Preparation of microsomal membranes for cotranslational protein translocation.

Secretory, lysosomal, and many integral membrane proteins are translocated across or asymmetrically integrated into the membrane of the rough endoplasmic reticulum.l,la The events of this translocation or integration process can be faithfully reproduced in vitro. 2-4 For this purpose, an in vitro protein translation system programmed with a suitable mRNA is supplemented with microsomal membranes, a fraction of closed vesicles derived from the rough endoplasmic reticulum. 2 In all cases investigated, translocation is a cotranslational process; i.e., the nascent chain is vectorially translocated across the membrane as it emerges from the ribosome. Consequently, the microsomal membrane fraction has to be present during protein synthesis. This also implies that  all the components added to study the translocation process have to be compatible with in vitro protein synthesis.: If the microsomal vesicles are added after protein synthesis is completed, a post-translational translocation is not observed.