Cotranslational Translocation of Proteins Into Microsomes: Methods


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 (PK) and the detergent (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.



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Further Reading

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