Kinetic analysis of the translocation of fluorescent precursor proteins into Escherichia coli membrane vesicles

Abstract

Protein secretion in Escherichia coli is mediated by translocase, a multi-subunit membrane protein complex with SecA as ATP-driven motor protein and the SecYEG complex as translocation pore. A fluorescent assay was developed to facilitate kinetic studies of protein translocation. Single cysteine mutants of proOmpA were sitespecific labeled with fluorescent dyes, and the SecA and ATP-dependent translocation into inner membrane vesicles and SecYEG proteoliposomes was monitored by means of protease accessibility and in gel fluorescent imaging. The translocation of fluorescently labeled proOmpA was largely independent on the position and the size of the fluorescent label (up to a size of 13-16 Å). A fluorophore at the +4 position blocked translocation, but inhibition was completely relieved in the Pr1A4 mutant. The kinetics of translocation of the fluorescently labeled proOmpA could be directly monitored by means of fluorescence quenching. Inner membrane vesicles containing wild-type SecYEG were found to translocate proOmpA with a turnover of 4.5 molecules proOmpA/SecYEG complex/min and an apparent Km of 180 nM, whereas the Pr1A4 mutant showed an almost 10-fold increase in turnover rate and a 3-fold increase of the apparent Km for proOmpA translocation.