Functional production of the Na+ F1FO ATP synthase from Acetobacterium woodii in Escherichia coli requires the native AtpI |
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Authors: | Karsten Brandt Daniel B Müller Jan Hoffmann Christine Hübert Bernd Brutschy Gabriele Deckers-Hebestreit Volker Müller |
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Institution: | 1. Molecular Microbiology & Bioenergetics, Institute of Molecular Biosciences, Johann Wolfgang Goethe University Frankfurt/Main, Max-von-Laue-Str. 9, 60438, Frankfurt, Germany 2. Institute for Physical and Theoretical Chemistry, Johann Wolfgang Goethe University Frankfurt/Main, Max-von-Laue Str. 7, 60438, Frankfurt, Germany 3. Microbiology, Department of Biology/Chemistry, University of Osnabrück, Barbarastra?e 11, 49069, Osnabrück, Germany
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Abstract: | The Na+ F1FO ATP synthase of the anaerobic, acetogenic bacterium Acetobacterium woodii has a unique FOVO hybrid rotor that contains nine copies of a FO-like c subunit and one copy of a VO-like c 1 subunit with one ion binding site in four transmembrane helices whose cellular function is obscure. Since a genetic system to address the role of different c subunits is not available for this bacterium, we aimed at a heterologous expression system. Therefore, we cloned and expressed its Na+ F1FO ATP synthase operon in Escherichia coli. A Δatp mutant of E. coli produced a functional, membrane-bound Na+ F1FO ATP synthase that was purified in a single step after inserting a His6-tag to its β subunit. The purified enzyme was competent in Na+ transport and contained the FOVO hybrid rotor in the same stoichiometry as in A. woodii. Deletion of the atpI gene from the A. woodii operon resulted in a loss of the c ring and a mis-assembled Na+ F1FO ATP synthase. AtpI from E. coli could not substitute AtpI from A. woodii. These data demonstrate for the first time a functional production of a FOVO hybrid rotor in E. coli and revealed that the native AtpI is required for assembly of the hybrid rotor. |
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