Chapter 7 In Vitro Protein Translocation into Escherichia coli Inverted Membrane Vesicles

Abstract

Publisher Summary The in vitro protein translocation system is composed of a soluble extract (S30) capable of synthesizing proteins when programmed with mRNA coded for secretory precursors, and a membrane fraction of inverted cytoplasmic membrane vesicles. In some cases, purified precursor molecules are used. In these systems, proteins are considered translocated across membranes, if processing of the signal sequences coincides with proteins resistant to proteolytic digestion and the processed proteins co-sediment with membrane vesicles. This is taken as an indication that the proteins are translocated into the lumen of the vesicles, although it is possible that some of the protein is embedded in the membranes. Protein translocation can be examined co-translationally by synthesizing proteins in the presence of membrane vesicles, or posttranslationally by blocking further synthesis prior to the addition of membrane vesicles. The concurrent genetic and biochemical approaches in protein secretion in E. coli make it possible to utilize the in vitro system to characterize the genetically identified translocation components and fractionation of the in vitro system facilitates defining the components of the translocation machinery and their interactions.