Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1978 Sep 1;78(3):874–893. doi: 10.1083/jcb.78.3.874

Spatial orientation of glycoproteins in membranes of rat liver rough microsomes. I. Localization of lectin-binding sites in microsomal membranes

PMCID: PMC2110186  PMID: 701363

Abstract

Carbohydrate-containing structures in rat liver rough microsomes (RM) were localized and characterized using iodinated lectins of defined specificity. Binding of [125I]Con A increased six- to sevenfold in the presence of low DOC (0.04--0.05%) which opens the vesicles and allows the penetration of the lectins. On the other hand, binding of [125I]WGA and [125I]RCA increased only slightly when the microsomal vesicles were opened by DOC. Sites available in the intact microsomal fraction had an affinity for [125I]Con A 14 times higher than sites for lectin binding which were exposed by the detergent treatment. Lectin-binding sites in RM were also localized electron microscopically with lectins covalently bound to biotin, which, in turn, were visualized after their reaction with ferritin-avidin (F-Av) markers. Using this method, it was demonstrated that in untreated RM samples, binding sites for lectins are not present on the cytoplasmic face of the microsomal vesicles, even after removal of ribosomes by treatment with high salt buffer and puromycin, but are located on smooth membranes which contaminate the rough microsomal fraction. Combining this technique with procedures which render the interior of the microsomal vesicles accessible to lectins and remove luminal proteins, it was found that RM membranes contain binding sites for Con A and for Lens culinaris agglutinin (LCA) located exclusively on the cisternal face of the membrane. No sites for WGA, RCA, soybean (SBA) and Lotus tetragonobulus (LTA) agglutinins were detected on either the cytoplasmic or the luminal faces of the rough microsomes. These observations demonstrate that: (a) sugar moieties of microsomal glycoproteins are exposed only on the luminal surface of the membranes and (b) microsomal membrane glycoproteins have incomplete carbohydrate chains without the characteristic terminal trisaccharides N-acetylglucosamine comes from galactose comes from sialic acid or fucose present in most glycoproteins secreted by the liver. The orientation and composition of the carbohydrate chains in microsomal glycoproteins indicate that the passage of these glycoproteins through the Golgi apparatus, followed by their return to the endoplasmic reticulum, is not required for their biogenesis and insertion into the endoplasmic reticulum (ER) membrane.

Full Text

The Full Text of this article is available as a PDF (4.5 MB).

Selected References

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

  1. Adelman M. R., Sabatini D. D., Blobel G. Ribosome-membrane interaction. Nondestructive disassembly of rat liver rough microsomes into ribosomal and membranous components. J Cell Biol. 1973 Jan;56(1):206–229. doi: 10.1083/jcb.56.1.206. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Agrawal B. B., Goldstein I. J. Physical and chemical characterization of concanavalin A, the hemagglutinin from jack bean (Canavalia ensiformis). Biochim Biophys Acta. 1967 Feb 21;133(2):376–379. doi: 10.1016/0005-2795(67)90081-5. [DOI] [PubMed] [Google Scholar]
  3. Autuori F., Svensson H., Dallner G. Biogenesis of microsomal membrane glycoproteins in rat liver. I. Presence of glycoproteins in microsomes and cytosol. J Cell Biol. 1975 Dec;67(3):687–699. doi: 10.1083/jcb.67.3.687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Autuori F., Svensson H., Dallner G. Biogenesis of microsomal membrane glycoproteins in rat liver. II. Purification of soluble glycoproteins and their incorporation into microsomal membranes. J Cell Biol. 1975 Dec;67(3):700–714. doi: 10.1083/jcb.67.3.700. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Behrens N. H., Carminatti H., Staneloni R. J., Leloir L. F., Cantarella A. I. Formation of lipid-bound oligosaccharides containing mannose. Their role in glycoprotein synthesis. Proc Natl Acad Sci U S A. 1973 Dec;70(12):3390–3394. doi: 10.1073/pnas.70.12.3390. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Borgese N., Mok W., Kreibich G., Sabatini D. D. Ribosomal-membrane interaction: in vitro binding of ribosomes to microsomal membranes. J Mol Biol. 1974 Sep 25;88(3):559–580. doi: 10.1016/0022-2836(74)90408-2. [DOI] [PubMed] [Google Scholar]
  7. Burger M. M., Goldberg A. R. Identification of a tumor-specific determinant on neoplastic cell surfaces. Proc Natl Acad Sci U S A. 1967 Feb;57(2):359–366. doi: 10.1073/pnas.57.2.359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chandramouli V., Williams S., Marshall J. S., Carter J. R., Jr Cell surface changes in diabetic rats. Studies of lectin binding to liver cell plasma membranes. Biochim Biophys Acta. 1977 Feb 14;465(1):19–33. doi: 10.1016/0005-2736(77)90352-2. [DOI] [PubMed] [Google Scholar]
  9. Edelman G. M., Millette C. F. Molecular probes of spermatozoan structures. Proc Natl Acad Sci U S A. 1971 Oct;68(10):2436–2440. doi: 10.1073/pnas.68.10.2436. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Elhammer A., Svensson H., Autuori F., Dallner G. Biogenesis of microsomal membrane glycoproteins in rat liver. III. Release of glycoproteins from the Golgi fraction and their transfer to microsomal membranes. J Cell Biol. 1975 Dec;67(3):715–724. doi: 10.1083/jcb.67.3.715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. FOSTER J. F., SOGAMI M., PETERSEN H. A., LEONARD W. J., Jr THE MICROHETEROGENEITY OF PLASMA ALBUMINS. I. CRITICAL EVIDENCE FOR AND DESCRIPTION OF THE MICROHETEROGENEITY MODEL. J Biol Chem. 1965 Jun;240:2495–2502. [PubMed] [Google Scholar]
  12. Fleischer B., Fleischer S. Preparation and characterization of golgi membranes from rat liver. Biochim Biophys Acta. 1970 Dec 1;219(2):301–319. doi: 10.1016/0005-2736(70)90209-9. [DOI] [PubMed] [Google Scholar]
  13. Greenaway P. J., LeVine D. Binding of N-acetyl-neuraminic acid by wheat-germ agglutinin. Nat New Biol. 1973 Feb 7;241(110):191–192. doi: 10.1038/newbio241191a0. [DOI] [PubMed] [Google Scholar]
  14. Guérin C., Zachowski A., Prigent B., Paraf A., Dunia I., Diawara M. A., Benedetti E. L. Correlation between the mobility of inner plasma membrane structure and agglutination by concanavalin A in two cell lines of MOPC 173 plasmocytoma cells. Proc Natl Acad Sci U S A. 1974 Jan;71(1):114–117. doi: 10.1073/pnas.71.1.114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. HELGELAND L. INCORPORATION OF RADIOACTIVE GLUCOSAMINE INTO SUBMICROSOMAL FRACTIONS ISOLATED FROM RAT LIVER. Biochim Biophys Acta. 1965 Mar 1;101:106–112. doi: 10.1016/0926-6534(65)90035-7. [DOI] [PubMed] [Google Scholar]
  16. Hallinan T., Murty C. N., Grant J. H. The exclusive function of reticulum bound ribosomes in glycoprotein biosynthesis. Life Sci. 1968 Mar 15;7(6):225–232. doi: 10.1016/0024-3205(68)90016-7. [DOI] [PubMed] [Google Scholar]
  17. Heitzmann H., Richards F. M. Use of the avidin-biotin complex for specific staining of biological membranes in electron microscopy. Proc Natl Acad Sci U S A. 1974 Sep;71(9):3537–3541. doi: 10.1073/pnas.71.9.3537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hirano H., Parkhouse B., Nicolson G. L., Lennox E. S., Singer S. J. Distribution of saccharide residues on membrane fragments from a myeloma-cell homogenate: its implications for membrane biogenesis. Proc Natl Acad Sci U S A. 1972 Oct;69(10):2945–2949. doi: 10.1073/pnas.69.10.2945. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kawasaki T., Yamashina I. Isolation and characterization of glycopeptides from rat liver nuclear membrane. J Biochem. 1972 Dec;72(6):1517–1525. doi: 10.1093/oxfordjournals.jbchem.a130043. [DOI] [PubMed] [Google Scholar]
  20. Kawasaki T., Yamashina I. Isolation and characterization of glycopeptides from rough and smooth microsomes of rat liver. J Biochem. 1973 Oct;74(4):639–647. doi: 10.1093/oxfordjournals.jbchem.a130288. [DOI] [PubMed] [Google Scholar]
  21. Keenan T. W., Franke W. W., Kartenbeck J. Concanavalin A binding by isolated plasma membranes and endomembranes from liver and mammary gland. FEBS Lett. 1974 Aug 30;44(3):274–278. doi: 10.1016/0014-5793(74)81156-7. [DOI] [PubMed] [Google Scholar]
  22. Knipe D. M., Baltimore D., Lodish H. F. Maturation of viral proteins in cells infected with temperature-sensitive mutants of vesicular stomatitis virus. J Virol. 1977 Mar;21(3):1149–1158. doi: 10.1128/jvi.21.3.1149-1158.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Knipe D. M., Baltimore D., Lodish H. F. Separate pathways of maturation of the major structural proteins of vesicular stomatitis virus. J Virol. 1977 Mar;21(3):1128–1139. doi: 10.1128/jvi.21.3.1128-1139.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Knipe D. M., Lodish H. F., Baltimore D. Localization of two cellular forms of the vesicular stomatitis viral glycoprotein. J Virol. 1977 Mar;21(3):1121–1127. doi: 10.1128/jvi.21.3.1121-1127.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kornfeld R., Ferris C. Interaction of immunoglobulin glycopeptides with concanavalin A. J Biol Chem. 1975 Apr 10;250(7):2614–2619. [PubMed] [Google Scholar]
  26. Kornfeld R., Keller J., Baenziger J., Kornfeld S. The structure of the glycopeptide of human gamma G myeloma proteins. J Biol Chem. 1971 May 25;246(10):3259–3268. [PubMed] [Google Scholar]
  27. Kornfeld R., Kornfeld S. The structure of a phytohemagglutinin receptor site from human erythrocytes. J Biol Chem. 1970 May 25;245(10):2536–2545. [PubMed] [Google Scholar]
  28. Kreibich G., Debey P., Sabatini D. D. Selective release of content from microsomal vesicles without membrane disassembly. I. Permeability changes induced by low detergent concentrations. J Cell Biol. 1973 Aug;58(2):436–462. doi: 10.1083/jcb.58.2.436. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kreibich G., Sabatini D. D. Selective release of content from microsomal vesicles without membrane disassembly. II. Electrophoretic and immunological characterization of microsomal subfractions. J Cell Biol. 1974 Jun;61(3):789–807. doi: 10.1083/jcb.61.3.789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Krusius T., Finne J., Rauvala H. The structural basis of the different affinities of two types of acidic N-glycosidic glycopeptides for concanavalin A--sepharose. FEBS Lett. 1976 Nov 15;72(1):117–120. doi: 10.1016/0014-5793(76)80911-8. [DOI] [PubMed] [Google Scholar]
  31. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  32. Lawford G. R., Schachter H. Biosynthesis of glycoprotein by liver. The incorporation in vivo of 14C-glucosamine into protein-bound hexosamine and sialic acid of rat liver subcellular fractions. J Biol Chem. 1966 Nov 25;241(22):5408–5418. [PubMed] [Google Scholar]
  33. Lennarz W. J. Lipid linked sugars in glycoprotein synthesis. Science. 1975 Jun 6;188(4192):986–991. doi: 10.1126/science.167438. [DOI] [PubMed] [Google Scholar]
  34. Lewis J. A., Tata J. R. A rapidly sedimenting fraction of rat liver endoplasmic reticulum. J Cell Sci. 1973 Sep;13(2):447–459. doi: 10.1242/jcs.13.2.447. [DOI] [PubMed] [Google Scholar]
  35. MELAMED M. D., GREEN N. M. AVIDIN. 2. PURIFICATION AND COMPOSITION. Biochem J. 1963 Dec;89:591–599. doi: 10.1042/bj0890591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. MOLNAR J., ROBINSON G. B., WINZLER R. J. BIOSYNTHESIS OF GLYCOPROTEINS. IV. THE SUBCELLULAR SITES OF INCORPORATION OF GLUCOSAMINE-1-14-C INTO GLYCOPROTEIN RAT LIVER. J Biol Chem. 1965 May;240:1882–1888. [PubMed] [Google Scholar]
  37. Molnar J., Sy D. Attachment of glucosamine to protein at the ribosomal site of rat liver. Biochemistry. 1967 Jul;6(7):1941–1947. doi: 10.1021/bi00859a009. [DOI] [PubMed] [Google Scholar]
  38. Nagata Y., Burger M. M. Wheat germ agglutinin. Molecular characteristics and specificity for sugar binding. J Biol Chem. 1974 May 25;249(10):3116–3122. [PubMed] [Google Scholar]
  39. Negishi M., Fujii-Kuriyama Y., Tashiro Y., Imai Y. Site of biosynthesis of cytochrome P450 in hepatocytes of phenobarbital treated rats. Biochem Biophys Res Commun. 1976 Aug 23;71(4):1153–1160. doi: 10.1016/0006-291x(76)90774-9. [DOI] [PubMed] [Google Scholar]
  40. Negishi M., Sawamura T., Morimoto T., Tashiro Y. Localization of nascent NADPH-cytochrome c reductase in rat liver microsomes. Biochim Biophys Acta. 1975 Jan 13;381(1):215–220. doi: 10.1016/0304-4165(75)90203-2. [DOI] [PubMed] [Google Scholar]
  41. Olsen B. R., Berg R. A., Kishida Y., Prockop D. J. Collagen synthesis: localization of prolyl hydroxylase in tendon cells detected with ferritin-labeled antibodies. Science. 1973 Nov 23;182(4114):825–827. doi: 10.1126/science.182.4114.825. [DOI] [PubMed] [Google Scholar]
  42. Palade G. Intracellular aspects of the process of protein synthesis. Science. 1975 Aug 1;189(4200):347–358. doi: 10.1126/science.1096303. [DOI] [PubMed] [Google Scholar]
  43. Redman C. M., Cherian M. G. The secretory pathways of rat serum glycoproteins and albumin. Localization of newly formed proteins within the endoplasmic reticulum. J Cell Biol. 1972 Feb;52(2):231–245. doi: 10.1083/jcb.52.2.231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Roseman S. The synthesis of complex carbohydrates by multiglycosyltransferase systems and their potential function in intercellular adhesion. Chem Phys Lipids. 1970 Oct;5(1):270–297. doi: 10.1016/0009-3084(70)90024-1. [DOI] [PubMed] [Google Scholar]
  45. SCHICK A. F., SINGER S. J. On the formation of covalent linkages between two protein molecules. J Biol Chem. 1961 Sep;236:2477–2485. [PubMed] [Google Scholar]
  46. SPIRO R. G. Studies on the monosaccharide sequence of the serum glycoprotein fetuin. J Biol Chem. 1962 Mar;237:646–652. [PubMed] [Google Scholar]
  47. Schachter H., Jabbal I., Hudgin R. L., Pinteric L., McGuire E. J., Roseman S. Intracellular localization of liver sugar nucleotide glycoprotein glycosyltransferases in a Golgi-rich fraction. J Biol Chem. 1970 Mar 10;245(5):1090–1100. [PubMed] [Google Scholar]
  48. Thomas D. B., Winzler R. J. Structure of glycoproteins of human erythrocytes. Alkali-stable oligosaccharides. Biochem J. 1971 Aug;124(1):55–59. doi: 10.1042/bj1240055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Toyoshima S., Fukuda M., Osawa T. Chemical nature of the receptor site for various phytomitogens. Biochemistry. 1972 Oct 10;11(21):4000–4005. doi: 10.1021/bi00771a025. [DOI] [PubMed] [Google Scholar]
  50. Wallach D. F., Schmidt-Ullrich R. Low- and high-affinity concanavalin a binding to thymocyte plasma membrane vesicles. J Cell Physiol. 1976 Dec;89(4):771–774. doi: 10.1002/jcp.1040890441. [DOI] [PubMed] [Google Scholar]

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

RESOURCES