Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1987 May 1;104(5):1261–1268. doi: 10.1083/jcb.104.5.1261

Sorting of endocytosed transferrin and asialoglycoprotein occurs immediately after internalization in HepG2 cells

PMCID: PMC2114480  PMID: 3032986

Abstract

After receptor-mediated uptake, asialoglycoproteins are routed to lysosomes, while transferrin is returned to the medium as apotransferrin. This sorting process was analyzed using 3,3'- diaminobenzidine (DAB) cytochemistry, followed by Percoll density gradient cell fractionation. A conjugate of asialoorosomucoid (ASOR) and horseradish peroxidase (HRP) was used as a ligand for the asialoglycoprotein receptor. Cells were incubated at 0 degree C in the presence of both 131I-transferrin and 125I-ASOR/HRP. Endocytosis of prebound 125I-ASOR/HRP and 131I-transferrin was monitored by cell fractionation on Percoll density gradients. Incubation of the cell homogenate in the presence of DAB and H2O2 before cell fractionation gave rise to a density shift of 125I-ASOR/HRP-containing vesicles due to HRP-catalyzed DAB polymerization. An identical change in density for 125I-transferrin and 125I-ASOR/HRP, induced by DAB cytochemistry, is taken as evidence for the concomitant presence of both ligands in the same compartment. At 37 degrees C, sorting of the two ligands occurred with a half-time of approximately 2 min, and was nearly completed within 10 min. The 125I-ASOR/HRP-induced shift of 131I-transferrin was completely dependent on the receptor-mediated uptake of 125I-ASOR/HRP in the same compartment. In the presence of a weak base (0.3 mM primaquine), the recycling of transferrin receptors was blocked. The cell surface transferrin receptor population was decreased within 6 min to 15% of its original size. DAB cytochemistry showed that sorting between endocytosed 131I-transferrin and 125I-ASOR/HRP was also blocked in the presence of primaquine. These results indicate that transferrin and asialoglycoprotein are taken up via the same compartments and that segregation of the transferrin-receptor complex and asialoglycoprotein occurs very efficiently soon after uptake.

Full Text

The Full Text of this article is available as a PDF (896.1 KB).

Selected References

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

  1. Berg T., Tolleshaug H. The effects of ammonium ions and chloroquine on uptake and degradation of 125I-labeled asialo-fetuin in isolated rat hepatocytes. Biochem Pharmacol. 1980 Mar 15;29(6):917–925. doi: 10.1016/0006-2952(80)90222-1. [DOI] [PubMed] [Google Scholar]
  2. Breitfeld P. P., Simmons C. F., Jr, Strous G. J., Geuze H. J., Schwartz A. L. Cell biology of the asialoglycoprotein receptor system: a model of receptor-mediated endocytosis. Int Rev Cytol. 1985;97:47–95. doi: 10.1016/s0074-7696(08)62348-7. [DOI] [PubMed] [Google Scholar]
  3. Bridges K., Harford J., Ashwell G., Klausner R. D. Fate of receptor and ligand during endocytosis of asialoglycoproteins by isolated hepatocytes. Proc Natl Acad Sci U S A. 1982 Jan;79(2):350–354. doi: 10.1073/pnas.79.2.350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Carpenter G., Cohen S. 125I-labeled human epidermal growth factor. Binding, internalization, and degradation in human fibroblasts. J Cell Biol. 1976 Oct;71(1):159–171. doi: 10.1083/jcb.71.1.159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Ciechanover A., Schwartz A. L., Dautry-Varsat A., Lodish H. F. Kinetics of internalization and recycling of transferrin and the transferrin receptor in a human hepatoma cell line. Effect of lysosomotropic agents. J Biol Chem. 1983 Aug 25;258(16):9681–9689. [PubMed] [Google Scholar]
  6. Ciechanover A., Schwartz A. L., Lodish H. F. The asialoglycoprotein receptor internalizes and recycles independently of the transferrin and insulin receptors. Cell. 1983 Jan;32(1):267–275. doi: 10.1016/0092-8674(83)90517-2. [DOI] [PubMed] [Google Scholar]
  7. Dautry-Varsat A., Ciechanover A., Lodish H. F. pH and the recycling of transferrin during receptor-mediated endocytosis. Proc Natl Acad Sci U S A. 1983 Apr;80(8):2258–2262. doi: 10.1073/pnas.80.8.2258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dean R. T., Jessup W., Roberts C. R. Effects of exogenous amines on mammalian cells, with particular reference to membrane flow. Biochem J. 1984 Jan 1;217(1):27–40. doi: 10.1042/bj2170027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Geuze H. J., Slot J. W., Strous G. J., Lodish H. F., Schwartz A. L. Intracellular site of asialoglycoprotein receptor-ligand uncoupling: double-label immunoelectron microscopy during receptor-mediated endocytosis. Cell. 1983 Jan;32(1):277–287. doi: 10.1016/0092-8674(83)90518-4. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Geuze H. J., Slot J. W., Strous G. J., Schwartz A. L. The pathway of the asialoglycoprotein-ligand during receptor-mediated endocytosis: a morphological study with colloidal gold/ligand in the human hepatoma cell line, Hep G2. Eur J Cell Biol. 1983 Nov;32(1):38–44. [PubMed] [Google Scholar]
  12. Goldstein J. L., Brown M. S., Anderson R. G., Russell D. W., Schneider W. J. Receptor-mediated endocytosis: concepts emerging from the LDL receptor system. Annu Rev Cell Biol. 1985;1:1–39. doi: 10.1146/annurev.cb.01.110185.000245. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Klausner R. D., Ashwell G., van Renswoude J., Harford J. B., Bridges K. R. Binding of apotransferrin to K562 cells: explanation of the transferrin cycle. Proc Natl Acad Sci U S A. 1983 Apr;80(8):2263–2266. doi: 10.1073/pnas.80.8.2263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Knowles B. B., Howe C. C., Aden D. P. Human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitis B surface antigen. Science. 1980 Jul 25;209(4455):497–499. doi: 10.1126/science.6248960. [DOI] [PubMed] [Google Scholar]
  16. Limet J. N., Schneider Y. J., Vaerman J. P., Trouet A. Binding, uptake and intracellular processing of polymeric rat immunoglobulin A by cultured rat hepatocytes. Eur J Biochem. 1982 Jul;125(2):437–443. doi: 10.1111/j.1432-1033.1982.tb06702.x. [DOI] [PubMed] [Google Scholar]
  17. Maxfield F. R. Weak bases and ionophores rapidly and reversibly raise the pH of endocytic vesicles in cultured mouse fibroblasts. J Cell Biol. 1982 Nov;95(2 Pt 1):676–681. doi: 10.1083/jcb.95.2.676. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Nakane P. K., Kawaoi A. Peroxidase-labeled antibody. A new method of conjugation. J Histochem Cytochem. 1974 Dec;22(12):1084–1091. doi: 10.1177/22.12.1084. [DOI] [PubMed] [Google Scholar]
  19. Ohkuma S., Poole B. Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents. Proc Natl Acad Sci U S A. 1978 Jul;75(7):3327–3331. doi: 10.1073/pnas.75.7.3327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Poole B., Ohkuma S. Effect of weak bases on the intralysosomal pH in mouse peritoneal macrophages. J Cell Biol. 1981 Sep;90(3):665–669. doi: 10.1083/jcb.90.3.665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Schwartz A. L., Bolognesi A., Fridovich S. E. Recycling of the asialoglycoprotein receptor and the effect of lysosomotropic amines in hepatoma cells. J Cell Biol. 1984 Feb;98(2):732–738. doi: 10.1083/jcb.98.2.732. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Schwartz A. L., Fridovich S. E., Knowles B. B., Lodish H. F. Characterization of the asialoglycoprotein receptor in a continuous hepatoma line. J Biol Chem. 1981 Sep 10;256(17):8878–8881. [PubMed] [Google Scholar]
  23. Schwartz A. L., Fridovich S. E., Lodish H. F. Kinetics of internalization and recycling of the asialoglycoprotein receptor in a hepatoma cell line. J Biol Chem. 1982 Apr 25;257(8):4230–4237. [PubMed] [Google Scholar]
  24. Schwartz A. L., Rup D., Lodish H. F. Difficulties in the quantification of asialoglycoprotein receptors on the rat hepatocyte. J Biol Chem. 1980 Oct 10;255(19):9033–9036. [PubMed] [Google Scholar]
  25. Schwartz A. L. The hepatic asialoglycoprotein receptor. CRC Crit Rev Biochem. 1984;16(3):207–233. doi: 10.3109/10409238409108716. [DOI] [PubMed] [Google Scholar]
  26. Simmons C. F., Jr, Schwartz A. L. Cellular pathways of galactose-terminal ligand movement in a cloned human hepatoma cell line. Mol Pharmacol. 1984 Nov;26(3):509–519. [PubMed] [Google Scholar]
  27. 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]
  28. Solari R., Kraehenbuhl J. P. Biosynthesis of the IgA antibody receptor: a model for the transepithelial sorting of a membrane glycoprotein. Cell. 1984 Jan;36(1):61–71. doi: 10.1016/0092-8674(84)90074-6. [DOI] [PubMed] [Google Scholar]
  29. Stein B. S., Bensch K. G., Sussman H. H. Complete inhibition of transferrin recycling by monensin in K562 cells. J Biol Chem. 1984 Dec 10;259(23):14762–14772. [PubMed] [Google Scholar]
  30. Strous G. J., Du Maine A., Zijderhand-Bleekemolen J. E., Slot J. W., Schwartz A. L. Effect of lysosomotropic amines on the secretory pathway and on the recycling of the asialoglycoprotein receptor in human hepatoma cells. J Cell Biol. 1985 Aug;101(2):531–539. doi: 10.1083/jcb.101.2.531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Tolleshaug H., Berg T. Chloroquine reduces the number of asialo-glycoprotein receptors in the hepatocyte plasma membrane. Biochem Pharmacol. 1979 Oct 1;28(19):2919–2922. doi: 10.1016/0006-2952(79)90586-0. [DOI] [PubMed] [Google Scholar]
  32. Tycko B., Keith C. H., Maxfield F. R. Rapid acidification of endocytic vesicles containing asialoglycoprotein in cells of a human hepatoma line. J Cell Biol. 1983 Dec;97(6):1762–1776. doi: 10.1083/jcb.97.6.1762. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. WARREN L. The thiobarbituric acid assay of sialic acids. J Biol Chem. 1959 Aug;234(8):1971–1975. [PubMed] [Google Scholar]
  34. Wall D. A., Wilson G., Hubbard A. L. The galactose-specific recognition system of mammalian liver: the route of ligand internalization in rat hepatocytes. Cell. 1980 Aug;21(1):79–93. doi: 10.1016/0092-8674(80)90116-6. [DOI] [PubMed] [Google Scholar]
  35. Weigel P. H., Oka J. A. Recycling of the hepatic asialoglycoprotein receptor in isolated rat hepatocytes. Receptor-ligand complexes in an intracellular slowly dissociating pool return to the cell surface prior to dissociation. J Biol Chem. 1984 Jan 25;259(2):1150–1154. [PubMed] [Google Scholar]
  36. Wibo M., Poole B. Protein degradation in cultured cells. II. The uptake of chloroquine by rat fibroblasts and the inhibition of cellular protein degradation and cathepsin B1. J Cell Biol. 1974 Nov;63(2 Pt 1):430–440. doi: 10.1083/jcb.63.2.430. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Willingham M. C., Hanover J. A., Dickson R. B., Pastan I. Morphologic characterization of the pathway of transferrin endocytosis and recycling in human KB cells. Proc Natl Acad Sci U S A. 1984 Jan;81(1):175–179. doi: 10.1073/pnas.81.1.175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Woods J. W., Doriaux M., Farquhar M. G. Transferrin receptors recycle to cis and middle as well as trans Golgi cisternae in Ig-secreting myeloma cells. J Cell Biol. 1986 Jul;103(1):277–286. doi: 10.1083/jcb.103.1.277. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES