Functional production of the Na+ F1F(O) ATP synthase from Acetobacterium woodii in Escherichia coli requires the native AtpI

• Published in: Journal of bioenergetics and biomembranes Vol. 45; no. 1-2; pp. 15 - 23
• Main Authors: Brandt, Karsten, Müller, Daniel B, Hoffmann, Jan, Hübert, Christine, Brutschy, Bernd, Deckers-Hebestreit, Gabriele, Müller, Volker
• Format: Journal Article
• Language: English
• Published: United States 01.02.2013
• Subjects: Acetobacterium - enzymology; Acetobacterium - genetics; Bacterial Proteins - biosynthesis; Bacterial Proteins - genetics; Escherichia coli - enzymology; Escherichia coli - genetics; Ion Transport - physiology; Proton-Translocating ATPases - biosynthesis; Proton-Translocating ATPases - genetics; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; Sodium - metabolism
• DOI: 10.1007/s10863-012-9474-8

The Na(+) F(1)F(O) ATP synthase of the anaerobic, acetogenic bacterium Acetobacterium woodii has a unique F(O)V(O) 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(+) F(1)F(O) ATP synthase operon in Escherichia coli. A Δatp mutant of E. coli produced a functional, membrane-bound Na(+) F(1)F(O) ATP synthase that was purified in a single step after inserting a His(6)-tag to its β subunit. The purified enzyme was competent in Na(+) transport and contained the F(O)V(O) 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(+) F(1)F(O) 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 F(O)V(O) hybrid rotor in E. coli and revealed that the native AtpI is required for assembly of the hybrid rotor.