In vitro translocation of bacterial proteins across the plasma membrane of Escherichia coli

Abstract

Precursors to two periplasmic proteins and one outer membrane protein were synthesized in a membrane-free extract from Escherichia coli programmed with plasmid DNA. In the presence of inverted plasma membrane vesicles from E. coli up to 25% of the precursor molecules were converted into their mature forms. Using externally added proteinase K as a probe, we found the processed proteins segregated within the membrane vesicles. By the same criteria, a small amount of each precursor also proved to be translocated, indicating that translocation and signal sequence cleavage are not necessarily coupled processes. Furthermore, we present conclusive evidence that the translocation step can occur post-translationally even as late as 60 min after the beginning of translation.