Skip to main content
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1983 Jun 1;96(6):1824–1828. doi: 10.1083/jcb.96.6.1824

Monensin inhibits intracellular dissociation of asialoglycoproteins from their receptor

PMCID: PMC2112432  PMID: 6304116

Abstract

Treatment of short-term monolayer cultures of rat hepatocytes with the proton ionophore, monensin, abolishes asialoglycoprotein degradation, despite little effect of the drug on either surface binding of ligand or internalization of prebound ligand. Centrifuging cell homogenates on Percoll density gradients indicates that, as a result of monensin treatment, ligand does not enter lysosomes but sediments instead in a lower density subcellular fraction that is likely an endocytic vesicle. Analyzing the degree of receptor association of intracellular ligand revealed that monensin prevents the dissociation of the receptor-ligand complex that normally occurs subsequent to endocytosis. The weak base, chloroquine, also blocks this intracellular dissociation. Evidence from sequential substitution experiments is presented, indicating that monensin and chloroquine act at the same point in the sequence of events leading to ligand dissociation. These data are discussed in terms of a pH-mediated dissociation of the receptor-ligand complex within a prelysosomal endocytic vesicle.

Full Text

The Full Text of this article is available as a PDF (717.1 KB).

Selected References

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

  1. Ashwell G., Harford J. Carbohydrate-specific receptors of the liver. Annu Rev Biochem. 1982;51:531–554. doi: 10.1146/annurev.bi.51.070182.002531. [DOI] [PubMed] [Google Scholar]
  2. Baenziger J. U., Fiete D. Recycling of the hepatocyte asialoglycoprotein receptor does not require delivery of ligand to lysosomes. J Biol Chem. 1982 Jun 10;257(11):6007–6009. [PubMed] [Google Scholar]
  3. Basu S. K., Goldstein J. L., Anderson R. G., Brown M. S. Monensin interrupts the recycling of low density lipoprotein receptors in human fibroblasts. Cell. 1981 May;24(2):493–502. doi: 10.1016/0092-8674(81)90340-8. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. Dunn W. A., Hubbard A. L., Aronson N. N., Jr Low temperature selectively inhibits fusion between pinocytic vesicles and lysosomes during heterophagy of 125I-asialofetuin by the perfused rat liver. J Biol Chem. 1980 Jun 25;255(12):5971–5978. [PubMed] [Google Scholar]
  7. Gonzalez-Noriega A., Grubb J. H., Talkad V., Sly W. S. Chloroquine inhibits lysosomal enzyme pinocytosis and enhances lysosomal enzyme secretion by impairing receptor recycling. J Cell Biol. 1980 Jun;85(3):839–852. doi: 10.1083/jcb.85.3.839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Haigler H. T., Maxfield F. R., Willingham M. C., Pastan I. Dansylcadaverine inhibits internalization of 125I-epidermal growth factor in BALB 3T3 cells. J Biol Chem. 1980 Feb 25;255(4):1239–1241. [PubMed] [Google Scholar]
  9. Hall C. W., Liebaers I., Di Natale P., Neufeld E. F. Enzymic diagnosis of the genetic mucopolysaccharide storage disorders. Methods Enzymol. 1978;50:439–456. doi: 10.1016/0076-6879(78)50048-7. [DOI] [PubMed] [Google Scholar]
  10. Harford J., Ashwell G. G. Immunological evidence for the transmembrane nature of the rat liver receptor for asialoglycoproteins. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1557–1561. doi: 10.1073/pnas.78.3.1557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Harford J., Lowe M., Tsunoo H., Ashwell G. Immunological approaches to the study of membrane receptors. A monoclonal antibody that inhibits the binding of asialoglycoproteins to the rat liver receptor. J Biol Chem. 1982 Nov 10;257(21):12685–12690. [PubMed] [Google Scholar]
  12. Hudgin R. L., Pricer W. E., Jr, Ashwell G., Stockert R. J., Morell A. G. The isolation and properties of a rabbit liver binding protein specific for asialoglycoproteins. J Biol Chem. 1974 Sep 10;249(17):5536–5543. [PubMed] [Google Scholar]
  13. Johnson D. C., Schlesinger M. J. Vesicular stomatitis virus and sindbis virus glycoprotein transport to the cell surface is inhibited by ionophores. Virology. 1980 Jun;103(2):407–424. doi: 10.1016/0042-6822(80)90200-7. [DOI] [PubMed] [Google Scholar]
  14. Kaplan J., Keogh E. A. Analysis of the effect of amines on inhibition of receptor-mediated and fluid-phase pinocytosis in rabbit alveolar macrophages. Cell. 1981 Jun;24(3):925–932. doi: 10.1016/0092-8674(81)90118-5. [DOI] [PubMed] [Google Scholar]
  15. Kawasaki T., Ashwell G. Isolation and characterization of an avian hepatic binding protein specific for N-acetylglucosamine-terminated glycoproteins. J Biol Chem. 1977 Sep 25;252(18):6536–6543. [PubMed] [Google Scholar]
  16. Kim K., Groman N. B. In vitro inhibition of diphtheria toxin action by ammonium salts and amines. J Bacteriol. 1965 Dec;90(6):1552–1556. doi: 10.1128/jb.90.6.1552-1556.1965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. King A. C., Hernaez-Davis L., Cuatrecasas P. Lysomotropic amines cause intracellular accumulation of receptors for epidermal growth factor. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3283–3287. doi: 10.1073/pnas.77.6.3283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Leppla S., Dorland R. B., Middlebrook J. L. Inhibition of diphtheria toxin degradation and cytotoxic action by chloroquine. J Biol Chem. 1980 Mar 25;255(6):2247–2250. [PubMed] [Google Scholar]
  19. Marnell M. H., Stookey M., Draper R. K. Monensin blocks the transport of diphtheria toxin to the cell cytoplasm. J Cell Biol. 1982 Apr;93(1):57–62. doi: 10.1083/jcb.93.1.57. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Marshall S., Green A., Olefsky J. M. Evidence for recycling of insulin receptors in isolated rat adipocytes. J Biol Chem. 1981 Nov 25;256(22):11464–11470. [PubMed] [Google Scholar]
  22. Matlin K. S., Reggio H., Helenius A., Simons K. Pathway of vesicular stomatitis virus entry leading to infection. J Mol Biol. 1982 Apr 15;156(3):609–631. doi: 10.1016/0022-2836(82)90269-8. [DOI] [PubMed] [Google Scholar]
  23. Mego J. L., Farb R. M., Barnes J. An adenosine triphosphate-dependent stabilization of proteolytic activity in heterolysosomes. Evidence for a proton pump. Biochem J. 1972 Jul;128(4):763–769. doi: 10.1042/bj1280763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Miller D. K., Lenard J. Inhibition of vesicular stomatitis virus infection by spike glycoprotein. Evidence for an intracellular, G protein-requiring step. J Cell Biol. 1980 Feb;84(2):430–437. doi: 10.1083/jcb.84.2.430. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Niedel J., Wilkinson S., Cuatrecasas P. Receptor-mediated uptake and degradation of 125I-chemotactic peptide by human neutrophils. J Biol Chem. 1979 Nov 10;254(21):10700–10706. [PubMed] [Google Scholar]
  26. 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]
  27. Posner B. I., Josefsberg Z., Bergeron J. J. Intracellular polypeptide hormone receptors. Characterization of insulin binding sites in Golgi fractions from the liver of female rats. J Biol Chem. 1978 Jun 10;253(11):4067–4073. [PubMed] [Google Scholar]
  28. Pressman B. C. Biological applications of ionophores. Annu Rev Biochem. 1976;45:501–530. doi: 10.1146/annurev.bi.45.070176.002441. [DOI] [PubMed] [Google Scholar]
  29. Sandvig K., Olsnes S. Diphtheria toxin entry into cells is facilitated by low pH. J Cell Biol. 1980 Dec;87(3 Pt 1):828–832. doi: 10.1083/jcb.87.3.828. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Schneider D. L. ATP-dependent acidification of intact and disrupted lysosomes. Evidence for an ATP-driven proton pump. J Biol Chem. 1981 Apr 25;256(8):3858–3864. [PubMed] [Google Scholar]
  31. Steer C. J., Ashwell G. Studies on a mammalian hepatic binding protein specific for asialoglycoproteins. Evidence for receptor recycling in isolated rat hepatocytes. J Biol Chem. 1980 Apr 10;255(7):3008–3013. [PubMed] [Google Scholar]
  32. Strous G. J., Lodish H. F. Intracellular transport of secretory and membrane proteins in hepatoma cells infected by vesicular stomatitis virus. Cell. 1980 Dec;22(3):709–717. doi: 10.1016/0092-8674(80)90547-4. [DOI] [PubMed] [Google Scholar]
  33. Tartakoff A. M., Vassalli P. Plasma cell immunoglobulin secretion: arrest is accompanied by alterations of the golgi complex. J Exp Med. 1977 Nov 1;146(5):1332–1345. doi: 10.1084/jem.146.5.1332. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Tartakoff A., Vassalli P., Détraz M. Comparative studies of intracellular transport of secretory proteins. J Cell Biol. 1978 Dec;79(3):694–707. doi: 10.1083/jcb.79.3.694. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Tietze C., Schlesinger P., Stahl P. Mannose-specific endocytosis receptor of alveolar macrophages: demonstration of two functionally distinct intracellular pools of receptor and their roles in receptor recycling. J Cell Biol. 1982 Feb;92(2):417–424. doi: 10.1083/jcb.92.2.417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Tycko B., Maxfield F. R. Rapid acidification of endocytic vesicles containing alpha 2-macroglobulin. Cell. 1982 Mar;28(3):643–651. doi: 10.1016/0092-8674(82)90219-7. [DOI] [PubMed] [Google Scholar]
  38. Uchida N., Smilowitz H., Ledger P. W., Tanzer M. L. Kinetic studies of the intracellular transport of procollagen and fibronectin in human fibroblasts. Effects of the monovalent ionophore, monensin. J Biol Chem. 1980 Sep 25;255(18):8638–8644. [PubMed] [Google Scholar]
  39. Van Lenten L., Ashwell G. The binding of desialylated glycoproteins by plasma membranes of rat liver. Development of a quantitative inhibition assay. J Biol Chem. 1972 Jul 25;247(14):4633–4640. [PubMed] [Google Scholar]
  40. Van Leuven F., Cassiman J. J., Van Den Berghe H. Primary amines inhibit recycling of alpha 2M receptors in fibroblasts. Cell. 1980 May;20(1):37–43. doi: 10.1016/0092-8674(80)90232-9. [DOI] [PubMed] [Google Scholar]
  41. Wilcox D. K., Kitson R. P., Widnell C. C. Inhibition of pinocytosis in rat embryo fibroblasts treated with monensin. J Cell Biol. 1982 Mar;92(3):859–864. doi: 10.1083/jcb.92.3.859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. van Renswoude J., Bridges K. R., Harford J. B., Klausner R. D. Receptor-mediated endocytosis of transferrin and the uptake of fe in K562 cells: identification of a nonlysosomal acidic compartment. Proc Natl Acad Sci U S A. 1982 Oct;79(20):6186–6190. doi: 10.1073/pnas.79.20.6186. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES