title: Proton motive force-dependent and -independent protein translocation revealed by an efficient in vitro assay system of Escherichia coli.
abstract: Inverted membrane vesicles prepared from Escherichia coli spheroplasts were fractionated by means of sucrose gradient centrifugation, and a vesicle preparation exhibiting efficient and quantitative translocation of secretory proteins was obtained. The translocation of OmpA and an uncleavable model protein, uncleavable OmpF-Lpp, took place almost completely in 2-3 min, whereas that of OmpF-Lpp, a chimeric secretory protein, required 20 min for completion. The requirement of the proton motive force (delta muH+) for in vitro translocation was then examined with these three proteins. The translocation of all these proteins was significantly inhibited by the addition of carbonyl cyanide m-chlorophenylhydrazone (CCCP) or when stripped membrane vesicles lacking F1-ATPase were used, suggesting that delta muH+ generally participates in the translocation reaction. The inhibition was complete with OmpF-Lpp, whereas significant amounts of uncleavable OmpF-Lpp and OmpA were translocated at a slower rate even with the stripped membrane vesicles in the presence of a high concentration of carbonyl cyanide m-chlorophenylhydrazone. The delta muH+-independent translocation was inhibited by a nonhydrolyzable ATP analogue. These results indicate that although translocation of OmpF-Lpp obligatory requires delta muH+, the latter two proteins can be translocated in not only a delta muH+-dependent manner but also a delta mu H+-independent manner.
authors.affiliation: Laboratory of Microbiology, School of Agriculture, Nagoya University, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Yamada
authors.forename: H
authors.initials: H
authors.lastname: Tokuda
authors.forename: S
authors.initials: S
authors.lastname: Mizushima
mesh_terms: D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008074:Lipoproteins / Q000378:metabolism*; D008566:Membranes / Q000378:metabolism; D008854:Microscopy, Electron; D009943:Organophosphorus Compounds / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D006180:Proton-Translocating ATPases / Q000378:metabolism; D011522:Protons
chemical_list: D008074:Lipoproteins; D009943:Organophosphorus Compounds; D011522:Protons; C008705:octamethyl pyrophosphoramide; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D006180:Proton-Translocating ATPases