In vitro studies with purified components reveal signal recognition particle (SRP) and SecA SecB as constituents of two independent protein-targetingpathways of Escherichia coli

The molecular requirements for the translocation of secretory proteins acro ss, and the integration of membrane proteins into, the plasma membrane of E scherichia coli were compared. This was achieved in a novel cell-free syste m from E. coli which, by extensive subfractionation, was simultaneously ren dered deficient in SecA/SecB and the signal recognition particle (SRP) comp onents, Ffh (P48), 4.5S RNA, and FtsY. The integration of two membrane prot eins into inside-out plasma membrane vesicles of E. coli required all three SRP components and could not be driven by SecA, SecB, and Delta mu H+. In contrast, these were the only components required for the translocation of secretory proteins into membrane vesicles, a process in which the SRP compo nents were completely inactive. Our results, while confirming previous in v ivo studies, provide the first in vitro evidence for the dependence of the integration of polytopic inner membrane proteins on SRP in E. coli. Further more, they suggest that SRP and SecA/SecB have different substrate specific ities resulting in two separate targeting mechanisms for membrane and secre tory proteins in E. coli. Both targeting pathways intersect at the transloc ation pore because they are equally affected by a blocked translocation cha nnel.