Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1988 Mar 1;106(3):617–628. doi: 10.1083/jcb.106.3.617

Intracellular movement of two mannose 6-phosphate receptors: return to the Golgi apparatus

PMCID: PMC2115106  PMID: 2964450

Abstract

We have used Chinese hamster ovary (CHO) cells and a murine lymphoma cell line to study the recycling of the 215-kD and the 46-kD mannose 6- phosphate receptors to various regions of the Golgi to determine the site where the receptors first encounter newly synthesized lysosomal enzymes. For assessing return to the trans-most Golgi compartments containing sialyltransferase (trans-cisternae and trans-Golgi network), the oligosaccharides of receptor molecules on the cell surface were labeled with [3H]galactose at 4 degrees C. Upon warming to 37 degrees C, the [3H]galactose residues on both receptors were substituted with sialic acid with a t1/2 approximately 3 hrs. Other glycoproteins acquired sialic acid at least 8-10 times slower. Return of the receptors to the trans-Golgi cisternae containing galactosyltransferase could not be detected. Return to the cis/middle Golgi cisternae containing alpha-mannosidase I was measured by adding deoxymannojirimycin, a mannosidase I inhibitor, during the initial posttranslational passage of [3H]mannose-labeled glycoproteins through the Golgi, thereby preserving oligosaccharides which would be substrates for alpha-mannosidase I. After removal of the inhibitor, return to the early Golgi with subsequent passage through the Golgi complex was measured by determining the conversion of the oligosaccharides from high mannose to complex-type units. This conversion was very slow for the receptors and other glycoproteins (t1/2 approximately 20 h). Exposure of the receptors and other glycoproteins to the dMM-sensitive alpha-mannosidase without movement through the Golgi apparatus was determined by measuring the loss of mannose residues from these proteins. This loss was also slow. These results indicate that both Man-6-P receptors routinely return to the Golgi compartment which contains sialyltransferase and recycle through other regions of the Golgi region less frequently. We infer that the trans-Golgi network is the major site for lysosomal enzyme sorting in CHO and murine lymphoma cells.

Full Text

The Full Text of this article is available as a PDF (1.6 MB).

Selected References

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

  1. Baenziger J. U., Fiete D. Structural determinants of Ricinus communis agglutinin and toxin specificity for oligosaccharides. J Biol Chem. 1979 Oct 10;254(19):9795–9799. [PubMed] [Google Scholar]
  2. Balch W. E., Elliott M. M., Keller D. S. ATP-coupled transport of vesicular stomatitis virus G protein between the endoplasmic reticulum and the Golgi. J Biol Chem. 1986 Nov 5;261(31):14681–14689. [PubMed] [Google Scholar]
  3. Balch W. E., Keller D. S. ATP-coupled transport of vesicular stomatitis virus G protein. Functional boundaries of secretory compartments. J Biol Chem. 1986 Nov 5;261(31):14690–14696. [PubMed] [Google Scholar]
  4. Balch W. E., Wagner K. R., Keller D. S. Reconstitution of transport of vesicular stomatitis virus G protein from the endoplasmic reticulum to the Golgi complex using a cell-free system. J Cell Biol. 1987 Mar;104(3):749–760. doi: 10.1083/jcb.104.3.749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bennett G., O'Shaughnessy D. The site of incorporation of sialic acid residues into glycoproteins and the subsequent fates of these molecules in various rat and mouse cell types as shown by radioautography after injection of [3H]N-acetylmannosamine. I. Observations in hepatocytes. J Cell Biol. 1981 Jan;88(1):1–15. doi: 10.1083/jcb.88.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Berger E. G., Thurnher M., Müller U. Galactosyltransferase and sialyltransferase are located in different subcellular compartments in HeLa cells. Exp Cell Res. 1987 Nov;173(1):267–273. doi: 10.1016/0014-4827(87)90352-1. [DOI] [PubMed] [Google Scholar]
  7. Bischoff J., Kornfeld R. The effect of 1-deoxymannojirimycin on rat liver alpha-mannosidases. Biochem Biophys Res Commun. 1984 Nov 30;125(1):324–331. doi: 10.1016/s0006-291x(84)80371-x. [DOI] [PubMed] [Google Scholar]
  8. Bischoff J., Liscum L., Kornfeld R. The use of 1-deoxymannojirimycin to evaluate the role of various alpha-mannosidases in oligosaccharide processing in intact cells. J Biol Chem. 1986 Apr 5;261(10):4766–4774. [PubMed] [Google Scholar]
  9. Braulke T., Gartung C., Hasilik A., von Figura K. Is movement of mannose 6-phosphate-specific receptor triggered by binding of lysosomal enzymes? J Cell Biol. 1987 Jun;104(6):1735–1742. doi: 10.1083/jcb.104.6.1735. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Briles E. B., Li E., Kornfeld S. Isolation of wheat germ agglutinin-resistant clones of Chinese hamster ovary cells deficient in membrane sialic acid and galactose. J Biol Chem. 1977 Feb 10;252(3):1107–1116. [PubMed] [Google Scholar]
  11. Brown W. J., Constantinescu E., Farquhar M. G. Redistribution of mannose-6-phosphate receptors induced by tunicamycin and chloroquine. J Cell Biol. 1984 Jul;99(1 Pt 1):320–326. doi: 10.1083/jcb.99.1.320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Brown W. J., Farquhar M. G. The mannose-6-phosphate receptor for lysosomal enzymes is concentrated in cis Golgi cisternae. Cell. 1984 Feb;36(2):295–307. doi: 10.1016/0092-8674(84)90223-x. [DOI] [PubMed] [Google Scholar]
  13. Brown W. J., Goodhouse J., Farquhar M. G. Mannose-6-phosphate receptors for lysosomal enzymes cycle between the Golgi complex and endosomes. J Cell Biol. 1986 Oct;103(4):1235–1247. doi: 10.1083/jcb.103.4.1235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dahms N. M., Lobel P., Breitmeyer J., Chirgwin J. M., Kornfeld S. 46 kd mannose 6-phosphate receptor: cloning, expression, and homology to the 215 kd mannose 6-phosphate receptor. Cell. 1987 Jul 17;50(2):181–192. doi: 10.1016/0092-8674(87)90214-5. [DOI] [PubMed] [Google Scholar]
  15. Deutscher S. L., Hirschberg C. B. Mechanism of galactosylation in the Golgi apparatus. A Chinese hamster ovary cell mutant deficient in translocation of UDP-galactose across Golgi vesicle membranes. J Biol Chem. 1986 Jan 5;261(1):96–100. [PubMed] [Google Scholar]
  16. Dunphy W. G., Brands R., Rothman J. E. Attachment of terminal N-acetylglucosamine to asparagine-linked oligosaccharides occurs in central cisternae of the Golgi stack. Cell. 1985 Feb;40(2):463–472. doi: 10.1016/0092-8674(85)90161-8. [DOI] [PubMed] [Google Scholar]
  17. Dunphy W. G., Fries E., Urbani L. J., Rothman J. E. Early and late functions associated with the Golgi apparatus reside in distinct compartments. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7453–7457. doi: 10.1073/pnas.78.12.7453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Dunphy W. G., Rothman J. E. Compartmental organization of the Golgi stack. Cell. 1985 Aug;42(1):13–21. doi: 10.1016/s0092-8674(85)80097-0. [DOI] [PubMed] [Google Scholar]
  19. Fedde K. N., Sly W. S. Ricin-binding properties of acid hydrolases from isolated lysosomes implies prior processing by terminal transferases of the trans-Golgi apparatus. Biochem Biophys Res Commun. 1985 Dec 17;133(2):614–620. doi: 10.1016/0006-291x(85)90949-0. [DOI] [PubMed] [Google Scholar]
  20. Fischer H. D., Gonzalez-Noriega A., Sly W. S. Beta-glucuronidase binding to human fibroblast membrane receptors. J Biol Chem. 1980 Jun 10;255(11):5069–5074. [PubMed] [Google Scholar]
  21. 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]
  22. Geuze H. J., Slot J. W., Strous G. J., Hasilik A., Von Figura K. Ultrastructural localization of the mannose 6-phosphate receptor in rat liver. J Cell Biol. 1984 Jun;98(6):2047–2054. doi: 10.1083/jcb.98.6.2047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Geuze H. J., Slot J. W., Strous G. J., Luzio J. P., Schwartz A. L. A cycloheximide-resistant pool of receptors for asialoglycoproteins and mannose 6-phosphate residues in the Golgi complex of hepatocytes. EMBO J. 1984 Nov;3(11):2677–2685. doi: 10.1002/j.1460-2075.1984.tb02193.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Gieselmann V., Pohlmann R., Hasilik A., Von Figura K. Biosynthesis and transport of cathepsin D in cultured human fibroblasts. J Cell Biol. 1983 Jul;97(1):1–5. doi: 10.1083/jcb.97.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Goldberg D. E., Gabel C. A., Kornfeld S. Studies of the biosynthesis of the mannose 6-phosphate receptor in receptor-positive and -deficient cell lines. J Cell Biol. 1983 Dec;97(6):1700–1706. doi: 10.1083/jcb.97.6.1700. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Goldberg D. E., Kornfeld S. Evidence for extensive subcellular organization of asparagine-linked oligosaccharide processing and lysosomal enzyme phosphorylation. J Biol Chem. 1983 Mar 10;258(5):3159–3165. [PubMed] [Google Scholar]
  29. 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]
  30. Hoflack B., Fujimoto K., Kornfeld S. The interaction of phosphorylated oligosaccharides and lysosomal enzymes with bovine liver cation-dependent mannose 6-phosphate receptor. J Biol Chem. 1987 Jan 5;262(1):123–129. [PubMed] [Google Scholar]
  31. Hoflack B., Kornfeld S. Lysosomal enzyme binding to mouse P388D1 macrophage membranes lacking the 215-kDa mannose 6-phosphate receptor: evidence for the existence of a second mannose 6-phosphate receptor. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4428–4432. doi: 10.1073/pnas.82.13.4428. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Hoflack B., Kornfeld S. Purification and characterization of a cation-dependent mannose 6-phosphate receptor from murine P388D1 macrophages and bovine liver. J Biol Chem. 1985 Oct 5;260(22):12008–12014. [PubMed] [Google Scholar]
  33. Kingsley D. M., Kozarsky K. F., Hobbie L., Krieger M. Reversible defects in O-linked glycosylation and LDL receptor expression in a UDP-Gal/UDP-GalNAc 4-epimerase deficient mutant. Cell. 1986 Mar 14;44(5):749–759. doi: 10.1016/0092-8674(86)90841-x. [DOI] [PubMed] [Google Scholar]
  34. Kornfeld K., Reitman M. L., Kornfeld R. The carbohydrate-binding specificity of pea and lentil lectins. Fucose is an important determinant. J Biol Chem. 1981 Jul 10;256(13):6633–6640. [PubMed] [Google Scholar]
  35. 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]
  36. Kornfeld S. Trafficking of lysosomal enzymes in normal and disease states. J Clin Invest. 1986 Jan;77(1):1–6. doi: 10.1172/JCI112262. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Kozutsumi Y., Kawasaki T., Yamashina I. Structures of galactose-containing oligosaccharides of alpha-mannosidase from porcine kidney. J Biochem. 1986 Aug;100(2):505–512. doi: 10.1093/oxfordjournals.jbchem.a121740. [DOI] [PubMed] [Google Scholar]
  38. Lobel P., Dahms N. M., Breitmeyer J., Chirgwin J. M., Kornfeld S. Cloning of the bovine 215-kDa cation-independent mannose 6-phosphate receptor. Proc Natl Acad Sci U S A. 1987 Apr;84(8):2233–2237. doi: 10.1073/pnas.84.8.2233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Narasimhan S., Wilson J. R., Martin E., Schachter H. A structural basis for four distinct elution profiles on concanavalin A--Sepharose affinity chromatography of glycopeptides. Can J Biochem. 1979 Jan;57(1):83–96. doi: 10.1139/o79-011. [DOI] [PubMed] [Google Scholar]
  40. Novikoff P. M., Tulsiani D. R., Touster O., Yam A., Novikoff A. B. Immunocytochemical localization of alpha-D-mannosidase II in the Golgi apparatus of rat liver. Proc Natl Acad Sci U S A. 1983 Jul;80(14):4364–4368. doi: 10.1073/pnas.80.14.4364. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Pfeffer S. R. The endosomal concentration of a mannose 6-phosphate receptor is unchanged in the absence of ligand synthesis. J Cell Biol. 1987 Jul;105(1):229–234. doi: 10.1083/jcb.105.1.229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Rosenfeld M. G., Kreibich G., Popov D., Kato K., Sabatini D. D. Biosynthesis of lysosomal hydrolases: their synthesis in bound polysomes and the role of co- and post-translational processing in determining their subcellular distribution. J Cell Biol. 1982 Apr;93(1):135–143. doi: 10.1083/jcb.93.1.135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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]
  44. 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]
  45. 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]
  46. Sahagian G. G., Distler J. J., Jourdian G. W. Membrane receptor for phosphomannosyl residues. Methods Enzymol. 1982;83:392–396. doi: 10.1016/0076-6879(82)83036-x. [DOI] [PubMed] [Google Scholar]
  47. Sahagian G. G., Distler J., Jourdian G. W. Characterization of a membrane-associated receptor from bovine liver that binds phosphomannosyl residues of bovine testicular beta-galactosidase. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4289–4293. doi: 10.1073/pnas.78.7.4289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Sahagian G. G., Neufeld E. F. Biosynthesis and turnover of the mannose 6-phosphate receptor in cultured Chinese hamster ovary cells. J Biol Chem. 1983 Jun 10;258(11):7121–7128. [PubMed] [Google Scholar]
  49. Sahagian G. G. The mannose 6-phosphate receptor: function, biosynthesis and translocation. Biol Cell. 1984;51(2):207–214. doi: 10.1111/j.1768-322x.1984.tb00300.x. [DOI] [PubMed] [Google Scholar]
  50. Sly W. S., Fischer H. D. The phosphomannosyl recognition system for intracellular and intercellular transport of lysosomal enzymes. J Cell Biochem. 1982;18(1):67–85. doi: 10.1002/jcb.1982.240180107. [DOI] [PubMed] [Google Scholar]
  51. 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]
  52. Snider M. D., Rogers O. C. Membrane traffic in animal cells: cellular glycoproteins return to the site of Golgi mannosidase I. J Cell Biol. 1986 Jul;103(1):265–275. doi: 10.1083/jcb.103.1.265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Steiner A. W., Rome L. H. Assay and purification of a solubilized membrane receptor that binds the lysosomal enzyme alpha-L-iduronidase. Arch Biochem Biophys. 1982 Apr 1;214(2):681–687. doi: 10.1016/0003-9861(82)90074-1. [DOI] [PubMed] [Google Scholar]
  54. Thilo L. Labeling of plasma membrane glycoconjugates by terminal glycosylation (galactosyltransferase and glycosidase). Methods Enzymol. 1983;98:415–421. doi: 10.1016/0076-6879(83)98169-7. [DOI] [PubMed] [Google Scholar]
  55. Torres C. R., Hart G. W. Topography and polypeptide distribution of terminal N-acetylglucosamine residues on the surfaces of intact lymphocytes. Evidence for O-linked GlcNAc. J Biol Chem. 1984 Mar 10;259(5):3308–3317. [PubMed] [Google Scholar]
  56. Trowbridge I. S., Hyman R., Ferson T., Mazauskas C. Expression of Thy-1 glycoprotein on lectin-resistant lymphoma cell lines. Eur J Immunol. 1978 Oct;8(10):716–723. doi: 10.1002/eji.1830081009. [DOI] [PubMed] [Google Scholar]
  57. Vladutiu G. D. Effect of the co-existence of galactosyl and phosphomannosyl residues on beta-hexosaminidase on the processing and transport of the enzyme in mucolipidosis I fibroblasts. Biochim Biophys Acta. 1983 Nov 8;760(3):363–370. doi: 10.1016/0304-4165(83)90374-4. [DOI] [PubMed] [Google Scholar]
  58. Willingham M. C., Pastan I. H., Sahagian G. G. Ultrastructural immunocytochemical localization of the phosphomannosyl receptor in Chinese hamster ovary (CHO) cells. J Histochem Cytochem. 1983 Jan;31(1):1–11. doi: 10.1177/31.1.6300218. [DOI] [PubMed] [Google Scholar]
  59. von Figura K., Gieselmann V., Hasilik A. Antibody to mannose 6-phosphate specific receptor induces receptor deficiency in human fibroblasts. EMBO J. 1984 Jun;3(6):1281–1286. doi: 10.1002/j.1460-2075.1984.tb01963.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. von Figura K., Hasilik A. Lysosomal enzymes and their receptors. Annu Rev Biochem. 1986;55:167–193. doi: 10.1146/annurev.bi.55.070186.001123. [DOI] [PubMed] [Google Scholar]

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

RESOURCES

OSZAR »