Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1993 Dec 2;123(6):1695–1706. doi: 10.1083/jcb.123.6.1695

Intracellular routing of GLcNAc-bearing molecules in thyrocytes: selective recycling through the Golgi apparatus

PMCID: PMC2290866  PMID: 7506265

Abstract

Previous experiments led us to speculate that thyrocytes contain a recycling system for GlcNAc-bearing immature thyroglobulin molecules which prevents these molecules from lysosomal degradation (Miquelis, R., C. Alquier, and M. Monsigny. 1987. J. Biol. Chem. 262:15291-15298). To confirm this hypothesis, the fate of GlcNAc-bearing proteins after internalization by thyrocytes was monitored and compared to that of fluid phase markers. Kinetic internalization studies were performed using 125I-GlcNAc-BSA and 131I-Man-BSA. We observed that the apparent intake rate as well as the amount of hydrolyzed GlcNAc-BSA are smaller than the corresponding values for Man-BSA. These differences were reduced by GlcNAc competitors (thyroglobulin and ovomucoid) or a weak base (chloroquine). Part of the internalized GlcNAc-BSA was released into the extracellular milieu at a higher rate and shorter half life (t1/2 = approximately 30 min) than the Man-BSA (t1/2 = approximately 8 h). Subcellular homing was first studied by cell fractionation after internalization using 125I-ovomucoid and 131I-BSA. During Percoll density gradient fractionation, endogenous thyroperoxidase was used to separate subsets of organelles involved in the biosynthetic exocytotic pathway. Incubation of the cell homogenate in the presence of DAB and H2O2 before cell fractionation give rise to a shift in the density of organelles containing 3.5 times more ovomucoid than BSA. Discontinuous sucrose gradient showed that: (a) thyroperoxidase was colocalized with galactosyltransferase-contraining organelles in Golgi-rich subfractions; and (b) that at every time studied from 10 to 100 min, the ovomucoid/BSA ratio was higher in these organelles than in other subfractions. Finally we also observed that: (a) ovomucoid sequestered in the Golgi-rich subfraction incorporated [3H]galactose; and (b) that part of internalized ovomucoid was localized on the Golgi stacks as well as elements of the trans-Golgi, as revealed by immunogold labeling on ultrathin cryosections. These data prove that in thyrocytes GlcNAc accessible sugar moieties on soluble internalized molecules are sufficient to trigger their recycling via the Golgi apparatus.

Full Text

The Full Text of this article is available as a PDF (2.1 MB).

Selected References

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

  1. Alquier C., Miquelis R., Monsigny M. Direct fluorescence localization of an endogenous N-acetyl-glucosamine-specific lectin in the thyroid gland. Histochemistry. 1988;89(2):171–176. doi: 10.1007/BF00489920. [DOI] [PubMed] [Google Scholar]
  2. Anderson R. G., Pathak R. K. Vesicles and cisternae in the trans Golgi apparatus of human fibroblasts are acidic compartments. Cell. 1985 Mar;40(3):635–643. doi: 10.1016/0092-8674(85)90212-0. [DOI] [PubMed] [Google Scholar]
  3. Bastiani P., Simon C., Penel C. Intrafollicular re-cycling of organic iodine in the hypophysectomized rat as observed by a long-term kinetic study. Acta Endocrinol (Copenh) 1980 May;94(1):71–75. doi: 10.1530/acta.0.0940071. [DOI] [PubMed] [Google Scholar]
  4. Bergeron J. J., Rachubinski R. A., Sikstrom R. A., Posner B. I., Paiement J. Galactose transfer to endogenous acceptors within Golgi fractions of rat liver. J Cell Biol. 1982 Jan;92(1):139–146. doi: 10.1083/jcb.92.1.139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Besterman J. M., Airhart J. A., Woodworth R. C., Low R. B. Exocytosis of pinocytosed fluid in cultured cells: kinetic evidence for rapid turnover and compartmentation. J Cell Biol. 1981 Dec;91(3 Pt 1):716–727. doi: 10.1083/jcb.91.3.716. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Braun M., Waheed A., von Figura K. Lysosomal acid phosphatase is transported to lysosomes via the cell surface. EMBO J. 1989 Dec 1;8(12):3633–3640. doi: 10.1002/j.1460-2075.1989.tb08537.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Buktenica S., Olenick S. J., Salgia R., Frankfater A. Degradation and regurgitation of extracellular proteins by cultured mouse peritoneal macrophages and baby hamster kidney fibroblasts. Kinetic evidence that the transfer of proteins to lysosomes is not irreversible. J Biol Chem. 1987 Jul 15;262(20):9469–9476. [PubMed] [Google Scholar]
  8. Chabaud O., Bouchilloux S., Ronin C., Ferrand M. Localization in a Golgi-rich thyroid fraction of sialyl-, galactosyl- and N-acetylglucosaminyltransferases. Biochimie. 1974;56(1):119–130. doi: 10.1016/s0300-9084(74)80362-7. [DOI] [PubMed] [Google Scholar]
  9. Consiglio E., Shifrin S., Yavin Z., Ambesi-Impiombato F. S., Rall J. E., Salvatore G., Kohn L. D. Thyroglobulin interactions with thyroid membranes. Relationship between receptor recognition of N-acetylglucosamine residues and the iodine content of thyroglobulin preparations. J Biol Chem. 1981 Oct 25;256(20):10592–10599. [PubMed] [Google Scholar]
  10. Cortese F., Schneider A. B., Salvatore G. Isopycnic centrifugation of thyroid iodoproteins: selectivity of endocytosis. Eur J Biochem. 1976 Sep;68(1):121–129. doi: 10.1111/j.1432-1033.1976.tb10770.x. [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. Duncan J. R., Kornfeld S. Intracellular movement of two mannose 6-phosphate receptors: return to the Golgi apparatus. J Cell Biol. 1988 Mar;106(3):617–628. doi: 10.1083/jcb.106.3.617. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ekholm R., Wollman S. H. Site of iodination in the rat thyroid gland deduced from electron microscopic autoradiographs. Endocrinology. 1975 Dec;97(6):1432–1444. doi: 10.1210/endo-97-6-1432. [DOI] [PubMed] [Google Scholar]
  14. Farquhar M. G. Multiple pathways of exocytosis, endocytosis, and membrane recycling: validation of a Golgi route. Fed Proc. 1983 May 15;42(8):2407–2413. [PubMed] [Google Scholar]
  15. Farquhar M. G. Progress in unraveling pathways of Golgi traffic. Annu Rev Cell Biol. 1985;1:447–488. doi: 10.1146/annurev.cb.01.110185.002311. [DOI] [PubMed] [Google Scholar]
  16. Freilich L. S., Richmond M. E., Reppucci A. C., Jr, Silbert J. E. A micro method for simultaneous determination of galactosyltransferase and 5'-nucleotidase activities in cell fractions. Biochem J. 1975 Mar;146(3):741–743. doi: 10.1042/bj1460741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Geuze H. J., Slot J. W., Strous G. J., Hasilik A., von Figura K. Possible pathways for lysosomal enzyme delivery. J Cell Biol. 1985 Dec;101(6):2253–2262. doi: 10.1083/jcb.101.6.2253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Goldstein J. L., Brown M. S. Binding and degradation of low density lipoproteins by cultured human fibroblasts. Comparison of cells from a normal subject and from a patient with homozygous familial hypercholesterolemia. J Biol Chem. 1974 Aug 25;249(16):5153–5162. [PubMed] [Google Scholar]
  19. 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]
  20. Gruenberg J., Howell K. E. Membrane traffic in endocytosis: insights from cell-free assays. Annu Rev Cell Biol. 1989;5:453–481. doi: 10.1146/annurev.cb.05.110189.002321. [DOI] [PubMed] [Google Scholar]
  21. Herzog V., Miller F. Membrane retrieval in epithelial cells of isolated thyroid follicles. Eur J Cell Biol. 1979 Aug;19(3):203–215. [PubMed] [Google Scholar]
  22. Herzog V., Neumüller W., Holzmann B. Thyroglobulin, the major and obligatory exportable protein of thyroid follicle cells, carries the lysosomal recognition marker mannose-6-phosphate. EMBO J. 1987 Mar;6(3):555–560. doi: 10.1002/j.1460-2075.1987.tb04790.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Howell K. E., Ito A., Palade G. E. Endoplasmic reticulum marker enzymes in Golgi fractions--what does this mean? J Cell Biol. 1978 Nov;79(2 Pt 1):581–589. doi: 10.1083/jcb.79.2.581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Huang K. M., Snider M. D. Glycoprotein recycling to the galactosyltransferase compartment of the Golgi complex. J Biol Chem. 1993 May 5;268(13):9302–9310. [PubMed] [Google Scholar]
  25. 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]
  26. Kornfeld R., Kornfeld S. Assembly of asparagine-linked oligosaccharides. Annu Rev Biochem. 1985;54:631–664. doi: 10.1146/annurev.bi.54.070185.003215. [DOI] [PubMed] [Google Scholar]
  27. Kornfeld S., Mellman I. The biogenesis of lysosomes. Annu Rev Cell Biol. 1989;5:483–525. doi: 10.1146/annurev.cb.05.110189.002411. [DOI] [PubMed] [Google Scholar]
  28. Kostrouch Z., Munari-Silem Y., Rajas F., Bernier-Valentin F., Rousset B. Thyroglobulin internalized by thyrocytes passes through early and late endosomes. Endocrinology. 1991 Oct;129(4):2202–2211. doi: 10.1210/endo-129-4-2202. [DOI] [PubMed] [Google Scholar]
  29. Miquelis R., Alquier C., Monsigny M. The N-acetylglucosamine-specific receptor of the thyroid. Binding characteristics, partial characterization, and potential role. J Biol Chem. 1987 Nov 5;262(31):15291–15298. [PubMed] [Google Scholar]
  30. Miquelis R., Simon C. In vivo sequential model for thyroglobulin iodination. Acta Endocrinol (Copenh) 1980 Dec;95(4):489–494. doi: 10.1530/acta.0.0950489. [DOI] [PubMed] [Google Scholar]
  31. Nilsson T., Lucocq J. M., Mackay D., Warren G. The membrane spanning domain of beta-1,4-galactosyltransferase specifies trans Golgi localization. EMBO J. 1991 Dec;10(12):3567–3575. doi: 10.1002/j.1460-2075.1991.tb04923.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Parente J. P., Wieruszeski J. M., Strecker G., Montreuil J., Fournet B., van Halbeek H., Dorland L., Vliegenthart J. F. A novel type of carbohydrate structure present in hen ovomucoid. J Biol Chem. 1982 Nov 25;257(22):13173–13176. [PubMed] [Google Scholar]
  33. Patzak A., Winkler H. Exocytotic exposure and recycling of membrane antigens of chromaffin granules: ultrastructural evaluation after immunolabeling. J Cell Biol. 1986 Feb;102(2):510–515. doi: 10.1083/jcb.102.2.510. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Paz-Parente J., Strecker G., Leroy Y., Montreuil J., Fournet B., van Halbeek H., Dorland L., Vliegenthart J. F. Primary structure of a novel N-glycosidic carbohydrate unit, derived from hen ovomucoid. A 500-MHz 1H-NMR study. FEBS Lett. 1983 Feb 21;152(2):145–152. doi: 10.1016/0014-5793(83)80367-6. [DOI] [PubMed] [Google Scholar]
  35. Pfeffer S. R., Rothman J. E. Biosynthetic protein transport and sorting by the endoplasmic reticulum and Golgi. Annu Rev Biochem. 1987;56:829–852. doi: 10.1146/annurev.bi.56.070187.004145. [DOI] [PubMed] [Google Scholar]
  36. Pratten M. K., Williams K. E., Lloyd J. B. A quantitative study of pinocytosis and intracellular proteolysis in rat peritoneal macrophages. Biochem J. 1977 Dec 15;168(3):365–372. doi: 10.1042/bj1680365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Roberts A. V., Williams K. E., Lloyd J. B. The pinocytosis of 125I-labelled poly(vinylpyrrolidone), [14C]sucrose and colloidal [198Au]gold by rat yolk sac cultured in vitro. Biochem J. 1977 Nov 15;168(2):239–244. doi: 10.1042/bj1680239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Roth J., Berger E. G. Immunocytochemical localization of galactosyltransferase in HeLa cells: codistribution with thiamine pyrophosphatase in trans-Golgi cisternae. J Cell Biol. 1982 Apr;93(1):223–229. doi: 10.1083/jcb.93.1.223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Roth J., Taatjes D. J., Lucocq J. M., Weinstein J., Paulson J. C. Demonstration of an extensive trans-tubular network continuous with the Golgi apparatus stack that may function in glycosylation. Cell. 1985 Nov;43(1):287–295. doi: 10.1016/0092-8674(85)90034-0. [DOI] [PubMed] [Google Scholar]
  40. Salacinski P. R., McLean C., Sykes J. E., Clement-Jones V. V., Lowry P. J. Iodination of proteins, glycoproteins, and peptides using a solid-phase oxidizing agent, 1,3,4,6-tetrachloro-3 alpha,6 alpha-diphenyl glycoluril (Iodogen). Anal Biochem. 1981 Oct;117(1):136–146. doi: 10.1016/0003-2697(81)90703-x. [DOI] [PubMed] [Google Scholar]
  41. Saraste J., Palade G. E., Farquhar M. G. Temperature-sensitive steps in the transport of secretory proteins through the Golgi complex in exocrine pancreatic cells. Proc Natl Acad Sci U S A. 1986 Sep;83(17):6425–6429. doi: 10.1073/pnas.83.17.6425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Shifrin S., Kohn L. D. Binding of thyroglobulin to bovine thyroid membranes. Role of specific amino acids in receptor recognition. J Biol Chem. 1981 Oct 25;256(20):10600–10605. [PubMed] [Google Scholar]
  43. Silverstein S. C., Steinman R. M., Cohn Z. A. Endocytosis. Annu Rev Biochem. 1977;46:669–722. doi: 10.1146/annurev.bi.46.070177.003321. [DOI] [PubMed] [Google Scholar]
  44. 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]
  45. 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]
  46. Stoorvogel W., Geuze H. J., Griffith J. M., Strous G. J. The pathways of endocytosed transferrin and secretory protein are connected in the trans-Golgi reticulum. J Cell Biol. 1988 Jun;106(6):1821–1829. doi: 10.1083/jcb.106.6.1821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Stoorvogel W., Geuze H. J., Strous G. J. Sorting of endocytosed transferrin and asialoglycoprotein occurs immediately after internalization in HepG2 cells. J Cell Biol. 1987 May;104(5):1261–1268. doi: 10.1083/jcb.104.5.1261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Stoorvogel W., Strous G. J., Geuze H. J., Oorschot V., Schwartz A. L. Late endosomes derive from early endosomes by maturation. Cell. 1991 May 3;65(3):417–427. doi: 10.1016/0092-8674(91)90459-c. [DOI] [PubMed] [Google Scholar]
  49. Thibault V., Blanck O., Courageot J., Pachetti C., Perrin C., de Mascarel A., Miquelis R. The N-acetylglucosamine-specific receptor of the thyroid: purification, further characterization, and expression patterns on normal and pathological glands. Endocrinology. 1993 Jan;132(1):468–476. doi: 10.1210/endo.132.1.8419143. [DOI] [PubMed] [Google Scholar]
  50. Wollman S. H. Turnover of plasma membrane in thyroid epithelium and review of evidence for the role of micropinocytosis. Eur J Cell Biol. 1989 Dec;50(2):247–256. [PubMed] [Google Scholar]
  51. Yamashita K., Kamerling J. P., Kobata A. Structural study of the carbohydrate moiety of hen ovomucoid. Occurrence of a series of pentaantennary complex-type asparagine-linked sugar chains. J Biol Chem. 1982 Nov 10;257(21):12809–12814. [PubMed] [Google Scholar]
  52. de Waard P., Koorevaar A., Kamerling J. P., Vliegenthart J. F. Structure determination by 1H NMR spectroscopy of (sulfated) sialylated N-linked carbohydrate chains released from porcine thyroglobulin by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase-F. J Biol Chem. 1991 Mar 5;266(7):4237–4243. [PubMed] [Google Scholar]
  53. van Meer G. Lipid traffic in animal cells. Annu Rev Cell Biol. 1989;5:247–275. doi: 10.1146/annurev.cb.05.110189.001335. [DOI] [PubMed] [Google Scholar]
  54. van den Hove M. F., Couvreur M., de Visscher M., Salvatore G. A new mechanism for the reabsorption of thyroid iodoproteins: selective fluid pinocytosis. Eur J Biochem. 1982 Feb;122(2):415–422. doi: 10.1111/j.1432-1033.1982.tb05896.x. [DOI] [PubMed] [Google Scholar]

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

RESOURCES