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. 1986 Jul 1;103(1):241–253. doi: 10.1083/jcb.103.1.241

Characterization of secretory protein translocation: ribosome-membrane interaction in endoplasmic reticulum

PMCID: PMC2113795  PMID: 3087996

Abstract

Secretory proteins are synthesized on ribosomes bound to the membrane of the endoplasmic reticulum (ER). After the selection of polysomes synthesizing secretory proteins and their direction to the membrane of the ER via signal recognition particle (SRP) and docking protein respectively, the polysomes become bound to the ER membrane via an unknown, protein-mediated mechanism. To identify proteins involved in protein translocation, beyond the (SRP-docking protein-mediated) recognition step, controlled proteolysis was used to functionally inactivate rough microsomes that had previously been depleted of docking protein. As the membranes were treated with increasing levels of protease, they lost their ability to be functionally reconstituted with the active cytoplasmic fragment of docking protein (DPf). This functional inactivation did not correlate with a loss of either signal peptidase activity, nor with the ability of the DPf to reassociate with the membrane. It did correlate, however, with a loss of the ability of the microsomes to bind ribosomes. Ribophorins are putative ribosome- binding proteins. Immunoblots developed with monoclonal antibodies against canine ribophorins I and II demonstrated that no correlation exists between the protease-induced inability to bind ribosomes and the integrity of the ribophorins. Ribophorin I was 85% resistant and ribophorin II 100% resistant to the levels of protease needed to totally eliminate ribosome binding. Moreover, no direct association was found between ribophorins and ribosomes; upon detergent solubilization at low salt concentrations, ribophorins could be sedimented in the presence or absence of ribosomes. Finally, the alkylating agent N- ethylmaleimide was shown to be capable of inhibiting translocation (beyond the SRP-docking protein-mediated recognition step), but had no affect on the ability of ribosomes to bind to ER membranes. We conclude that potentially two additional proteinaceous components, as yet unidentified, are involved in protein translocation. One is protease sensitive and possibly involved in ribosome binding, the other is N- ethylmaleimide sensitive and of unknown function.

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Selected References

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  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. 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]
  3. Ansorge W. Fast and sensitive detection of protein and DNA bands by treatment with potassium permanganate. J Biochem Biophys Methods. 1985 May;11(1):13–20. doi: 10.1016/0165-022x(85)90037-5. [DOI] [PubMed] [Google Scholar]
  4. Aulinskas T. H., Burden T. S. Hepatic membrane proteins involved in ribosome binding: identification by three procedures. Hoppe Seylers Z Physiol Chem. 1979 Jun;360(6):709–720. doi: 10.1515/bchm2.1979.360.1.709. [DOI] [PubMed] [Google Scholar]
  5. Baglioni C., Bleiberg I., Zauderer M. Assembly of membrane-bound polyribosomes. Nat New Biol. 1971 Jul 7;232(27):8–12. doi: 10.1038/newbio232008a0. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  9. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  10. Fujiki Y., Hubbard A. L., Fowler S., Lazarow P. B. Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum. J Cell Biol. 1982 Apr;93(1):97–102. doi: 10.1083/jcb.93.1.97. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fujita S., Ogata F., Nakamura J., Omata S., Sugano H. Isolation and characterization of membrane proteins responsible for attachment of polyribosomes to rough microsomal fraction of rat liver. Biochem J. 1977 Apr 15;164(1):53–66. doi: 10.1042/bj1640053. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gilmore R., Blobel G. Transient involvement of signal recognition particle and its receptor in the microsomal membrane prior to protein translocation. Cell. 1983 Dec;35(3 Pt 2):677–685. doi: 10.1016/0092-8674(83)90100-9. [DOI] [PubMed] [Google Scholar]
  13. Gilmore R., Blobel G. Translocation of secretory proteins across the microsomal membrane occurs through an environment accessible to aqueous perturbants. Cell. 1985 Sep;42(2):497–505. doi: 10.1016/0092-8674(85)90107-2. [DOI] [PubMed] [Google Scholar]
  14. Gilmore R., Walter P., Blobel G. Protein translocation across the endoplasmic reticulum. II. Isolation and characterization of the signal recognition particle receptor. J Cell Biol. 1982 Nov;95(2 Pt 1):470–477. doi: 10.1083/jcb.95.2.470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Griffiths G., Warren G., Quinn P., Mathieu-Costello O., Hoppeler H. Density of newly synthesized plasma membrane proteins in intracellular membranes. I. Stereological studies. J Cell Biol. 1984 Jun;98(6):2133–2141. doi: 10.1083/jcb.98.6.2133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hamilton M. G., Pavlovec A., Petermann M. L. Molecular weight, buoyant density, and composition of active subunits of rat liver ribosomes. Biochemistry. 1971 Aug 31;10(18):3424–3427. doi: 10.1021/bi00794a017. [DOI] [PubMed] [Google Scholar]
  17. Hortsch M., Avossa D., Meyer D. I. A structural and functional analysis of the docking protein. Characterization of active domains by proteolysis and specific antibodies. J Biol Chem. 1985 Aug 5;260(16):9137–9145. [PubMed] [Google Scholar]
  18. Hortsch M., Meyer D. I. Immunochemical analysis of rough and smooth microsomes from rat liver. Segregation of docking protein in rough membranes. Eur J Biochem. 1985 Aug 1;150(3):559–564. doi: 10.1111/j.1432-1033.1985.tb09057.x. [DOI] [PubMed] [Google Scholar]
  19. Hortsch M., Meyer D. I. Transfer of secretory proteins through the membrane of the endoplasmic reticulum. Int Rev Cytol. 1986;102:215–242. doi: 10.1016/s0074-7696(08)61276-0. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Jackson R. C., Walter P., Blobel G. Secretion requires a cytoplasmically disposed sulphydryl of the RER membrane. Nature. 1980 Jul 10;286(5769):174–176. doi: 10.1038/286174a0. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. 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]
  25. Lambert N., Freedman R. B. The latency of rat liver microsomal protein disulphide-isomerase. Biochem J. 1985 Jun 15;228(3):635–645. doi: 10.1042/bj2280635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lewis M. J., Mazzarella R. A., Green M. Structure and assembly of the endoplasmic reticulum. The synthesis of three major endoplasmic reticulum proteins during lipopolysaccharide-induced differentiation of murine lymphocytes. J Biol Chem. 1985 Mar 10;260(5):3050–3057. [PubMed] [Google Scholar]
  27. 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]
  28. Marcantonio E. E., Grebenau R. C., Sabatini D. D., Kreibich G. Identification of ribophorins in rough microsomal membranes from different organs of several species. Eur J Biochem. 1982 May;124(1):217–222. doi: 10.1111/j.1432-1033.1982.tb05928.x. [DOI] [PubMed] [Google Scholar]
  29. Meyer D. I., Dobberstein B. A membrane component essential for vectorial translocation of nascent proteins across the endoplasmic reticulum: requirements for its extraction and reassociation with the membrane. J Cell Biol. 1980 Nov;87(2 Pt 1):498–502. doi: 10.1083/jcb.87.2.498. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Meyer D. I., Dobberstein B. Identification and characterization of a membrane component essential for the translocation of nascent proteins across the membrane of the endoplasmic reticulum. J Cell Biol. 1980 Nov;87(2 Pt 1):503–508. doi: 10.1083/jcb.87.2.503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Meyer D. I., Dobberstein B. Proteins mediating vectorial translocation: purification of the active domain of the endoplasmic reticulum docking protein. Methods Enzymol. 1983;96:692–700. doi: 10.1016/s0076-6879(83)96058-5. [DOI] [PubMed] [Google Scholar]
  32. Meyer D. I., Krause E., Dobberstein B. Secretory protein translocation across membranes-the role of the "docking protein'. Nature. 1982 Jun 24;297(5868):647–650. doi: 10.1038/297647a0. [DOI] [PubMed] [Google Scholar]
  33. Ohba H., Harano T., Omura T. Intracellular and intramembranous localization of a protein disulfide isomerase in rat liver. J Biochem. 1981 Mar;89(3):889–900. doi: 10.1093/oxfordjournals.jbchem.a133272. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. Prehn S., Nürnberg P., Rapoport T. A. A receptor for single segments of secretory proteins in rough endoplasmic reticulum membranes. FEBS Lett. 1981 Jan 12;123(1):79–84. doi: 10.1016/0014-5793(81)80024-5. [DOI] [PubMed] [Google Scholar]
  36. Prehn S., Tsamaloukas A., Rapoport T. A. Demonstration of specific receptors of the rough endoplasmic membrane for the signal sequence of carp preproinsulin. Eur J Biochem. 1980;107(1):185–195. doi: 10.1111/j.1432-1033.1980.tb04639.x. [DOI] [PubMed] [Google Scholar]
  37. Redman C. M., Sabatini D. D. Vectorial discharge of peptides released by puromycin from attached ribosomes. Proc Natl Acad Sci U S A. 1966 Aug;56(2):608–615. doi: 10.1073/pnas.56.2.608. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Rees-Jones R., Al-Awqati Q. Proton-translocating adenosinetriphosphatase in rough and smooth microsomes from rat liver. Biochemistry. 1984 May 8;23(10):2236–2240. doi: 10.1021/bi00305a022. [DOI] [PubMed] [Google Scholar]
  39. Sabatini D. D., Tashiro Y., Palade G. E. On the attachment of ribosomes to microsomal membranes. J Mol Biol. 1966 Aug;19(2):503–524. doi: 10.1016/s0022-2836(66)80019-0. [DOI] [PubMed] [Google Scholar]
  40. Shires T. K., Ekren T., Narurkar L. M., Pitot H. C. Protein synthesis on rat liver polysome-membrane complexes formed in vitro and disposition of the discharges chains. Nat New Biol. 1973 Apr 18;242(120):198–201. doi: 10.1038/newbio242198a0. [DOI] [PubMed] [Google Scholar]
  41. Shires T. K., Narurkar L., Pitot H. C. The association in vitro of polyribosomes with ribonuclease-treated derivatives of hepatic rough endoplasmic reticulum. Characteristics of the membrane binding sites and factors influencing association. Biochem J. 1971 Nov;125(1):67–79. doi: 10.1042/bj1250067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Walter P., Blobel G. Translocation of proteins across the endoplasmic reticulum. II. Signal recognition protein (SRP) mediates the selective binding to microsomal membranes of in-vitro-assembled polysomes synthesizing secretory protein. J Cell Biol. 1981 Nov;91(2 Pt 1):551–556. doi: 10.1083/jcb.91.2.551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Walter P., Gilmore R., Blobel G. Protein translocation across the endoplasmic reticulum. Cell. 1984 Aug;38(1):5–8. doi: 10.1016/0092-8674(84)90520-8. [DOI] [PubMed] [Google Scholar]
  44. Walter P., Ibrahimi I., Blobel G. Translocation of proteins across the endoplasmic reticulum. I. Signal recognition protein (SRP) binds to in-vitro-assembled polysomes synthesizing secretory protein. J Cell Biol. 1981 Nov;91(2 Pt 1):545–550. doi: 10.1083/jcb.91.2.545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Walter P., Jackson R. C., Marcus M. M., Lingappa V. R., Blobel G. Tryptic dissection and reconstitution of translocation activity for nascent presecretory proteins across microsomal membranes. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1795–1799. doi: 10.1073/pnas.76.4.1795. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Warren G., Dobberstein B. Protein transfer across microsomal membranes reassembled from separated membrane components. Nature. 1978 Jun 15;273(5663):569–571. doi: 10.1038/273569a0. [DOI] [PubMed] [Google Scholar]
  47. Wickner W. T., Lodish H. F. Multiple mechanisms of protein insertion into and across membranes. Science. 1985 Oct 25;230(4724):400–407. doi: 10.1126/science.4048938. [DOI] [PubMed] [Google Scholar]

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