In Vitro Dissection of Protein Translocation into the Mammalian Endoplasmic Reticulum

  • Ajay Sharma
  • Malaiyalam Mariappan
  • Suhila Appathurai
  • Ramanujan S. Hegde
Part of the Methods in Molecular Biology book series (MIMB, volume 619)


In eukaryotic cells, roughly one-fourth of all mRNAs code for secretory and membrane proteins. This class of proteins must first be segregated to the endoplasmic reticulum, where they are either translocated into the lumen or inserted into the lipid bilayer. The study of these processes has long relied on their successful reconstitution in cell-free systems. The high manipulability of such in vitro systems has allowed the identification of key machinery, elucidation of their functional roles in translocation, and dissection of their mechanisms of action. Here, we provide the basic methodology for (i) setting up robust mammalian-based in vitro translation and translocation systems, (ii) assays for protein translocation, insertion, and topology, and (iii) methods to solubilize, fractionate, and reconstitute ER membranes. Variations of these methods should be applicable not only to forward protein translocation systems but also for dissecting other poorly understood membrane-associated processes such as retrotranslocation.

Key words

in vitro translation microsomes membrane proteins reconstitution proteoliposomes protease protection protein topology 



Work in the Hegde lab is supported by the NICHD Intramural Research Program of the National Institutes of Health.


  1. 1.
    Walter, P., and Blobel, G. (1983) Preparation of microsomal membranes for cotranslational protein translocation. Methods Enzymol 96, 84–93.CrossRefPubMedGoogle Scholar
  2. 2.
    Adelman, M.R., Blobel, G., and Sabatini, D.D. (1973) An improved cell fractionation procedure for the preparation of rat liver membrane-bound ribosomes. J Cell Biol 56, 191–205.CrossRefPubMedGoogle Scholar
  3. 3.
    Wilson, R., Allen, A.J., Oliver, J., Brookman, J.L., High, S., and Bulleid, N.J. (1995) The translocation, folding, assembly and redox-dependent degradation of secretory and membrane proteins in semi-permeabilized mammalian cells. Biochem J 307, 679–687.PubMedGoogle Scholar
  4. 4.
    Stefanovic, S., and Hegde, R.S. (2007) Identification of a targeting factor for posttranslational membrane protein insertion into the ER. Cell 128, 1147–1159.CrossRefPubMedGoogle Scholar
  5. 5.
    Schuldiner, M., Metz, J., Schmid, V., Denic, V., Rakwalska, M., Schmitt, H.D., Schwappach, B., and Weissman, J.S. (2008) The GET complex mediates insertion of tail-anchored proteins into the ER membrane. Cell 134, 634–645.CrossRefPubMedGoogle Scholar
  6. 6.
    Daniels, R., Kurowski, B., Johnson, A.E., and Hebert, D.N. (2003) N-linked glycans direct the cotranslational folding pathway of influenza hemagglutinin. Mol Cell 11, 79–90.CrossRefPubMedGoogle Scholar
  7. 7.
    Oberdorf, J., and Skach, W.R. (2002) In vitro reconstitution of CFTR biogenesis and degradation. Methods Mol Med 70, 295–310.PubMedGoogle Scholar
  8. 8.
    Brambillasca, S., Yabal, M., Soffientini, P., Stefanovic, S., Makarow, M., Hegde, R.S., and Borgese, N. (2005) Transmembrane topogenesis of a tail-anchored protein is modulated by membrane lipid composition. EMBO J 24, 2533–2542.CrossRefPubMedGoogle Scholar
  9. 9.
    Fons, R.D., Bogert, B.A., and Hegde, R.S. (2003) Substrate-specific function of the translocon-associated protein complex during translocation across the ER membrane. J Cell Biol 160, 529–539.CrossRefPubMedGoogle Scholar
  10. 10.
    Garrison, J.L., Kunkel, E.J., Hegde, R.S., and Taunton J. (2005) A substrate-specific inhibitor of protein translocation into the endoplasmic reticulum. Nature 436, 285–289.CrossRefPubMedGoogle Scholar
  11. 11.
    Görlich, D., and Rapoport, T.A. (1993) Protein translocation into proteoliposomes reconstituted from purified components of the endoplasmic reticulum membrane. Cell 75, 615–630.CrossRefPubMedGoogle Scholar
  12. 12.
    Jackson, R.J., and Hunt, T. (1983) Preparation and use of nuclease-treated rabbit reticulocyte lysates for the translation of eukaryotic messenger RNA. Methods Enzymol 96, 50–74.CrossRefPubMedGoogle Scholar
  13. 13.
    Trachsel, H., Ranu, R.S., and London, I.M. (1978) Regulation of protein synthesis in rabbit reticulocyte lysates: purification and characterization of heme-reversible translational inhibitor. Proc Natl Acad Sci USA 75, 3654–3658.CrossRefPubMedGoogle Scholar
  14. 14.
    Helenius, A., and Simons, K. (1975) Solubilization of membranes by detergents. Biochim Biophys Acta 415, 29–79.PubMedGoogle Scholar
  15. 15.
    Perara, E., Rothman, R.E., and Lingappa, V.R. (1986) Uncoupling translocation from translation: implications for transport of proteins across membranes. Science 232, 348–352.CrossRefPubMedGoogle Scholar
  16. 16.
    Wiedmann, M., Kurzchalia, T.V., Hartmann, E., and Rapoport, T.A. (1987) A signal sequence receptor in the endoplasmic reticulum membrane. Nature 328, 830–833.CrossRefPubMedGoogle Scholar
  17. 17.
    Görlich, D., Hartmann, E., Prehn, S., and Rapoport, T.A. (1992) A protein of the endoplasmic reticulum involved early in polypeptide translocation. Nature 357, 47–52.CrossRefPubMedGoogle Scholar
  18. 18.
    Crowley, K.S., Reinhart, G.D., and Johnson, A.E. (1993) The signal sequence moves through a ribosomal tunnel into a noncytoplasmic aqueous environment at the ER membrane early in translocation. Cell 73, 1101–1115.CrossRefPubMedGoogle Scholar
  19. 19.
    Hegde, R.S., and Lingappa, V.R. (1996) Sequence-specific alteration of the ribosome-membrane junction exposes nascent secretory proteins to the cytosol. Cell 85, 217–228.CrossRefPubMedGoogle Scholar
  20. 20.
    Hegde, R.S., Mastrianni, J.A., Scott, M.R., DeFea, K.A., Tremblay, P., Torchia, M., DeArmond, S.J., Prusiner, S.B., and Lingappa, V.R. (1998) A transmembrane form of the prion protein in neurodegenerative disease. Science 279, 827–834.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Ajay Sharma
    • 1
  • Malaiyalam Mariappan
    • 1
  • Suhila Appathurai
    • 1
  • Ramanujan S. Hegde
    • 1
  1. 1.Cell Biology and Metabolism ProgramNational Institute of Child Health and Human Development, National Institutes of HealthBethesdaUSA

Personalised recommendations