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.