Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1987 Apr 1;104(4):875–886. doi: 10.1083/jcb.104.4.875

Rapid analytical and preparative isolation of functional endosomes by free flow electrophoresis

PMCID: PMC2114435  PMID: 3031085

Abstract

Endosomes are prelysosomal organelles that serve as an intracellular site for the sorting, distribution, and processing of receptors, ligands, fluid phase components, and membrane proteins internalized by endocytosis. Whereas the overall functions of endosomes are increasingly understood, little is known about endosome structure, composition, or biogenesis. In this paper, we describe a rapid procedure that permits analytical and preparative isolation of endosomes from a variety of tissue culture cells. The procedure relies on a combination of density gradient centrifugation and free flow electrophoresis. It yields a fraction of highly purified, functionally intact organelles. As markers for endosomes in Chinese hamster ovary cells, we used endocytosed horseradish peroxidase, FITC-conjugated dextran, and [35S]methionine-labeled Semliki Forest virus. Total postnuclear supernatants, crude microsomal pellets, or partially purified Golgi fractions were subjected to free flow electrophoresis. Endosomes and lysosomes migrated together as a single anodally deflected peak separated from most other organelles (plasma membrane, mitochondria, endoplasmic reticulum, and Golgi). The endosomes and lysosomes were then resolved by centrifugation in Percoll density gradients. Endosomes prepared in this way were enriched up to 70-fold relative to the initial homogenate and were still capable of ATP- dependent acidification. By electron microscopy, the isolated organelles were found to consist of electron lucent vacuoles and tubules, many of which could be shown to contain an endocytic tracer (e.g., horseradish peroxidase). SDS PAGE analysis of integral and peripheral membrane proteins (separated from each other by condensation in Triton X-114) revealed a unique and restricted subset of proteins when compared with lysosomes, the unshifted free flow electrophoresis peak, and total cell protein. Altogether, the purification procedure takes 5-6 h and yields amounts of endosomes (150-200 micrograms protein) sufficient for biochemical, immunological, and functional analysis.

Full Text

The Full Text of this article is available as a PDF (2.7 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abrahamson D. R., Rodewald R. Evidence for the sorting of endocytic vesicle contents during the receptor-mediated transport of IgG across the newborn rat intestine. J Cell Biol. 1981 Oct;91(1):270–280. doi: 10.1083/jcb.91.1.270. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Balch W. E., Dunphy W. G., Braell W. A., Rothman J. E. Reconstitution of the transport of protein between successive compartments of the Golgi measured by the coupled incorporation of N-acetylglucosamine. Cell. 1984 Dec;39(2 Pt 1):405–416. doi: 10.1016/0092-8674(84)90019-9. [DOI] [PubMed] [Google Scholar]
  3. Balch W. E., Rothman J. E. 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. 1985 Jul;240(1):413–425. doi: 10.1016/0003-9861(85)90046-3. [DOI] [PubMed] [Google Scholar]
  4. Beguinot L., Lyall R. M., Willingham M. C., Pastan I. Down-regulation of the epidermal growth factor receptor in KB cells is due to receptor internalization and subsequent degradation in lysosomes. Proc Natl Acad Sci U S A. 1984 Apr;81(8):2384–2388. doi: 10.1073/pnas.81.8.2384. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bergeron J. J., Cruz J., Khan M. N., Posner B. I. Uptake of insulin and other ligands into receptor-rich endocytic components of target cells: the endosomal apparatus. Annu Rev Physiol. 1985;47:383–403. doi: 10.1146/annurev.ph.47.030185.002123. [DOI] [PubMed] [Google Scholar]
  6. Bordier C. Phase separation of integral membrane proteins in Triton X-114 solution. J Biol Chem. 1981 Feb 25;256(4):1604–1607. [PubMed] [Google Scholar]
  7. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  8. Brew K., Shaper J. H., Olsen K. W., Trayer I. P., Hill R. L. Cross-linking of the components of lactose synthetase with dimethylpimelimidate. J Biol Chem. 1975 Feb 25;250(4):1434–1444. [PubMed] [Google Scholar]
  9. Brown M. S., Anderson R. G., Goldstein J. L. Recycling receptors: the round-trip itinerary of migrant membrane proteins. Cell. 1983 Mar;32(3):663–667. doi: 10.1016/0092-8674(83)90052-1. [DOI] [PubMed] [Google Scholar]
  10. Chamberlain J. P. Fluorographic detection of radioactivity in polyacrylamide gels with the water-soluble fluor, sodium salicylate. Anal Biochem. 1979 Sep 15;98(1):132–135. doi: 10.1016/0003-2697(79)90716-4. [DOI] [PubMed] [Google Scholar]
  11. Courtoy P. J., Quintart J., Baudhuin P. 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. 1984 Mar;98(3):870–876. doi: 10.1083/jcb.98.3.870. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Debanne M. T., Evans W. H., Flint N., Regoeczi E. Receptor-rich intracellular membrane vesicles transporting asialotransferrin and insulin in liver. Nature. 1982 Jul 22;298(5872):398–400. doi: 10.1038/298398a0. [DOI] [PubMed] [Google Scholar]
  13. Dickson R. B., Beguinot L., Hanover J. A., Richert N. D., Willingham M. C., Pastan I. Isolation and characterization of a highly enriched preparation of receptosomes (endosomes) from a human cell line. Proc Natl Acad Sci U S A. 1983 Sep;80(17):5335–5339. doi: 10.1073/pnas.80.17.5335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Diment S., Stahl P. Macrophage endosomes contain proteases which degrade endocytosed protein ligands. J Biol Chem. 1985 Dec 5;260(28):15311–15317. [PubMed] [Google Scholar]
  15. Draper R. K., Simon M. I. The entry of diphtheria toxin into the mammalian cell cytoplasm: evidence for lysosomal involvement. J Cell Biol. 1980 Dec;87(3 Pt 1):849–854. doi: 10.1083/jcb.87.3.849. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Forbush B., 3rd Assay of Na,K-ATPase in plasma membrane preparations: increasing the permeability of membrane vesicles using sodium dodecyl sulfate buffered with bovine serum albumin. Anal Biochem. 1983 Jan;128(1):159–163. doi: 10.1016/0003-2697(83)90356-1. [DOI] [PubMed] [Google Scholar]
  17. Galloway C. J., Dean G. E., Marsh M., Rudnick G., Mellman I. Acidification of macrophage and fibroblast endocytic vesicles in vitro. Proc Natl Acad Sci U S A. 1983 Jun;80(11):3334–3338. doi: 10.1073/pnas.80.11.3334. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Geuze H. J., Slot J. W., Strous G. J., Peppard J., von Figura K., Hasilik A., Schwartz A. L. Intracellular receptor sorting during endocytosis: comparative immunoelectron microscopy of multiple receptors in rat liver. Cell. 1984 May;37(1):195–204. doi: 10.1016/0092-8674(84)90315-5. [DOI] [PubMed] [Google Scholar]
  19. Green J., Griffiths G., Louvard D., Quinn P., Warren G. Passage of viral membrane proteins through the Golgi complex. J Mol Biol. 1981 Nov 15;152(4):663–698. doi: 10.1016/0022-2836(81)90122-4. [DOI] [PubMed] [Google Scholar]
  20. Griffiths G., Pfeiffer S., Simons K., Matlin K. Exit of newly synthesized membrane proteins from the trans cisterna of the Golgi complex to the plasma membrane. J Cell Biol. 1985 Sep;101(3):949–964. doi: 10.1083/jcb.101.3.949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Griffiths G., Simons K. The trans Golgi network: sorting at the exit site of the Golgi complex. Science. 1986 Oct 24;234(4775):438–443. doi: 10.1126/science.2945253. [DOI] [PubMed] [Google Scholar]
  22. Harford J., Bridges K., Ashwell G., Klausner R. D. Intracellular dissociation of receptor-bound asialoglycoproteins in cultured hepatocytes. A pH-mediated nonlysosomal event. J Biol Chem. 1983 Mar 10;258(5):3191–3197. [PubMed] [Google Scholar]
  23. Harms E., Kartenbeck J., Darai G., Schneider J. Purification and characterization of human lysosomes from EB-virus transformed lymphoblasts. Exp Cell Res. 1981 Feb;131(2):251–266. doi: 10.1016/0014-4827(81)90230-5. [DOI] [PubMed] [Google Scholar]
  24. Harms E., Kern H., Schneider J. A. Human lysosomes can be purified from diploid skin fibroblasts by free-flow electrophoresis. Proc Natl Acad Sci U S A. 1980 Oct;77(10):6139–6143. doi: 10.1073/pnas.77.10.6139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Helenius A., Kartenbeck J., Simons K., Fries E. On the entry of Semliki forest virus into BHK-21 cells. J Cell Biol. 1980 Feb;84(2):404–420. doi: 10.1083/jcb.84.2.404. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Hopkins C. R. Intracellular routing of transferrin and transferrin receptors in epidermoid carcinoma A431 cells. Cell. 1983 Nov;35(1):321–330. doi: 10.1016/0092-8674(83)90235-0. [DOI] [PubMed] [Google Scholar]
  27. Hornick C. A., Hamilton R. L., Spaziani E., Enders G. H., Havel R. J. Isolation and characterization of multivesicular bodies from rat hepatocytes: an organelle distinct from secretory vesicles of the Golgi apparatus. J Cell Biol. 1985 May;100(5):1558–1569. doi: 10.1083/jcb.100.5.1558. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Hubbard A. L., Cohn Z. A. Externally disposed plasma membrane proteins. I. Enzymatic iodination of mouse L cells. J Cell Biol. 1975 Feb;64(2):438–460. doi: 10.1083/jcb.64.2.438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lewis V., Green S. A., Marsh M., Vihko P., Helenius A., Mellman I. Glycoproteins of the lysosomal membrane. J Cell Biol. 1985 Jun;100(6):1839–1847. doi: 10.1083/jcb.100.6.1839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Marsh M., Bolzau E., Helenius A. Penetration of Semliki Forest virus from acidic prelysosomal vacuoles. Cell. 1983 Mar;32(3):931–940. doi: 10.1016/0092-8674(83)90078-8. [DOI] [PubMed] [Google Scholar]
  31. Marsh M., Griffiths G., Dean G. E., Mellman I., Helenius A. Three-dimensional structure of endosomes in BHK-21 cells. Proc Natl Acad Sci U S A. 1986 May;83(9):2899–2903. doi: 10.1073/pnas.83.9.2899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Marsh M., Helenius A. Adsorptive endocytosis of Semliki Forest virus. J Mol Biol. 1980 Sep 25;142(3):439–454. doi: 10.1016/0022-2836(80)90281-8. [DOI] [PubMed] [Google Scholar]
  33. Marsh M., Wellsteed J., Kern H., Harms E., Helenius A. Monensin inhibits Semliki Forest virus penetration into culture cells. Proc Natl Acad Sci U S A. 1982 Sep;79(17):5297–5301. doi: 10.1073/pnas.79.17.5297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Mellman I. S., Steinman R. M., Unkeless J. C., Cohn Z. A. Selective iodination and polypeptide composition of pinocytic vesicles. J Cell Biol. 1980 Sep;86(3):712–722. doi: 10.1083/jcb.86.3.712. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Mellman I., Fuchs R., Helenius A. Acidification of the endocytic and exocytic pathways. Annu Rev Biochem. 1986;55:663–700. doi: 10.1146/annurev.bi.55.070186.003311. [DOI] [PubMed] [Google Scholar]
  36. Mellman I., Plutner H. Internalization and degradation of macrophage Fc receptors bound to polyvalent immune complexes. J Cell Biol. 1984 Apr;98(4):1170–1177. doi: 10.1083/jcb.98.4.1170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Mellman I., Plutner H., Ukkonen P. Internalization and rapid recycling of macrophage Fc receptors tagged with monovalent antireceptor antibody: possible role of a prelysosomal compartment. J Cell Biol. 1984 Apr;98(4):1163–1169. doi: 10.1083/jcb.98.4.1163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Merion M., Schlesinger P., Brooks R. M., Moehring J. M., Moehring T. J., Sly W. S. Defective acidification of endosomes in Chinese hamster ovary cell mutants "cross-resistant" to toxins and viruses. Proc Natl Acad Sci U S A. 1983 Sep;80(17):5315–5319. doi: 10.1073/pnas.80.17.5315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Merion M., Sly W. S. The role of intermediate vesicles in the adsorptive endocytosis and transport of ligand to lysosomes by human fibroblasts. J Cell Biol. 1983 Mar;96(3):644–650. doi: 10.1083/jcb.96.3.644. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Mueller S. C., Hubbard A. L. Receptor-mediated endocytosis of asialoglycoproteins by rat hepatocytes: receptor-positive and receptor-negative endosomes. J Cell Biol. 1986 Mar;102(3):932–942. doi: 10.1083/jcb.102.3.932. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Murphy R. F., Powers S., Cantor C. R. Endosome pH measured in single cells by dual fluorescence flow cytometry: rapid acidification of insulin to pH 6. J Cell Biol. 1984 May;98(5):1757–1762. doi: 10.1083/jcb.98.5.1757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Neville D. M., Jr, Glossmann H. Molecular weight determination of membrane protein and glycoprotein subunits by discontinuous gel electrophoresis in dodecyl sulfate. Methods Enzymol. 1974;32:92–102. doi: 10.1016/0076-6879(74)32012-5. [DOI] [PubMed] [Google Scholar]
  43. Pool R. R., Jr, Maurey K. M., Storrie B. Characterization of pinocytic vesicles from CHO cells: resolution of pinosomes from lysosomes by analytical centrifugation. Cell Biol Int Rep. 1983 May;7(5):361–367. doi: 10.1016/0309-1651(83)90076-0. [DOI] [PubMed] [Google Scholar]
  44. Posner B. I., Patel B., Verma A. K., Bergeron J. J. Uptake of insulin by plasmalemma and Golgi subcellular fractions of rat liver. J Biol Chem. 1980 Jan 25;255(2):735–741. [PubMed] [Google Scholar]
  45. Robbins A. R., Peng S. S., Marshall J. L. Mutant Chinese hamster ovary cells pleiotropically defective in receptor-mediated endocytosis. J Cell Biol. 1983 Apr;96(4):1064–1071. doi: 10.1083/jcb.96.4.1064. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Roth J., Taatjes D. J., Weinstein J., Paulson J. C., Greenwell P., Watkins W. M. Differential subcompartmentation of terminal glycosylation in the Golgi apparatus of intestinal absorptive and goblet cells. J Biol Chem. 1986 Oct 25;261(30):14307–14312. [PubMed] [Google Scholar]
  47. Sims N. R., Carnegie P. R. Use of fluorescamine for the estimation of protein in the presence of detergent Triton X-100. Anal Biochem. 1975 May 12;65(1-2):578–580. doi: 10.1016/0003-2697(75)90550-3. [DOI] [PubMed] [Google Scholar]
  48. Snider M. D., Rogers O. C. Intracellular movement of cell surface receptors after endocytosis: resialylation of asialo-transferrin receptor in human erythroleukemia cells. J Cell Biol. 1985 Mar;100(3):826–834. doi: 10.1083/jcb.100.3.826. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Steinman R. M., Brodie S. E., Cohn Z. A. Membrane flow during pinocytosis. A stereologic analysis. J Cell Biol. 1976 Mar;68(3):665–687. doi: 10.1083/jcb.68.3.665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Steinman R. M., Mellman I. S., Muller W. A., Cohn Z. A. Endocytosis and the recycling of plasma membrane. J Cell Biol. 1983 Jan;96(1):1–27. doi: 10.1083/jcb.96.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Ukkonen P., Lewis V., Marsh M., Helenius A., Mellman I. Transport of macrophage Fc receptors and Fc receptor-bound ligands to lysosomes. J Exp Med. 1986 Apr 1;163(4):952–971. doi: 10.1084/jem.163.4.952. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Wall D. A., Hubbard A. L. Receptor-mediated endocytosis of asialoglycoproteins by rat liver hepatocytes: biochemical characterization of the endosomal compartments. J Cell Biol. 1985 Dec;101(6):2104–2112. doi: 10.1083/jcb.101.6.2104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Yamashiro D. J., Tycko B., Fluss S. R., Maxfield F. R. Segregation of transferrin to a mildly acidic (pH 6.5) para-Golgi compartment in the recycling pathway. Cell. 1984 Jul;37(3):789–800. doi: 10.1016/0092-8674(84)90414-8. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES