An oligodeoxynucleotide-dependent method to generate nascent polypeptide chains was adopted for use in a cell-free translation system prepared from Escherichia coli. In this way, NH 2 -terminal pOmpA fragments of distinct sizes were synthesized. Because most of these pOmpA fragments could be covalently linked to puromycin, precipitated with cetyltrimethylammonium bromide, and were enriched by sedimentation, they represent a population of elongation-arrested, ribosomeassociated nascent chains. Translocation of these nascent pOmpA chains into inside-out membrane vesicles of E. coli required SecA and (depending on size) SecB. Whereas their translocation was strictly dependent on the H ؉ -motive force of the vesicles, no indication for the involvement of the bacterial signal recognition particle was obtained. SecA and SecB, although required for translocation, did not mediate binding of the ribosome-associated pOmpA to membrane vesicles. However, SecA and SecB cotranslationally associated with nascent pOmpA, since they could be co-isolated with the ribosome-associated nascent chains and as such catalyzed translocation subsequent to the release of the ribosome. These results indicate that in E. coli, SecA also functionally interacts with preproteins before they are targeted to the translocase of the plasma membrane.Protein export across the plasma (cytoplasmic, inner) membrane of Escherichia coli is achieved by the concerted action of a distinct set of Sec proteins (summarized in Ref. 1). Most of them are integral membrane proteins of the plasma membrane. SecY and SecE most likely are core constituents of the translocation pore in the membrane; SecG appears to change its membrane topography during polypeptide translocation (2); the exact roles of SecD, SecF, and YajC are yet to be established. SecA on the other hand, is a peripheral membrane protein that binds to SecYE (3) probably serving as a membrane receptor for a preprotein-SecB complex. SecB functions as a chaperone (4) and as a targeting factor (5). Due to its ATPase activity, SecA inserts in, and deinserts from, the membrane in a cyclic manner (6, 7), which leads to the stepwise translocation of the precursor across the membrane bilayer (8, 9). In addition to ATP, the H ϩ -motive force (⌬H ϩ ) is utilized as an energy source for translocation.This model does not ascribe a function to the soluble form of SecA, which in E. coli partitions roughly equally between cytosol/ribosomes and the plasma membrane (10, 11). The occurrence of cytosolic complexes between precursor proteins and SecA (12, 13) suggests that SecA actually might interact with its protein substrate well before it has been targeted to the membrane. Besides the Sec proteins, E. coli possesses a signal recognition particle (SRP) 1 /SRP receptor system whose eukaryotic equivalents mediate the cotranslational targeting of nascent secretory and membrane proteins to the endoplasmic reticulum (14). Whereas more recent results (15-18) indicate that the bacterial SRP has a specialized role in the integrati...