogle Scholar
  9. 9.
    Ponnambalam, S., Robinson, M. S., Jackson, A. P., Peiperl, L., and Parham, P. (1990) Conservation and diversity in families of coated vesicle adaptins. J. Biol. Chem. 265, 4814–4820.PubMedGoogle Scholar
  10. 10.
    Robinson, M. S. (1990) Cloning and expression of γ-adaptin, a component of clathrin-coated vesicles associated with the Golgi apparatus. J. Cell Biol. III, 2319–2326.CrossRefGoogle Scholar
  11. 11.
    Pearse, B. M. F. (1975) Coated vesicles from pig brain: Purification and biochemical characterization. J. Mol. Biol. 97, 93–98.PubMedCrossRefGoogle Scholar
  12. 12.
    Jackson, A. P., Seow, H-F., Holmes, N., Drickamer, K., and Parham, P. (1987) Clathrin light chains contain brain-specific insertion sequences and a region of homology with intermediate filaments. Nature 326, 154–159.PubMedCrossRefGoogle Scholar
  13. 13.
    Kirchhausen, T., Scarmato, P., Harrison, S. C., Monroe, J. J., Chow, E. P., Mattaliano, R. J., Ramachandran, K. L., Smart, J. E., Ahn, A. H., and Brosius, J. (1987) Clathrin light-chains LCA and LCB are similar, polymorphic, and share repeated heptad motifs. Science 236, 320–324.PubMedCrossRefGoogle Scholar
  14. 14.
    Jackson, A. P. and Parham, P. (1988) Structure of human clathrin light-chains: conservation of light-chain polymorphism in three mammalian species. J. Biol. Chem. 263, 16,688–16,695.PubMedGoogle Scholar
  15. 15.
    Ahle, S. and Ungewickell, E. (1986) Purification and properties of a new clathrin assembly protein. EMBO J. 5, 3143–3149.PubMedGoogle Scholar
  16. 16.
    Ahle, S. and Ungewickell, E. (1990) Auxilin, a newly identified clathrin-associated protein in coated vesicles from bovine brain. J. Cell Biol. III, 19–21.CrossRefGoogle Scholar
  17. 17.
    Kelly, W. G., Passaniti, A., Woods, J. W., Daiss, J. L., and Roth, T. F. (1983) Tubulin is a molecular component of coated vesicles. J. Cell Biol. 97, 1191–1199.PubMedCrossRefGoogle Scholar
  18. 18.
    Keen, J. H., Willingham, M. C., and Pastan, I. H. (1979) Clathrin-coated vesicles: isolation, dissociation and factor-dependent reassociation of clathrin baskets. Cell 16, 303–312.PubMedCrossRefGoogle Scholar
  19. 19.
    Pearse, B. M. F. (1982) Coated vesicles from human placenta carry ferritin, transferrin and immunoglobulin G. Proc. Natl. Acad. Sci. USA 79, 451–455.PubMedCrossRefGoogle Scholar
  20. 20.
    Campbell, C., Squicciarini, J., Shia, M., Pilch, P. F., and Fine, R. E. (1984) Identification of protein kinase as an intrinsic component of rat liver coated vesicle. Biochemistry 23, 4420–4426.PubMedCrossRefGoogle Scholar
  21. 21.
    Bar-Zvi, D. and Branton, D. (1986) Clathrin-coated vesicles contain two protein kinase activities. J. Biol. Chem. 261, 9614–9621.PubMedGoogle Scholar
  22. 22.
    Lisanti, M. P., Shapiro, L. S., Moskowitz, N., Hua, E. L., Puszkin, S., and Schook, W. (1982) Isolation and preliminary characterization of clathrin-associated proteins. Eur. J. Biochem. 125, 463–470.PubMedCrossRefGoogle Scholar
  23. 23.
    Brodsky, F. M., Holmes, N. J., and Parham, P. (1983) Tropomysin-like properties of clathrin light chains allow a rapid, high-yield purification. J. Cell Biol. 96, 911–914.PubMedCrossRefGoogle Scholar
  24. 24.
    Keen, J. H. (1987) Clathrin assembly proteins: affinity purification and a model for coat assembly. J. Cell Biol. 105, 1989–1998.PubMedCrossRefGoogle Scholar
  25. 25.
    Parham, P., Brodsky, F. M., and Drickamer, K. (1989) The occurrence of disulphide bonds in purified clathrin light chains. Biochem. J. 257, 775–781.PubMedGoogle Scholar
  26. 26.
    Ungewickell, E. (1983) Biochemical and immunological studies on clathrin light chains and their binding sites on clathrin triskelions. EMBO J. 2, 1401–1408.PubMedGoogle Scholar

Copyright information

© Humana Press Inc. 1993

Authors and Affiliations

  • Antony P. Jackson
    • 1
  1. 1.Department of BiochemistryUniversity of CambridgeUK

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