Cotranslational Translocation of Proteins Into Microsomes: Methods

Abstract

Translocation of secretory and membrane proteins through the membrane of the endoplasmic reticulum involves a signal sequence on the nascent protein, a cytosolic receptor system and a translocation channel (translocon). The process can be reconstituted using a protein‐synthesising system and rough microsomes derived from the endoplasmic reticulum (ER) of eukaryotic cells. Applications of this system have realised substantial insights into the mechanisms of protein targeting to the ER membrane and the insertion of a nascent polypeptide into the membrane. The arrest of polypeptides at distinct stages of the targeting or insertion process yielded snapshots from the movement of a polypeptide across the membrane. This has allowed identification of components of the translocation machinery and the characterisation of their sequential involvement in targeting and translocation.

Key Concepts:

  • Many membrane transport processes can be studied with fragmented membranes.

  • Mechanical shearing of cellular membranes always results in closed vesicles.

  • Cellular membranes are self‐organising structures without corners and breaks.

  • Reconstitution of cellular subfunctions is a very efficient way to characterise cellular processes at the molecular level.

  • When a system is too complex, then study first its parts.

  • Generating reaction intermediates blocked at defined steps allows complex processes to be broken down into simpler and more tractable subreactions.

Keywords: signal sequence; endoplasmic reticulum; secretion; secretory proteins; membrane proteins; protein biosynthesis

Figure 1.

Assay for the translocation of preprolactin (PPL) across rough microsomal membranes (RMs). [35S]Methionine‐labelled PPL was synthesised in a reticulocyte cell‐free system in the absence (lane 1) or presence of RMs (lanes 2–4). After translation, proteinase K (lane 3) or proteinase K (PK) and the detergent Triton X‐100 (TX) (lane 4) were added and incubation continued. Proteins were then separated by SDS‐polyacrylamide gel electrophoresis and radioactively labelled proteins visualised by autoradiography. PL, prolactin from which the signal sequence has been cleaved.

close

References

Becker T, Bhushan S, Jarasch A et al. (2009) Structure of monomeric yeast and mammalian Sec61 complexes interacting with the translating ribosome. Science 326: 1369–1373.

Bernstein HD (2001) Cotranslational translocation of proteins into canine rough microsomes. Current Protocols in Cell Biology. Chapter 11: Unit 11.4, 11.4.1–11.4.11.

Blobel G and Dobberstein B (1975) Transfer of proteins across membranes I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane‐bound ribosomes of murine myeloma. Journal of Cell Biology 67: 835–851.

Halic M, Becker T, Pool MR et al. (2004) Structure of the signal recognition particle interacting with the elongation‐arrested ribosome. Nature 427: 808–814.

Halic M, Gartmann M, Schlenker O et al. (2006) Signal recognition particle receptor exposes the ribosomal translocon binding site. Science 312: 745–747.

Hegde RS and Kang SW (2008) The concept of translocational regulation. Journal of Cell Biology 182: 225–232.

Martoglio B, Hauser S and Dobberstein B (1997) Cotranslational translocation of proteins into microsomes derived from the rough endoplasmic reticulum of mammalian cells. In: Celis JE (ed.) Cell Biology: A Laboratory Handbook, pp. 265–273. San Diego, CA: Academic Press.

Ménétret JF, Hegde RS, Aguiar M et al. (2008) Single copies of Sec61 and TRAP associate with a nontranslating mammalian ribosome. Structure 28: 1083–1092.

Rapoport T, Jungnickel B and Kutay U (1996) Protein translocation across the eukaryotic endoplasmic reticulum and bacterial inner membranes. Annual Review of Biochemistry 65: 271–303.

Schatz G and Dobberstein B (1996) Common principles of protein translocation across membranes. Science 271: 1519–1526.

Uemura S, Aitken CE, Korlach J et al. (2010) Real‐time tRNA transit on single translating ribosomes at codon resolution. Nature 464: 1012–1017.

Walter P and Blobel G (1983) Preparation of microsomal membranes for cotranslational protein translocation. Methods in Enzymology 96: 84–93.

Walter P and Johnson AE (1994) Signal sequence recognition and protein targeting to the endoplasmic reticulum membrane. Annual Review of Cell Biology 10: 87–119.

Zimmermann R, Müller L and Wullich B (2006) Protein transport into the endoplasmic reticulum: mechanisms and pathologies. Trends in Molecular Medicine 12: 567–573.

Further Reading

Johnson AE (1997) Protein translocation at the ER membrane: a complex process becomes more so. Trends in Cell Biology 7: 90–95.

Martoglio B and Dobberstein B (1996) Snapshots of membrane‐translocating proteins. Trends in Cell Biology 6: 142–147.

Rapoport TA (2007) Protein translocation across the eukaryotic endoplasmic reticulum and bacterial plasma membranes. Nature 450: 663–669.

Contact Editor close
Submit a note to the editor about this article by filling in the form below.

* Required Field

How to Cite close
Pool, Martin, and Dobberstein, Bernhard(Mar 2011) Cotranslational Translocation of Proteins Into Microsomes: Methods. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0002612.pub2]