Toward a Biochemical Definition of the Endosomal Compartment

Studies Using Free Flow Electrophoresis
  • Sandra L. Schmid
Part of the Subcellular Biochemistry book series (SCBI, volume 19)


The eucaryotic cell is highly compartmentalized, containing many functionally and structurally distinct membrane-bound organelles. Subcellular fractionation is a powerful technique being applied to the purification of individual organelles so that they might be studied in biochemical detail. A complete biochemical definition of organelles of the vacuolar system involved in membrane transport along either the endocytic or exocytic pathways requires their definition in both static and dynamic terms. An organelle is defined in static terms by its own specific set of resident proteins, which give it its structural and functional identity. An organelle is defined in dynamic terms by its temporal location along an intracellular membrane traffic route based on the kinetics of protein transit into and through each organelle along either the endocytic or exocytic pathway. To completely define an organelle in static terms would require its purification to homogeneity and subsequent biochemical characterization. To adequately define an organelle in dynamic terms does not necessarily require that an organelle be purified to homogeneity but instead requires a rapid, high-yield procedure that clearly resolves the organelle from other organelles along a given pathway. This review discusses the novel method of free flow electrophoresis that we have applied to both the preparation of highly purified endosomal fractions for their biochemical characterization and to the analytical characterization of the kinetics of transit of endocytic tracers through the organelles of the endocytic pathway.


Early Endosome Late Endosome Endocytic Pathway Endosomal Compartment Dynamic Term 
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  1. Ajioka, R. S., and Kaplan, J., 1987, Characterization of endocytic compartments using the horse- radish peroxidase—diaminobenzidine density shift technique, J. Cell Biol. 104: 77–85.PubMedCrossRefGoogle Scholar
  2. 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
  3. Bartlett, S. F., and Smith, A. D., 1974, Adrenal chromattin granules: Isolation and disassembly, Meth. Enzym. 31: 379–389.PubMedCrossRefGoogle Scholar
  4. Beardmore, J., Howell, K. E., Miller, K., and Hopkins, C. R., 1987, Isolation of an endocytic compartment from A431 cells using a density modification procedure employing a receptor- specific monoclonal antibody complexed with colloidal gold, J. Cell Sci. 87: 495–506.PubMedGoogle Scholar
  5. Belcher, J. D., Hamilton, R. L., Brady, S. E., Homick, C. A., Jaeckle, S., Schneider, W. J., and Havel, R. J., 1987, Isolation and characterization of three endosomal fractions from the liver of estradiol-treated rats, Proc. Natl. Acad. Sci. USA, 84: 6785–6789.PubMedCrossRefGoogle Scholar
  6. Bocek, P., Gebauer, P., Dolnik, V., and Foret, F., 1985, Recent developments in isotachophoresis, J. Chrom. 334: 157–195.CrossRefGoogle Scholar
  7. Braell, W. A., 1987, Fusion between endocytic vesicles in a cell-free system, Proc. Natl. Acad. Sci. USA, 84: 1137–1141.PubMedCrossRefGoogle Scholar
  8. Cameron, R. S. and Castle, J. D., 1984, Isolation and compositional analysis of secretion granules and their membrane subfractionation from the rat parotid gland, J. Membr. Biol. 79: 127–144.PubMedCrossRefGoogle Scholar
  9. Carlson, S. S., Wagner, J. A., and Kelly, R. B., 1978, Purification of synaptic vesicles from elasmobranch electric organ and the use of biophysical criteria to demonstrate purity, Biochem. 17: 1188–1199.CrossRefGoogle Scholar
  10. Ciechanover, A., Schwartz, A. L., Dautry-Varsat, A., and Lodish, H. F., 1983, Kinetics of internalization and recycling of transferrin and the transferrin receptor in a human hepatoma cell line, J. Biol. Chem. 258: 9681–9689.PubMedGoogle Scholar
  11. Courtoy, P. J., Quintart, J., and Baudhuin, P., 1984, Shift of equilibrium density induced by 3,3’diaminobenzidine cytochemistry: a new procedure for the analysis and purification of peroxidase-containing organelles, J. Cell Biol. 98: 870–876.PubMedCrossRefGoogle Scholar
  12. Crawford, N., 1985, Use of free flow electrophoresis (PPE) in studies of surface and intracellular membranes and specific surface functional domains in blood platelets and leucocytes, in Cell Electrophoresis ( W. Schutt and H. Klinkmann, eds.) pp. 225–246, Walter deGuyter and Co., Berlin, New York.Google Scholar
  13. Daiss, J. L., and Roth, T. F., 1983, Isolation of coated vesicles: comparative studies, Meth. Enzym. 98: 337–349.PubMedCrossRefGoogle Scholar
  14. Davey, J., Hurtley, S. M., and Warren, G., 1985, Reconstitution of an endocytic fusion event in a cell-free system, Cell, 43: 643–652.PubMedCrossRefGoogle Scholar
  15. Diaz, R., Mayorga, L., and Stahl, P., 1988, In vitro fusion of endosomes following receptor-mediated endocytosis, J. Biol. Chem. 263: 6093–6100.Google Scholar
  16. Dickson, R. B., Beguinot, L., Hanover, J. A., Richert, N. D., Willingham, M. C., and Pastan, I., 1983, Isolation and characterization of a highly enriched preparation of receptosomes (endosomes) from a human cell line, Proc. Natl. Acad. Sci., USA, 80: 5335–5339.CrossRefGoogle Scholar
  17. Dunn, W. A., Hubbard, A. L., and Aronson, N. N., Jr., 1980, Low temperature selectively inhibits fusion between pinocytic vesicles and lysosomes during heterophagy of’251-asialofetuin by the perfused rat liver, J. Biol. Chem. 255: 5971–5978.PubMedGoogle Scholar
  18. Dunphy, W. G., and Rothman, J. E., 1985, Compartmental organization of the Golgi stack, Cell, 42: 13–21.PubMedCrossRefGoogle Scholar
  19. Evans, W. H., and Flint, N., 1985, Subfractionation of hepatic endosomes in Nycodenz gradients and by free flow electrophoresis, Biochem. J. 232: 25–32.PubMedGoogle Scholar
  20. Farquhar, M. G., 1985, Progress in unraveling pathways of Golgi traffic, Ann. Rev. Cell Biol. 1: 447–488.PubMedCrossRefGoogle Scholar
  21. Fleischer, S., and Kervina, M., 1974, Subcellular fractionation of rat liver. Meth. Enzym. 31: 6–40.PubMedCrossRefGoogle Scholar
  22. Fuchs, R., Male, P., and Mellman, I., 1989, Acidification and ion permeabilities of highly purified rat liver endosomes, J. Biol. Chem. 264: 2212–2220.PubMedGoogle Scholar
  23. Fuchs, R., Schmid, S., and Mellman, I., 1989, A possible role for Nat, K±-ATPase in regulating ATP-dependent endosome acidification, Proc. Natl. Acad. Sci. USA, 86: 539–543.PubMedCrossRefGoogle Scholar
  24. Geuze, H. J., Slot, J. W., and Strous, G. J. A. M., 1983, Intracellular site of asialoglycoprotein receptor-ligand uncoupling: double-label immunoelectron microscopy during receptor-mediated endocytosis, Cell, 32: 277–287.PubMedCrossRefGoogle Scholar
  25. Geuze, H. J., Slot, J. W., Strous, G. J. A. M., Peppard, J., von Figura, K., Hasilik, A., and Schwartz, A. L., 1984, Intracellular receptor sorting during endocytosis: comparative itnmunoelectron microscopy of multiple receptors in rat liver, Cell, 37: 195–204.PubMedCrossRefGoogle Scholar
  26. Geuze, H. J., Stoorvogel, W., Strous, G. J. A. M., Slot, J. W., Bleekemolen, J. E., and Mellman, L., 1988, Sorting of mannose 6-phosphate receptors and lysosomal membrane proteins in endocytic vesicles, J. Cell Biol. 107: 2491–2501.PubMedCrossRefGoogle Scholar
  27. Goldstein, J. L., Anderson, R. G. W., and Brown, M. S., 1979, Coated pits, coated vesicles and receptor-mediated endocytosis. Nature. 279: 679–685.PubMedCrossRefGoogle Scholar
  28. Griffiths, G., Hoflack, B., Simons, K., Mellman, I., and Kornfeld, S., 1988, The mannose 6-phosphate receptor and the biogenesis of lysosomes, Cell, 52: 329–341.PubMedCrossRefGoogle Scholar
  29. Gruenberg, J. E., and Howell, K. E., 1985, Immun-isolation of vesicles using antigenic sites either located on the cytoplasmic or the exoplasmic domain of an implanted viral protein. A quantitative analysis. Eur. J. Cell Biol. 38: 312–321.PubMedGoogle Scholar
  30. Gruenberg, J. E., and Howell, K. E., 1986, Reconstitution of vesicle fusions occurring in endocytosis with a cell-free system, EMBO J. 5: 3091–3101.PubMedGoogle Scholar
  31. Gruenberg, J. E., and Howell, K. E., 1989, Membrane traffic in endocytosis: insights from cell-free assays, Ann. Rev. Cell Biol. 5: 453–482.PubMedCrossRefGoogle Scholar
  32. Greenberg, J. E., Griffiths, G., and Howell, K. E., 1989, Characterization of the early endosome and putative endocytic camer vesicles in vivo and with an assay of vesicle fusion in vitro, J. Cell Biol., 108: 1301–1316.CrossRefGoogle Scholar
  33. Guerra, F. C., 1974, Rapid isolation techniques for mitochondria: Technique for rat liver mitochondria, Meth. Enzym. 31: 299–305.PubMedCrossRefGoogle Scholar
  34. Hannig, K., and Heidrich, H.-G., 1974, The use of continuous preparative free flow electrophoresis for dissociating cell fractions and isolation of membranous components, Meth. Enzym. 31: 746761.Google Scholar
  35. Hannig, K., and Heidrich, H.-G., 1977, Continuous free flow electrophoresis and its application in biology, in Cell Separation Methods Part IV. Electrophoretic Methods ( H. Bloemendal, ed.), pp. 93–116, Elsevier/North Holland, Biomedical Press, Amsterdam.Google Scholar
  36. Harms, E., Kern, H., and Schneider, J. A., 1980, Human lysosomes can be purified from diploid skin fibroblasts by free flow electrophoresis, Proc. Natl. Acad. Sci. USA, 77: 6139–6143.PubMedCrossRefGoogle Scholar
  37. Helenius, A., Mellman, I., Wall, D., and Hubbard, A., 1983, Endosomes, Trends in Biochem. Sci. 8: 245–250.CrossRefGoogle Scholar
  38. Heydt, A., Wagner, H., and Paul, H. L., 1988, Concentration and purification of plant pathogenic viruses by field step electrophoresis, J. Virol. Meth., 9: 13–22.CrossRefGoogle Scholar
  39. Holloway, C. J., and Battersby, R. V., 1984, Preparative isotachophoresis, Meth. Enzym. 104: 28 1301.Google Scholar
  40. Hubbard, A. L., Wall, D. A., and Mah, A., 1983, Isolation of rat hepatocyte plasma membranes. I. Presence of the three major domains, J. Cell Biol. 96: 217–229.PubMedCrossRefGoogle Scholar
  41. Jamieson, J. D., and Palade, G. E., 1967a, Intracellular transport of secretory proteins in the pancreatic exocrine cell. I. Role of the peripheral elements of the Golgi complex. J. Cell Biol. 34: 577–596.PubMedCrossRefGoogle Scholar
  42. Jamieson, J. D., and Palade, G. E., 1967b, Intracellular transport of secretory proteins in the pancreatic exocrine cell. II. Transport to condensing vacuoles and zymogen granules, J. Cell Biol. 34: 597–616.PubMedCrossRefGoogle Scholar
  43. Klausner, R. D., van Renswoude, J., Ashwell, G., Kempf, C., Schechter, A. N., Dean, A., and Bridges, K. R., 1983, Receptor-mediated endocytosis of transferrin in K562 cells., J. Biol Chem. 258: 4715–4724.PubMedGoogle Scholar
  44. Kopwillem, A., Merriman, W. G., Cuddeback, R. M., Smolka, A. J. K., and Bier, M., 1976, Serum protein fractionation by isotachophoresis using amino acid spacers, J. Chrom. 118: 35–46.Google Scholar
  45. Kornfeld, R., and Kornfeld, S., 1985, Assembly of asparagine-linked oligosaccharides, Ann. Rev. Biochem. 54: 631–664.PubMedCrossRefGoogle Scholar
  46. Kornfeld, S., and Mellman, I., 1989, The biogenesis of lysosomes, Ann. Rev. Cell Biol. 5:483–525. Lewis, V., Green, S. A., Marsh, M., Vihko, P., Helenius, A., and Mellman, I., 1985, Glycoproteins of the lysosomal membrane, J. Cell Biol. 100: 1839–1847.Google Scholar
  47. Lippincott-Schwartz, J., and Fambrough, D. M., 1987, Cycling of the integral membrane glycoprotein, LEP100, between plasma membrane and lysosomes: kinetic and morphological analysis, Cell 49: 669–677.PubMedCrossRefGoogle Scholar
  48. Louvard, D., Reggio, H., and Warren, G., 1982, Antibodies to the Golgi complex and the rough endoplasmic reticulum, J. Cell Biol. 92: 92–107.PubMedCrossRefGoogle Scholar
  49. Marsh, M., Schmid, S. L., Kern, H., Harms, E., Male, P., Mellman, I., and Helenius, A., 1987, Rapid analytical and preparative isolation of functional endosomes by free flow electrophoresis, J. Cell Biol. 104: 875–886.PubMedCrossRefGoogle Scholar
  50. Matteoni, R., and Kreis, T. E., 1987, Translocation and clustering of endosomes and lysosomes depends on microtubules, J. Cell Biol., 105: 1253–1265.PubMedCrossRefGoogle Scholar
  51. Mayorga, L. S., Diaz, R., and Stahl, P. D., 1988, Plasma membrane-derived vesicles containing receptor-ligand complexes are fusogenic with early endosomes in a cell-free system, J. Biol. Chem. 263: 17213–17216.PubMedGoogle Scholar
  52. Mueller, S. C., and Hubbard, A. L., 1986, Receptor-mediated endocytosis of asialoglycoproteins by rat hepatocytes: receptor-positive and receptor-negative endosomes, J. Cell Biol. 102: 932–942.PubMedCrossRefGoogle Scholar
  53. Mullock, B. M., Branch, W. J., van Schaid, M., Gilbert, L. K., and Luzio, J. P., 1989, Reconstitu-tion of an endosome-lysosome interaction in a cell-free system, J. Cell Biol. 108: 2093–2099.PubMedCrossRefGoogle Scholar
  54. Pearse, B. M. F., 1983, Isolation of coated vesicles, Meth. Enzym. 98: 320–326.PubMedCrossRefGoogle Scholar
  55. Quintart, J., Courtoy, P. J., and Baudhuin, P., 1984, Receptor-mediated endocytosis in rat liver: purification and enzymic characterization of low density organelles involved in uptake of galactose-exposing proteins, J. Cell Biol. 98: 877–884.PubMedCrossRefGoogle Scholar
  56. Sato, S. B., Sako, Y., Yamashina, S., and Ohnishi, S.-I., 1986, A novel method of isolating specific endocytic vesicles using very fine ferrite particles coated with biological ligands and the high-gradient magnetic separation technique, J. Biochem. 100: 1481–1492.PubMedGoogle Scholar
  57. Schmid, S. L., and Mellman, I., 1987, Isolation of functionally distinct endosome subpopulations by free flow electrophoresis, in Cell Free Analysis of Membrane Traffic ( D. J. Morre, K. E. Howell, and G. M. W. Cook, eds.), pp. 35–49, Alan R. Liss, New York.Google Scholar
  58. Schmid, S. L., Fuchs, R., Male, P., and Mellman, I., 1988, Two distinct subpopulations of endosomes involved in membrane recycling and transport to lysosomes, Cell, 52: 73–83.PubMedCrossRefGoogle Scholar
  59. Schmid, S. L., Fuchs, R., Kielian, M., Helenius, A., and Mellman, I., 1989, Acidification of endosome subpopulations in wild-type Chinese hamster ovary cells and temperature-sensitive acidification-defective mutants, J. Cell Biol. 108: 1291–1300.PubMedCrossRefGoogle Scholar
  60. Steinman, R. M., Brodie, S. E., and Cohn, Z. A., 1976, Membrane flow during pinocytosis. A stereological analysis. J. Cell Bio. 68: 665–687.CrossRefGoogle Scholar
  61. Steinman, R. M., Mellman, I. S., Muller, W. A., and Cohn, Z. A., 1983, Endocytosis and the recycling of plasma membrane, J. Cell Biol. 96: 1–27.PubMedCrossRefGoogle Scholar
  62. Stoorvogel, W., Geuze, H. J., and Strous, G. J. A. M., 1987, Sorting of endocytosed transferrin and asialoglycoprotein occurs immediately after internalization in HepG2 cells, J. Cell Biol., 104: 1261–1268.PubMedCrossRefGoogle Scholar
  63. Storrie, B., Pool, R. R., Sachdeva, M., Maurey, K. M., and Oliver, C., 1984, Evidence for both prelysosomal and lysosomal intermediates in endocytic pathways, J. Cell Biol. 98: 108–115.PubMedCrossRefGoogle Scholar
  64. Tata, J. R., 1974, Isolation of nuclei from liver and other tissues, Meth. Enzym. 31: 253–262.PubMedCrossRefGoogle Scholar
  65. Townsend, R. R., Wall, D. A., Hubbard, A. L., and Lee, Y. C., 1984, Rapid release of galactose-terminated ligands after endocytosis by hepatic parenchymal cells: Evidence for a role of carbohydrate structure in the release of internalized ligand from receptor, Proc. Natl. Acad. Sci. USA 81: 466–470.PubMedCrossRefGoogle Scholar
  66. Ukkonen, P., Lewis, V., Marsh, M., Helenius, A., and Mellman, I., 1986, Transport of macrophage Fc Receptors and Fc receptor-bound ligands to lysosomes, J. Exp. Med. 163: 952–971.PubMedCrossRefGoogle Scholar
  67. Woodman, P. G., and Warren, G., 1988, Fusion between vesicles from the pathway of receptor-mediated endocytosis in a cell-free system, Eur. J. Biochem. 173: 101–108.PubMedCrossRefGoogle Scholar
  68. Yamashiro, D. J., Tycko, B., Fluss, S. R., and Maxfield, F. R., 1984, Segregation of transferrin to a mildly acidic (pH 6.5) para-Golgi compartment in the recycling pathway, Cell, 37: 789–800.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 1993

Authors and Affiliations

  • Sandra L. Schmid
    • 1
  1. 1.Departments of Cell and Molecular BiologyThe Scripps Research InstituteLa JollaUSA

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