Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1983 Aug 1;97(2):340–350. doi: 10.1083/jcb.97.2.340

Alteration of membrane barrier in stripped rough microsomes from rat liver on incubation with GTP: its relevance to the stimulation by this nucleotide of the dolichol pathway for protein glycosylation

PMCID: PMC2112521  PMID: 6309858

Abstract

The membrane barrier of stripped rough microsomes from rat liver is markedly altered on incubation with GTP at 37 degrees C: after 30 min the structure-linked latency of mannose-6-phosphatase was considerably reduced, and esterase and nucleoside diphosphatase were partly released into the suspension medium. This phenomenon was already maximal with 30 microM GTP and was specific for this nucleotide. Similar conditions enhance the dolichol-mediated glycosylation of protein in microsomes incubated with uridine diphosphate N-acetylglucosamine and guanosine diphosphate mannose (Godelaine, D., H. Beaufay, M. Wibo, and A. Amar- Costesec, 1979, Eur. J. Biochem., 96:17-26; Godelaine, D., H. Beaufay, and M. Wibo, 1979, Eur. J. Biochem., 96:27-34). The GTP-induced permeability and glycosylation activities evolved in parallel in rough microsomes subjected to various treatments to detach the ribosomes and were maximal after removal of congruent to 60% of the RNA. In addition, GTP had no effect of this type in smooth microsome subfractions. Triton X-100, in spite of complex inhibitory effects on glycosylation reactions, mimicked the action of GTP by increasing the amount of microsomal dolichylphosphate that reacts with uridine diphosphate N- acetylglucosamine and by enhancing synthesis of dolichylpyrophosphoryl- chitobiose at concentrations greater than 2 mg/ml. Thus, GTP may activate dolichol-mediated glycosylation reactions in stripped microsomes by lowering the permeability barrier that prevents access of sugar nucleotides to the inner aspect of the membrane. The genuine role of GTP in the functioning of the endoplasmic reticulum membrane in situ remains unknown. Because GTP seems to act only on rough microsomes, we hypothesize that this role is somehow related to biosynthesis of protein by the rough endoplasmic reticulum.

Full Text

The Full Text of this article is available as a PDF (1.2 MB).

Selected References

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

  1. Beaufay H., Amar-Costesec A., Feytmans E., Thinès-Sempoux D., Wibo M., Robbi M., Berthet J. Analytical study of microsomes and isolated subcellular membranes from rat liver. I. Biochemical methods. J Cell Biol. 1974 Apr;61(1):188–200. doi: 10.1083/jcb.61.1.188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bergman A., Dallner G. Incorporation of N-acetylglucosamine from UDP-N-acetylglucosamine into proteins and lipid intermediates in microsomal and Golgi membranes from rat liver. Biochim Biophys Acta. 1978 Sep 11;512(1):123–135. doi: 10.1016/0005-2736(78)90223-7. [DOI] [PubMed] [Google Scholar]
  3. Coates S. W., Gurney T., Jr, Sommers L. W., Yeh M., Hirschberg C. B. Subcellular localization of sugar nucleotide synthetases. J Biol Chem. 1980 Oct 10;255(19):9225–9229. [PubMed] [Google Scholar]
  4. Czichi U., Lennarz W. J. Localization of the enzyme system for glycosylation of proteins via the lipid-linked pathway in rough endoplasmic reticulum. J Biol Chem. 1977 Nov 25;252(22):7901–7904. [PubMed] [Google Scholar]
  5. De Duve C. The separation and characterization of subcellular particles. Harvey Lect. 1965;59:49–87. [PubMed] [Google Scholar]
  6. Eggens I., Dallner G. Transverse localization of glycosyl transferases in rough and smooth microsomes. FEBS Lett. 1980 Dec 29;122(2):247–250. doi: 10.1016/0014-5793(80)80449-2. [DOI] [PubMed] [Google Scholar]
  7. FLECK A., MUNRO H. N. The precision of ultraviolet absorption measurements in the Schmidt-Thannhauser procedure for nucleic acid estimation. Biochim Biophys Acta. 1962 May 14;55:571–583. doi: 10.1016/0006-3002(62)90836-3. [DOI] [PubMed] [Google Scholar]
  8. Godelaine D., Beaufay H. The dolichol pathway of protein glycosylation in rat liver. Evidence that GTP promotes transformation of endogenous dolichyl phosphate into dolichylpyrophosphoryl-N-acetylglucosamine in stripped rough microsomes. Eur J Biochem. 1983 Apr 5;131(3):667–670. doi: 10.1111/j.1432-1033.1983.tb07315.x. [DOI] [PubMed] [Google Scholar]
  9. Godelaine D., Beaufay H., Wibo M., Amar-Costesec A. The dolichol pathway of protein glycosylation in rat liver. Stimulation by GTP of the incorporation of N-acetylglucosamine in endogenous lipids and proteins of rough microsomes treated with pyrophosphate. Eur J Biochem. 1979 May 2;96(1):17–26. doi: 10.1111/j.1432-1033.1979.tb13008.x. [DOI] [PubMed] [Google Scholar]
  10. Godelaine D., Beaufay H., Wibo M. Incorporation of N-acetylglucosamine into endogenous acceptors of rough microsomes from rat liver: stimulation by GTP after treatment with pyrophosphate. Proc Natl Acad Sci U S A. 1977 Mar;74(3):1095–1099. doi: 10.1073/pnas.74.3.1095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Godelaine D., Beaufay H., Wibo M. The dolichol pathway of protein glycosylation in rat liver. Incorporation of mannose into endogenous lipids and proteins of rough microsomes. Eur J Biochem. 1979 May 2;96(1):27–34. doi: 10.1111/j.1432-1033.1979.tb13009.x. [DOI] [PubMed] [Google Scholar]
  12. Hanover J. A., Lennarz W. J. N-Linked glycoprotein assembly. Evidence that oligosaccharide attachment occurs within the lumen of the endoplasmic reticulum. J Biol Chem. 1980 Apr 25;255(8):3600–3604. [PubMed] [Google Scholar]
  13. Hanover J. A., Lennarz W. J. The topological orientation of N,N'-diacetylchitobiosylpyrophosphoryldolichol in artificial and natural membranes. J Biol Chem. 1979 Sep 25;254(18):9237–9246. [PubMed] [Google Scholar]
  14. Hanover J. A., Lennarz W. J. Transmembrane assembly of N-linked glycoproteins. Studies on the topology of saccharide synthesis. J Biol Chem. 1982 Mar 25;257(6):2787–2794. [PubMed] [Google Scholar]
  15. Hanover J. A., Lennarz W. J. Transmembrane assembly of membrane and secretory glycoproteins. Arch Biochem Biophys. 1981 Oct 1;211(1):1–19. doi: 10.1016/0003-9861(81)90423-9. [DOI] [PubMed] [Google Scholar]
  16. Katz F. N., Rothman J. E., Lingappa V. R., Blobel G., Lodish H. F. Membrane assembly in vitro: synthesis, glycosylation, and asymmetric insertion of a transmembrane protein. Proc Natl Acad Sci U S A. 1977 Aug;74(8):3278–3282. doi: 10.1073/pnas.74.8.3278. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kreibich G., Ojakian G., Rodriguez-Boulan E., Sabatini D. D. Recovery of ribophorins and ribosomes in "inverted rough" vesicles derived from rat liver rough microsomes. J Cell Biol. 1982 Apr;93(1):111–121. doi: 10.1083/jcb.93.1.111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. LIttle J. S., Thiers D. R., Widnell C. C. Latency of inosine-5'-diphosphatase in microsomes isolated from rat liver. J Biol Chem. 1976 Dec 25;251(24):7821–7825. [PubMed] [Google Scholar]
  19. 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]
  20. Lehle L., Tanner W. The specific site of tunicamycin inhibition in the formation of dolichol-bound N-acetylglucosamine derivatives. FEBS Lett. 1976 Nov 15;72(1):167–170. doi: 10.1016/0014-5793(76)80922-2. [DOI] [PubMed] [Google Scholar]
  21. Lingappa V. R., Lingappa J. R., Prasad R., Ebner K. E., Blobel G. Coupled cell-free synthesis, segregation, and core glycosylation of a secretory protein. Proc Natl Acad Sci U S A. 1978 May;75(5):2338–2342. doi: 10.1073/pnas.75.5.2338. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lodish H. F., Braell W. A., Schwartz A. L., Strous G. J., Zilberstein A. Synthesis and assembly of membrane and organelle proteins. Int Rev Cytol Suppl. 1981;12:247–307. doi: 10.1016/b978-0-12-364373-5.50016-0. [DOI] [PubMed] [Google Scholar]
  23. Lucas J. J., Waechter J., Lennarz W. J. The participation of lipid-linked oligosaccharide in synthesis of membrane glycoproteins. J Biol Chem. 1975 Mar 25;250(6):1992–2002. [PubMed] [Google Scholar]
  24. Nilsson O. S., De Tomás M. E., Peterson E., Bergman A., Dallner G., Hemming F. W. Mannosylation of endogenous proteins of rough and smooth endoplasmic reticulum and of Golgi membranes. Eur J Biochem. 1978 Sep 1;89(2):619–628. doi: 10.1111/j.1432-1033.1978.tb12566.x. [DOI] [PubMed] [Google Scholar]
  25. Paiement J., Beaufay H., Godelaine D. Coalescence of microsomal vesicles from rat liver: a phenomenon occurring in parallel with enhancement of the glycosylation activity during incubation of stripped rough microsomes with GTP. J Cell Biol. 1980 Jul;86(1):29–37. doi: 10.1083/jcb.86.1.29. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Parodi A. J., Leloir L. F. The role of lipid intermediates in the glycosylation of proteins in the eucaryotic cell. Biochim Biophys Acta. 1979 Apr 23;559(1):1–37. doi: 10.1016/0304-4157(79)90006-6. [DOI] [PubMed] [Google Scholar]
  27. Ravoet A. M., Amar-Costesec A., Godelaine D., Beaufay H. Quantitative assay and subcellular distribution of enzymes acting on dolichyl phosphate in rat liver. J Cell Biol. 1981 Dec;91(3 Pt 1):679–688. doi: 10.1083/jcb.91.3.679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Sabatini D. D., Kreibich G., Morimoto T., Adesnik M. Mechanisms for the incorporation of proteins in membranes and organelles. J Cell Biol. 1982 Jan;92(1):1–22. doi: 10.1083/jcb.92.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Snider M. D., Robbins P. W. Transmembrane organization of protein glycosylation. Mature oligosaccharide-lipid is located on the luminal side of microsomes from Chinese hamster ovary cells. J Biol Chem. 1982 Jun 25;257(12):6796–6801. [PubMed] [Google Scholar]
  31. Snider M. D., Sultzman L. A., Robbins P. W. Transmembrane location of oligosaccharide-lipid synthesis in microsomal vesicles. Cell. 1980 Sep;21(2):385–392. doi: 10.1016/0092-8674(80)90475-4. [DOI] [PubMed] [Google Scholar]
  32. Snider M. D. Transport of sugars during glycoprotein synthesis. Nature. 1982 Jul 8;298(5870):117–118. doi: 10.1038/298117a0. [DOI] [PubMed] [Google Scholar]
  33. Waechter C. J., Lucas J. J., Lennarz W. J. Membrane glycoproteins. I. Enzymatic synthesis of mannosyl phosphoryl polyisoprenol and its role as a mannosyl donor in glycoprotein synthesis. J Biol Chem. 1973 Nov 10;248(21):7570–7579. [PubMed] [Google Scholar]
  34. Wibo M., Thinès-Sempoux D., Amar-Costesec A., Beaufay H., Godelaine D. Analytical study of microsomes and isolated subcellular membranes from rat liver VIII. Subfractionation of preparations enriched with plasma membranes, outer mitochondrial membranes, or Golgi complex membranes. J Cell Biol. 1981 Jun;89(3):456–474. doi: 10.1083/jcb.89.3.456. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES