Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1982 Apr 1;93(1):111–121. doi: 10.1083/jcb.93.1.111

Recovery of ribophorins and ribosomes in "inverted rough" vesicles derived from rat liver rough microsomes

PMCID: PMC2112111  PMID: 7068749

Abstract

Treatment of rat liver rough microsomes (3.5 mg of protein/ml) with sublytical concentrations (0.08%) of the neutral detergent Triton X-100 caused a lateral displacement of bound ribosomes and the formation of ribosomal aggregates on the microsomal surface. At slightly higher detergent concentrations (0.12-0.16%) membrane areas bearing ribosomal aggregates invaginated into the microsomal lumen and separated from the rest of the membrane. Two distinct classes of vesicles could be isolated by density gradient centrifugation from microsomes treated with 0.16% Triton X-100: one with ribosomes bound to the inner membrane surfaces ("inverted rough" vesicles) and another with no ribosomes attached to the membranes. Analysis of the fractions showed that approximately 30% of the phospholipids and 20-30% of the total membrane protein were released from the membranes by this treatment. Labeling with avidin-ferritin conjugates demonstrated that concanavalin A binding sites, which in native rough microsomes are found in the luminal face of the membranes, were present on the outer surface of the inverted rough vesicles. Freeze-fracture electron microscopy showed that both fracture faces had similar concentrations of intramembrane particles. SDS PAGE analysis of the two vesicle subfractions demonstrated that, of all the integral microsomal membrane proteins, only ribophorins I and II were found exclusively in the inverted rough vesicles bearing ribosomes. These observations are consistent with the proposal that ribophorins are associated with the ribosomal binding sites characteristic of rough microsomal membranes.

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. AMES B. N., DUBIN D. T. The role of polyamines in the neutralization of bacteriophage deoxyribonucleic acid. J Biol Chem. 1960 Mar;235:769–775. [PubMed] [Google Scholar]
  2. Adelman M. R., Blobel G., Sabatini D. D. An improved cell fractionation procedure for the preparation of rat liver membrane-bound ribosomes. J Cell Biol. 1973 Jan;56(1):191–205. doi: 10.1083/jcb.56.1.191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Blobel G., Dobberstein B. Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma. J Cell Biol. 1975 Dec;67(3):835–851. doi: 10.1083/jcb.67.3.835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Blobel G., Dobberstein B. Transfer of proteins across membranes. II. Reconstitution of functional rough microsomes from heterologous components. J Cell Biol. 1975 Dec;67(3):852–862. doi: 10.1083/jcb.67.3.852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. De Lorenzo F., Goldberger R. F., Steers E., Jr, Givol D., Anfinsen B. Purification and properties of an enzyme from beef liver which catalyzes sulfhydryl-disulfide interchange in proteins. J Biol Chem. 1966 Apr 10;241(7):1562–1567. [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. FOLCH J., LEES M., SLOANE STANLEY G. H. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem. 1957 May;226(1):497–509. [PubMed] [Google Scholar]
  9. 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]
  10. 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]
  11. Jackson R. C., Blobel G. Post-translational cleavage of presecretory proteins with an extract of rough microsomes from dog pancreas containing signal peptidase activity. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5598–5602. doi: 10.1073/pnas.74.12.5598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kreibich G., Czakó-Graham M., Grebenau R. C., Sabatini D. D. Functional and structural characteristics of endoplasmic reticulum proteins associated with ribosome binding sites. Ann N Y Acad Sci. 1980;343:17–33. doi: 10.1111/j.1749-6632.1980.tb47239.x. [DOI] [PubMed] [Google Scholar]
  13. Kreibich G., Czakó-Graham M., Grebenau R., Mok W., Rodriguez-Boulan E., Sabatini D. D. Characterization of the ribosomal binding site in rat liver rough microsomes: ribophorins I and II, two integral membrane proteins related to ribosome binding. J Supramol Struct. 1978;8(3):279–302. doi: 10.1002/jss.400080307. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. Kreibich G., Sabatini D. D. Procedure for the selective release of content from microsomal vesicles without membrane disassembly. Methods Enzymol. 1974;31:215–225. doi: 10.1016/0076-6879(74)31023-3. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. Kruppa J., Sabatini D. D. Release of poly A(+) messenger RNA from rat liver rough microsomes upon disassembly of bound polysomes. J Cell Biol. 1977 Aug;74(2):414–427. doi: 10.1083/jcb.74.2.414. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. OMURA T., SATO R. THE CARBON MONOXIDE-BINDING PIGMENT OF LIVER MICROSOMES. I. EVIDENCE FOR ITS HEMOPROTEIN NATURE. J Biol Chem. 1964 Jul;239:2370–2378. [PubMed] [Google Scholar]
  22. Ojakian G. K., Kreibich G., Sabatini D. D. Mobility of ribosomes bound to microsomal membranes. A freeze-etch and thin-section electron microscope study of the structure and fluidity of the rough endoplasmic reticulum. J Cell Biol. 1977 Mar;72(3):530–551. doi: 10.1083/jcb.72.3.530. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Omura T., Siekevitz P., Palade G. E. Turnover of constituents of the endoplasmic reticulum membranes of rat hepatocytes. J Biol Chem. 1967 May 25;242(10):2389–2396. [PubMed] [Google Scholar]
  24. 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]
  25. Peterkofsky B., Assad R. Localization of chick embryo limb bone microsomal lysyl hydroxylase at intracisternal and intramembrane sites. J Biol Chem. 1979 Jun 10;254(11):4714–4720. [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. 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]
  29. Schmidt M. F., Schlesinger M. J. Relation of fatty acid attachment to the translation and maturation of vesicular stomatitis and Sindbis virus membrane glycoproteins. J Biol Chem. 1980 Apr 25;255(8):3334–3339. [PubMed] [Google Scholar]
  30. Sheetz M. P., Singer S. J. Biological membranes as bilayer couples. A molecular mechanism of drug-erythrocyte interactions. Proc Natl Acad Sci U S A. 1974 Nov;71(11):4457–4461. doi: 10.1073/pnas.71.11.4457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Steiner D. F., Quinn P. S., Chan S. J., Marsh J., Tager H. S. Processing mechanisms in the biosynthesis of proteins. Ann N Y Acad Sci. 1980;343:1–16. doi: 10.1111/j.1749-6632.1980.tb47238.x. [DOI] [PubMed] [Google Scholar]
  32. Studier F. W. Analysis of bacteriophage T7 early RNAs and proteins on slab gels. J Mol Biol. 1973 Sep 15;79(2):237–248. doi: 10.1016/0022-2836(73)90003-x. [DOI] [PubMed] [Google Scholar]
  33. TASHIRO Y., SIEKEVITZ P. ULTRACENTRIFUGAL STUDIES ON THE DISSOCIATION OF HEPATIC RIBOSOMES. J Mol Biol. 1965 Feb;11:149–165. doi: 10.1016/s0022-2836(65)80047-x. [DOI] [PubMed] [Google Scholar]

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

RESOURCES