Kinetic Analysis of the Translocation of Fluorescent Precursor Proteins into Escherichia coli Membrane Vesicles

Abstract

Protein secretion in Escherichia coliis 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 site-specific 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 PrlA4 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 K m of 180 nm, whereas the PrlA4 mutant showed an almost 10-fold increase in turnover rate and a 3-fold increase of the apparent K m for proOmpA translocation. Protein secretion in Escherichia coliis 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 site-specific 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 PrlA4 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 K m of 180 nm, whereas the PrlA4 mutant showed an almost 10-fold increase in turnover rate and a 3-fold increase of the apparent K m for proOmpA translocation. Translocase is a membrane-bound enzyme complex that mediates the translocation across and integration of proteins into the cytoplasmic membrane of Escherichia coli (for a review see Ref. 1Manting E.H. Driessen A.J.M. Mol. Microbiol. 2000; 37: 226-238Crossref PubMed Scopus (210) Google Scholar). Secretory proteins are synthesized in the cytosol as precursors with an amino-terminal signal sequence targeted to the membrane and subsequently transported across the membrane in an ATP-dependent manner via a proteinaceous channel formed by a heterotrimeric membrane protein complex composed of SecY, SecE, and SecG (2Brundage L. Fimmel C.J. Mizushima S. Wickner W. J. Biol. Chem. 1992; 267: 4166-4170Abstract Full Text PDF PubMed Google Scholar, 3Manting E.H. van der Does C. Remigy H. Engel A. Driessen A.J.M. EMBO J. 2000; 19: 852-861Crossref PubMed Scopus (167) Google Scholar). SecA is a soluble ATPase that associates with the SecYEG complex (4Hendrick J.P. Wickner W. J. Biol. Chem. 1991; 266: 24596-24600Abstract Full Text PDF PubMed Google Scholar) where it serves both as a receptor for precursor proteins (5Hartl F.U. Lecker S. Schiebel E. Hendrick J.P. Wickner W. Cell. 1990; 63: 269-279Abstract Full Text PDF PubMed Scopus (444) Google Scholar) and as an ATP-driven molecular motor (6Economou A. Wickner W. Cell. 1994; 78: 835-843Abstract Full Text PDF PubMed Scopus (479) Google Scholar, 7Schiebel E. Driessen A.J.M. Hartl F.U. Wickner W. Cell.