Characterization of a Bacillus subtilis SecA mutant protein deficient in translocation ATPase and release from the membrane |
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Authors: | J van der Wolk M Klose E Breukink R A Demel B de Kruijff R Freudl A J M Driessen |
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Institution: | Department of Microbiology, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands.;Institut für Biotechnologie 1, Forschungszentrum Jülich GmbH, Postfach 1913, 5170 Jülich, Germany.;Centre for Biomembranes and Lipid Enzymology, Department of Biochemistry of Membranes, and Institute of Molecular Biology and Medical Biotechnology, University of Utrecht, Padualaan 8, 3584 CH Utrecht. The Netherlands. |
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Abstract: | SecA is the precursor protein binding subunit of the bacterial precursor protein translocase, which consists of the SecY/E protein as integral membrane domain. SecA is an ATPase, and couples the hydrolysis of ATP to the release of bound precursor proteins to allow their proton-motive-force-driven translocation across the cytoplasmic membrane. A putative ATP-binding motif can be predicted from the amino acid sequence of SecA with homology to the consensus Walker A-type motif. The role of this domain is not known. A lysine residue at position 106 at the end of the glycine-rich loop in the A motif of the Bacillus subtilis SecA was replaced by an asparagine through site-directed mutagenesis (K106N SecA). A similar replacement was introduced at an adjacent lysine residue at position 101 (K101N SecA). Wild-type and mutant SecA proteins were expressed to a high level and purified to homogeneity. The catalytic efficacy (kcat/km) of the K106N SecA for lipid-stimulated ATP hydrolysis was only 1% of that of the wild-type and K101N SecA. K106N SecA retained the ability to bind ATP, but its ATPase activity was not stimulated by precursor proteins. Mutant and wild-type SecA bind with similar affinity to Escherichia coli inner membrane vesicles and insert into a phospholipid mono-layer, in contrast to the wild type, membrane insertion of the K106N SecA was not prevented by ATP. K106N SecA blocks the ATP and proton-motive-force-dependent chase of a translocation intermediate to fully translocated proOmpA. It is concluded that the GKT motif in the amino-terminal domain of SecA is part of the catalytic ATP-binding site. This site may be involved in the ATP-driven protein recycling function of SecA which allows the release of SecA from its association with precursor proteins, and the phospholipid bilayer. |
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