Functional production of the Na+ F1FO ATP synthase from Acetobacterium woodii in Escherichia coli requires the native AtpI
Autor(en): | Brandt, Karsten Mueller, Daniel B. Hoffmann, Jan Huebert, Christine Brutschy, Bernd Deckers-Hebestreit, Gabriele Mueller, Volker |
Stichwörter: | ATP synthase; AtpI; Bioenergetics; Biophysics; Cell Biology; ENERGY-CONSERVATION; Hybrid rotor; IDENTIFICATION; INTERMEDIATE STEP; Membrane enzymes; OLIGOMER; OPERON; PROTEIN; PURIFICATION; RING; Sodium transport; SUBUNIT; TRANSLOCATING F1F0-ATPASE | Erscheinungsdatum: | 2013 | Herausgeber: | SPRINGER/PLENUM PUBLISHERS | Journal: | JOURNAL OF BIOENERGETICS AND BIOMEMBRANES | Volumen: | 45 | Ausgabe: | 1-2 | Startseite: | 15 | Seitenende: | 23 | Zusammenfassung: | The Na+ F1FO ATP synthase of the anaerobic, acetogenic bacterium Acetobacterium woodii has a unique FOVO hybrid rotor that contains nine copies of a F-O-like c subunit and one copy of a V-O-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 Delta atp mutant of E. coli produced a functional, membrane-bound Na+ F1FO ATP synthase that was purified in a single step after inserting a His(6)-tag to its beta 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. |
ISSN: | 0145479X | DOI: | 10.1007/s10863-012-9474-8 |
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geprüft am 01.06.2024