We have separately analyzed membrane‐targeting and membrane translocation of an exported bacterial protein. The precursor of the outer membrane protein LamB of Escherichia coli was synthesized in vitro and translocated into inverted plasma membrane vesicles under co‐ and post‐translational conditions. The translation/translocation products of LamB were subsequently resolved into soluble and membrane‐associated material. Dissipation of the H(+)‐motive force, depletion of ATP and treatment of membranes with N‐ethylmaleimide each inhibited processing and translocation of preLamB without preventing its binding to the membranes. Hence, all three conditions block transmembrane passage rather than membrane‐targeting. The latter was abolished by pretreatment of salt‐extracted membrane vesicles with trypsin. It was also drastically reduced when preLamB was synthesized in cell extracts derived from either a secA amber or a secB null mutant. Membrane‐targeting of preLamB therefore requires soluble SecA and SecB as well as a protease‐sensitive membrane receptor. The finding that SecA is involved in targeting whereas ATP is required for the transmembrane passage suggests that SecA, which harbors an ATPase activity [Lill et al. (1989), EMBO J., 8, 961‐966], might have a dual function in bacterial protein export.
SecY is an integral plasma-membrane protein of Escherichia coli which is essential for the export of periplasmic and outer-membrane proteins containing cleavable signal sequences. We have synthesized SecY in vitro using an E. coli transcription/translation system. In the absence of membranes, SecY remained largely soluble but cosedimented on sucrose gradients with the membrane fraction when inside-out plasma-membrane vesicles (INV) had been added cotranslationally. Membrane association of SecY was unaffected if the endogenous SecY of the INV had been inactivated by either antibodies, a mutation or trypsin treatment. In contrast, inactivation of the INV SecY interfered with membrane targeting and, consequently, the processing of precursors to p-lactamase and , I receptor. When SecYdeprived INV were, however, first functionally reconstituted with in-vitro-synthesized SecY, targeting and translocation of the , I receptor were partially restored. Thus, the assembly of SecY into INV in vitro leads to an active enzyme. In addition, we show that the prlA4 allele of the sec Y gene suppresses signal-sequence mutations of the , I receptor in vitro. Collectively, our results demonstrate that SecY, while functioning as a membrane-located receptor for precursors of exported proteins, appears to be virtually independent of pre-existing SecY for its own membrane integration.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.