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. 1992 Jun 1;117(5):949–958. doi: 10.1083/jcb.117.5.949

O-glycosylation of intact and truncated ribophorins in brefeldin A- treated cells: newly synthesized intact ribophorins are only transiently accessible to the relocated glycosyltransferases

PMCID: PMC2289488  PMID: 1577870

Abstract

Ribophorins I and II are type I transmembrane glycoproteins of the ER that are segregated to the rough domains of this organelle. Both ribophorins appear to be part of the translocation apparatus for nascent polypeptides that is associated with membrane-bound ribosomes and participate in the formation of a proteinaceous network within the ER membrane that also includes other components of the translocation apparatus. The ribophorins are both highly stable proteins that lack O- linked sugars but each contains one high mannose N-linked oligosaccharide that remains endo H sensitive throughout their lifetimes. We have previously shown (Tsao, Y. S., N. E. Ivessa, M. Adesnik, D. D. Sabatini, and G. Kreibich. 1992. J. Cell Biol. 116:57- 67) that a COOH-terminally truncated variant of ribophorin I that contains only the first 332 amino acids of the luminal domain (RI332), when synthesized in permanent transformants of HeLa cells, undergoes a rapid degradation with biphasic kinetics in the ER itself and in a second, as yet unidentified nonlysosomal pre-Golgi compartment. We now show that in cells treated with brefeldin A (BFA) RI332 molecules undergo rapid O-glycosylation in a multistep process that involves the sequential addition of N-acetylgalactosamine, galactose, and terminal sialic acid residues. Addition of O-linked sugars affected all newly synthesized RI332 molecules and was completed soon after synthesis with a half time of about 10 min. In the same cells, intact ribophorins I and II also underwent O-linked glycosylation in the presence of BFA, but these molecules were modified only during a short time period immediately after their synthesis was completed, and the modification affected only a fraction of the newly synthesized polypeptides. More important, these molecules synthesized before the addition of BFA were not modified by O-glycosylation. The same is true for ribophorin I when overexpressed in HeLa cells although it is significantly less stable than the native polypeptide in control cells. We, therefore, conclude that soon after their synthesis, ribophorins lose their susceptibility to the relocated Golgi enzymes that effect the O-glycosylation, most likely as a consequence of a conformational change in the ribophorins that occurs during their maturation, although it cannot be excluded that rapid integration of these molecules into a supramolecular complex in the ER membrane leads to their inaccessibility to these enzymes.

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Selected References

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  1. Amar-Costesec A., Todd J. A., Kreibich G. Segregation of the polypeptide translocation apparatus to regions of the endoplasmic reticulum containing ribophorins and ribosomes. I. Functional tests on rat liver microsomal subfractions. J Cell Biol. 1984 Dec;99(6):2247–2253. doi: 10.1083/jcb.99.6.2247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Bhavanandan V. P., Katlic A. W. The interaction of wheat germ agglutinin with sialoglycoproteins. The role of sialic acid. J Biol Chem. 1979 May 25;254(10):4000–4008. [PubMed] [Google Scholar]
  4. Bonifacino J. S., Lippincott-Schwartz J. Degradation of proteins within the endoplasmic reticulum. Curr Opin Cell Biol. 1991 Aug;3(4):592–600. doi: 10.1016/0955-0674(91)90028-w. [DOI] [PubMed] [Google Scholar]
  5. Brands R., Snider M. D., Hino Y., Park S. S., Gelboin H. V., Rothman J. E. Retention of membrane proteins by the endoplasmic reticulum. J Cell Biol. 1985 Nov;101(5 Pt 1):1724–1732. doi: 10.1083/jcb.101.5.1724. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Capasso J. M., Keenan T. W., Abeijon C., Hirschberg C. B. Mechanism of phosphorylation in the lumen of the Golgi apparatus. Translocation of adenosine 5'-triphosphate into Golgi vesicles from rat liver and mammary gland. J Biol Chem. 1989 Mar 25;264(9):5233–5240. [PubMed] [Google Scholar]
  7. Chege N. W., Pfeffer S. R. Compartmentation of the Golgi complex: brefeldin-A distinguishes trans-Golgi cisternae from the trans-Golgi network. J Cell Biol. 1990 Sep;111(3):893–899. doi: 10.1083/jcb.111.3.893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Crimaudo C., Hortsch M., Gausepohl H., Meyer D. I. Human ribophorins I and II: the primary structure and membrane topology of two highly conserved rough endoplasmic reticulum-specific glycoproteins. EMBO J. 1987 Jan;6(1):75–82. doi: 10.1002/j.1460-2075.1987.tb04721.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cummings R. D., Kornfeld S., Schneider W. J., Hobgood K. K., Tolleshaug H., Brown M. S., Goldstein J. L. Biosynthesis of N- and O-linked oligosaccharides of the low density lipoprotein receptor. J Biol Chem. 1983 Dec 25;258(24):15261–15273. [PubMed] [Google Scholar]
  10. De Lemos-Chiarandini C., Alvarez F., Bernard O., Homberg J. C., Kreibich G. Anti-liver-kidney microsome antibody is a marker for the rat hepatocyte endoplasmic reticulum. Hepatology. 1987 May-Jun;7(3):468–475. doi: 10.1002/hep.1840070310. [DOI] [PubMed] [Google Scholar]
  11. Doms R. W., Russ G., Yewdell J. W. Brefeldin A redistributes resident and itinerant Golgi proteins to the endoplasmic reticulum. J Cell Biol. 1989 Jul;109(1):61–72. doi: 10.1083/jcb.109.1.61. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Donaldson J. G., Lippincott-Schwartz J., Klausner R. D. Guanine nucleotides modulate the effects of brefeldin A in semipermeable cells: regulation of the association of a 110-kD peripheral membrane protein with the Golgi apparatus. J Cell Biol. 1991 Feb;112(4):579–588. doi: 10.1083/jcb.112.4.579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Elhammer A., Kornfeld S. Two enzymes involved in the synthesis of O-linked oligosaccharides are localized on membranes of different densities in mouse lymphoma BW5147 cells. J Cell Biol. 1984 Jul;99(1 Pt 1):327–331. doi: 10.1083/jcb.99.1.327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fujiwara T., Oda K., Yokota S., Takatsuki A., Ikehara Y. Brefeldin A causes disassembly of the Golgi complex and accumulation of secretory proteins in the endoplasmic reticulum. J Biol Chem. 1988 Dec 5;263(34):18545–18552. [PubMed] [Google Scholar]
  15. Griffiths G., Brands R., Burke B., Louvard D., Warren G. Viral membrane proteins acquire galactose in trans Golgi cisternae during intracellular transport. J Cell Biol. 1982 Dec;95(3):781–792. doi: 10.1083/jcb.95.3.781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Harnik-Ort V., Prakash K., Marcantonio E., Colman D. R., Rosenfeld M. G., Adesnik M., Sabatini D. D., Kreibich G. Isolation and characterization of cDNA clones for rat ribophorin I: complete coding sequence and in vitro synthesis and insertion of the encoded product into endoplasmic reticulum membranes. J Cell Biol. 1987 Apr;104(4):855–863. doi: 10.1083/jcb.104.4.855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Huttner W. B. Tyrosine sulfation and the secretory pathway. Annu Rev Physiol. 1988;50:363–376. doi: 10.1146/annurev.ph.50.030188.002051. [DOI] [PubMed] [Google Scholar]
  18. Jackson M. R., Nilsson T., Peterson P. A. Identification of a consensus motif for retention of transmembrane proteins in the endoplasmic reticulum. EMBO J. 1990 Oct;9(10):3153–3162. doi: 10.1002/j.1460-2075.1990.tb07513.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jamieson J. D., Palade G. E. Intracellular transport of secretory proteins in the pancreatic exocrine cell. I. Role of the peripheral elements of the Golgi complex. J Cell Biol. 1967 Aug;34(2):577–596. doi: 10.1083/jcb.34.2.577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kaufman R. J. Vectors used for expression in mammalian cells. Methods Enzymol. 1990;185:487–511. doi: 10.1016/0076-6879(90)85041-l. [DOI] [PubMed] [Google Scholar]
  21. Keller G. A., Tokuyasu K. T., Dutton A. H., Singer S. J. An improved procedure for immunoelectron microscopy: ultrathin plastic embedding of immunolabeled ultrathin frozen sections. Proc Natl Acad Sci U S A. 1984 Sep;81(18):5744–5747. doi: 10.1073/pnas.81.18.5744. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Kreibich G., Freienstein C. M., Pereyra B. N., Ulrich B. L., Sabatini D. D. Proteins of rough microsomal membranes related to ribosome binding. II. Cross-linking of bound ribosomes to specific membrane proteins exposed at the binding sites. J Cell Biol. 1978 May;77(2):488–506. doi: 10.1083/jcb.77.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kreibich G., Marcantonio E. E., Sabatini D. D. Ribophorins I and II: membrane proteins characteristic of the rough endoplasmic reticulum. Methods Enzymol. 1983;96:520–530. doi: 10.1016/s0076-6879(83)96045-7. [DOI] [PubMed] [Google Scholar]
  25. Kreibich G., Ulrich B. L., Sabatini D. D. Proteins of rough microsomal membranes related to ribosome binding. I. Identification of ribophorins I and II, membrane proteins characteristics of rough microsomes. J Cell Biol. 1978 May;77(2):464–487. doi: 10.1083/jcb.77.2.464. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lippincott-Schwartz J., Donaldson J. G., Schweizer A., Berger E. G., Hauri H. P., Yuan L. C., Klausner R. D. Microtubule-dependent retrograde transport of proteins into the ER in the presence of brefeldin A suggests an ER recycling pathway. Cell. 1990 Mar 9;60(5):821–836. doi: 10.1016/0092-8674(90)90096-w. [DOI] [PubMed] [Google Scholar]
  27. Lippincott-Schwartz J., Yuan L. C., Bonifacino J. S., Klausner R. D. Rapid redistribution of Golgi proteins into the ER in cells treated with brefeldin A: evidence for membrane cycling from Golgi to ER. Cell. 1989 Mar 10;56(5):801–813. doi: 10.1016/0092-8674(89)90685-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Marcantonio E. E., Amar-Costesec A., Kreibich G. Segregation of the polypeptide translocation apparatus to regions of the endoplasmic reticulum containing ribophorins and ribosomes. II. Rat liver microsomal subfractions contain equimolar amounts of ribophorins and ribosomes. J Cell Biol. 1984 Dec;99(6):2254–2259. doi: 10.1083/jcb.99.6.2254. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Nilsson T., Jackson M., Peterson P. A. Short cytoplasmic sequences serve as retention signals for transmembrane proteins in the endoplasmic reticulum. Cell. 1989 Aug 25;58(4):707–718. doi: 10.1016/0092-8674(89)90105-0. [DOI] [PubMed] [Google Scholar]
  30. Pelham H. R. Control of protein exit from the endoplasmic reticulum. Annu Rev Cell Biol. 1989;5:1–23. doi: 10.1146/annurev.cb.05.110189.000245. [DOI] [PubMed] [Google Scholar]
  31. Pirozzi G., Zhou Z. M., D'Eustachio P., Sabatini D. D., Kreibich G. Rat ribophorin II: molecular cloning and chromosomal localization of a highly conserved transmembrane glycoprotein of the rough endoplasmic reticulum. Biochem Biophys Res Commun. 1991 May 15;176(3):1482–1486. doi: 10.1016/0006-291x(91)90454-f. [DOI] [PubMed] [Google Scholar]
  32. Päbo S., Bhat B. M., Wold W. S., Peterson P. A. A short sequence in the COOH-terminus makes an adenovirus membrane glycoprotein a resident of the endoplasmic reticulum. Cell. 1987 Jul 17;50(2):311–317. doi: 10.1016/0092-8674(87)90226-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rodriguez Boulan E., Kreibich G., Sabatini D. D. Spatial orientation of glycoproteins in membranes of rat liver rough microsomes. I. Localization of lectin-binding sites in microsomal membranes. J Cell Biol. 1978 Sep;78(3):874–893. doi: 10.1083/jcb.78.3.874. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rodriguez Boulan E., Sabatini D. D., Pereyra B. N., Kreibich G. Spatial orientation of glycoproteins in membranes of rat liver rough microsomes. II. Transmembrane disposition and characterization of glycoproteins. J Cell Biol. 1978 Sep;78(3):894–909. doi: 10.1083/jcb.78.3.894. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Rosenfeld M. G., Marcantonio E. E., Hakimi J., Ort V. M., Atkinson P. H., Sabatini D., Kreibich G. Biosynthesis and processing of ribophorins in the endoplasmic reticulum. J Cell Biol. 1984 Sep;99(3):1076–1082. doi: 10.1083/jcb.99.3.1076. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Tamura G., Ando K., Suzuki S., Takatsuki A., Arima K. Antiviral activity of brefeldin A and verrucarin A. J Antibiot (Tokyo) 1968 Feb;21(2):160–161. doi: 10.7164/antibiotics.21.160. [DOI] [PubMed] [Google Scholar]
  37. Tartakoff A. M., Vassalli P. Lectin-binding sites as markers of Golgi subcompartments: proximal-to-distal maturation of oligosaccharides. J Cell Biol. 1983 Oct;97(4):1243–1248. doi: 10.1083/jcb.97.4.1243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Tokuyasu K. T. Immunochemistry on ultrathin frozen sections. Histochem J. 1980 Jul;12(4):381–403. doi: 10.1007/BF01011956. [DOI] [PubMed] [Google Scholar]
  39. Tooze S. A., Tooze J., Warren G. Site of addition of N-acetyl-galactosamine to the E1 glycoprotein of mouse hepatitis virus-A59. J Cell Biol. 1988 May;106(5):1475–1487. doi: 10.1083/jcb.106.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Tsao Y. S., Ivessa N. E., Adesnik M., Sabatini D. D., Kreibich G. Carboxy terminally truncated forms of ribophorin I are degraded in pre-Golgi compartments by a calcium-dependent process. J Cell Biol. 1992 Jan;116(1):57–67. doi: 10.1083/jcb.116.1.57. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Ulmer J. B., Palade G. E. Targeting and processing of glycophorins in murine erythroleukemia cells: use of brefeldin A as a perturbant of intracellular traffic. Proc Natl Acad Sci U S A. 1989 Sep;86(18):6992–6996. doi: 10.1073/pnas.86.18.6992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Umemoto J., Bhavanandan V. P., Davidson E. A. Purification and properties of an endo-alpha-N-acetyl-D-galactosaminidase from Diplococcus pneumoniae. J Biol Chem. 1977 Dec 10;252(23):8609–8614. [PubMed] [Google Scholar]
  43. Wiedmann M., Kurzchalia T. V., Hartmann E., Rapoport T. A. A signal sequence receptor in the endoplasmic reticulum membrane. 1987 Aug 27-Sep 2Nature. 328(6133):830–833. doi: 10.1038/328830a0. [DOI] [PubMed] [Google Scholar]
  44. Yamamoto A., Masaki R., Tashiro Y. Is cytochrome P-450 transported from the endoplasmic reticulum to the Golgi apparatus in rat hepatocytes? J Cell Biol. 1985 Nov;101(5 Pt 1):1733–1740. doi: 10.1083/jcb.101.5.1733. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Yu Y. H., Sabatini D. D., Kreibich G. Antiribophorin antibodies inhibit the targeting to the ER membrane of ribosomes containing nascent secretory polypeptides. J Cell Biol. 1990 Oct;111(4):1335–1342. doi: 10.1083/jcb.111.4.1335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Yu Y. H., Zhang Y. Y., Sabatini D. D., Kreibich G. Reconstitution of translocation-competent membrane vesicles from detergent-solubilized dog pancreas rough microsomes. Proc Natl Acad Sci U S A. 1989 Dec;86(24):9931–9935. doi: 10.1073/pnas.86.24.9931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Yuan L., Barriocanal J. G., Bonifacino J. S., Sandoval I. V. Two integral membrane proteins located in the cis-middle and trans-part of the Golgi system acquire sialylated N-linked carbohydrates and display different turnovers and sensitivity to cAMP-dependent phosphorylation. J Cell Biol. 1987 Jul;105(1):215–227. doi: 10.1083/jcb.105.1.215. [DOI] [PMC free article] [PubMed] [Google Scholar]

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