Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1984 Jun 15;220(3):665–675. doi: 10.1042/bj2200665

Monensin inhibits recycling of macrophage mannose-glycoprotein receptors and ligand delivery to lysosomes.

T Wileman, R L Boshans, P Schlesinger, P Stahl
PMCID: PMC1153682  PMID: 6087792

Abstract

Binding studies with cells that had been permeabilized with saponin indicate that alveolar macrophages have an intracellular pool of mannose-specific binding sites which is about 4-fold greater than the cell surface pool. Monensin, a carboxylic ionophore which mediates proton movement across membranes, has no effect on binding of ligand to macrophages but blocks receptor-mediated uptake of 125I-labelled beta-glucuronidase. Inhibition of uptake was concentration- and time-dependent. Internalization of receptor-bound ligand, after warming to 37 degrees C, was unaffected by monensin. Moreover, internalization of ligand in the presence of monensin resulted in an intracellular accumulation of receptor-ligand complexes. The monensin effect was not dependent on the presence of ligand, since incubation of macrophages with monensin at 37 degrees C without ligand resulted in a substantial decrease in cell-surface binding activity. However, total binding activity, measured in the presence of saponin, was much less affected by monensin treatment. Removal of monensin followed by a brief incubation at pH 6.0 and 37 degrees C, restored both cell-surface binding and uptake activity. Fractionation experiments indicate that ligands enter a low-density (endosomal) fraction within the first few minutes of uptake, and within 20 min transfer to the lysosomal fraction has occurred. Monensin blocks the transfer from endosomal to lysosomal fraction. Lysosomal pH, as measured by the fluorescein-dextran method, was increased by monensin in the same concentration range that blocked ligand uptake. The results indicate that monensin blockade of receptor-mediated endocytosis of mannose-terminated ligands by macrophages is due to entrapment of receptor-ligand complexes and probably receptors in the pre-lysosomal compartment. The inhibition is linked with an increase in the pH of acid intracellular vesicles.

Full text

PDF
665

Selected References

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

  1. Amar-Costesec A., Beaufay H., Wibo M., Thinès-Sempoux D., Feytmans E., Robbi M., Berthet J. Analytical study of microsomes and isolated subcellular membranes from rat liver. II. Preparation and composition of the microsomal fraction. J Cell Biol. 1974 Apr;61(1):201–212. doi: 10.1083/jcb.61.1.201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Cohn Z. A., Steinman R. M. Phagocytosis and fluid-phase pinocytosis. Ciba Found Symp. 1982;(92):15–34. doi: 10.1002/9780470720745.ch2. [DOI] [PubMed] [Google Scholar]
  4. Fiete D., Brownell M. D., Baenziger J. U. Evidence for transmembrane modulation of the ligand-binding site of the hepatocyte galactose/N-acetylgalactosamine-specific receptor. J Biol Chem. 1983 Jan 25;258(2):817–823. [PubMed] [Google Scholar]
  5. Fischer H. D., Gonzalez-Noriega A., Sly W. S., Morré D. J. Phosphomannosyl-enzyme receptors in rat liver. Subcellular distribution and role in intracellular transport of lysosomal enzymes. J Biol Chem. 1980 Oct 25;255(20):9608–9615. [PubMed] [Google Scholar]
  6. 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]
  7. Goldstein J. L., Anderson R. G., Brown M. S. Coated pits, coated vesicles, and receptor-mediated endocytosis. Nature. 1979 Jun 21;279(5715):679–685. doi: 10.1038/279679a0. [DOI] [PubMed] [Google Scholar]
  8. Harford J., Wolkoff A. W., Ashwell G., Klausner R. D. Monensin inhibits intracellular dissociation of asialoglycoproteins from their receptor. J Cell Biol. 1983 Jun;96(6):1824–1828. doi: 10.1083/jcb.96.6.1824. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hoppe C. A., Lee Y. C. Stimulation of mannose-binding activity in the rabbit alveolar macrophage by simple sugars. J Biol Chem. 1982 Nov 10;257(21):12831–12834. [PubMed] [Google Scholar]
  10. Jessup W., Dean R. T. Secretion by mononuclear phagocytes of lysosomal hydrolases bearing ligands for the mannose-6-phosphate receptor system of fibroblasts: evidence for a second mechanism of spontaneous secretion? Biochem Biophys Res Commun. 1982 Apr 14;105(3):922–927. doi: 10.1016/0006-291x(82)91058-0. [DOI] [PubMed] [Google Scholar]
  11. Keller R. K., Touster O. Physical and chemical properties of beta-glucuronidase from the preputial gland of the female rat. J Biol Chem. 1975 Jun 25;250(12):4765–4769. [PubMed] [Google Scholar]
  12. Konish M., Shepherd V., Holt G., Stahl P. Uptake of glycoproteins and glycoconjugates by macrophages. Methods Enzymol. 1983;98:301–304. doi: 10.1016/0076-6879(83)98157-0. [DOI] [PubMed] [Google Scholar]
  13. Lee Y. C., Stowell C. P., Krantz M. J. 2-Imino-2-methoxyethyl 1-thioglycosides: new reagents for attaching sugars to proteins. Biochemistry. 1976 Sep 7;15(18):3956–3963. doi: 10.1021/bi00663a008. [DOI] [PubMed] [Google Scholar]
  14. Lin H. S., Gordon S. Secretion of plasminogen activator by bone marrow-derived mononuclear phagocytes and its enhancement by colony-stimulating factor. J Exp Med. 1979 Aug 1;150(2):231–245. doi: 10.1084/jem.150.2.231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. Maynard Y., Baenziger J. U. Oligosaccharide specific endocytosis by isolated rat hepatic reticuloendothelial cells. J Biol Chem. 1981 Aug 10;256(15):8063–8068. [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. Shepherd V. L., Lee Y. C., Schlesinger P. H., Stahl P. D. L-Fucose-terminated glycoconjugates are recognized by pinocytosis receptors on macrophages. Proc Natl Acad Sci U S A. 1981 Feb;78(2):1019–1022. doi: 10.1073/pnas.78.2.1019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Stahl P. D., Rodman J. S., Miller M. J., Schlesinger P. H. Evidence for receptor-mediated binding of glycoproteins, glycoconjugates, and lysosomal glycosidases by alveolar macrophages. Proc Natl Acad Sci U S A. 1978 Mar;75(3):1399–1403. doi: 10.1073/pnas.75.3.1399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Stahl P., Gordon S. Expression of a mannosyl-fucosyl receptor for endocytosis on cultured primary macrophages and their hybrids. J Cell Biol. 1982 Apr;93(1):49–56. doi: 10.1083/jcb.93.1.49. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Stahl P., Schlesinger P. H., Sigardson E., Rodman J. S., Lee Y. C. Receptor-mediated pinocytosis of mannose glycoconjugates by macrophages: characterization and evidence for receptor recycling. Cell. 1980 Jan;19(1):207–215. doi: 10.1016/0092-8674(80)90402-x. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Tietze C., Schlesinger P., Stahl P. Chloroquine and ammonium ion inhibit receptor-mediated endocytosis of mannose-glycoconjugates by macrophages: apparent inhibition of receptor recycling. Biochem Biophys Res Commun. 1980 Mar 13;93(1):1–8. doi: 10.1016/s0006-291x(80)80237-3. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. Weigel P. H., Oka J. A. The large intracellular pool of asialoglycoprotein receptors functions during the endocytosis of asialoglycoproteins by isolated rat hepatocytes. J Biol Chem. 1983 Apr 25;258(8):5095–5102. [PubMed] [Google Scholar]
  29. 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]
  30. Willingham M. C., Pastan I. The receptosome: an intermediate organelle of receptor mediated endocytosis in cultured fibroblasts. Cell. 1980 Aug;21(1):67–77. doi: 10.1016/0092-8674(80)90115-4. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES