Functional production of the Na+ F1FO ATP synthase from Acetobacterium woodii in Escherichia coli requires the native AtpI

Abstract

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.

Roles of AtpI and Two YidC-Type Proteins from Alkaliphilic Bacillus pseudofirmus OF4 in ATP Synthase Assembly and Nonfermentative Growth

“…Similar results were observed with the Na ϩ -coupled rotor of Acetobacterium woodii (24). The current observations were closer to those of Matthies et al (15), who reported that the absence of AtpI reduced the yield, but did not preclude formation, of functional ATP synthase obtained from a cell-free expression system using a plasmid with atp genes from Caldalkalibacillus thermarum TA2.A1.…”

“…The single YidC found in E. coli can be depleted but deletion of yidC is lethal (20,21), and Bacillus subtilis, which has two yidC homologues, yqjG and spoIIIJ, can spare one of them but not both without losing viability (22,23). It is possible that AtpI plays a more indispensable role in assembly of Na ϩ -coupled than H ϩ -coupled ATP synthases; a requirement for AtpI similar to that described by the Yoshida group was recently reported for the Na ϩ -coupled ATP synthase of Acetobacterium woodii in a heterologous system (24). Further, a plausible explanation for dispensability of YidC homologues for c-rotor or ATP synthase synthesis in vitro is that the use of a multicopy, inducible plasmid to express atpI and the subunit c-encoding atpE gene or the whole atp operon may artificially bypass the need for additional proteins that play critical roles in vivo (25,26).…”

An Electron-bifurcating Caffeyl-CoA Reductase

“…Thus, the internal Rnf loop accounts for up to 60% of the total amount of ions translocated during this respiration. In sum, 3 mol of Na Considering that the Na ϩ F 1 F 0 -ATP synthase of A. woodii has 10 ion-binding sites (14,16,51), this makes 3.3 ions/ATP, and thus, caffeate respiration yields 0.9 mol of ATP/mol of caffeate. This is in good agreement with the experimental data (21).…”

The Arabidopsis Protein CONSERVED ONLY IN THE GREEN LINEAGE160 Promotes the Assembly of the Membranous Part of the Chloroplast ATP Synthase

“…Thus, a hybrid F 1 F o (F 1 from Bacillus PS3 and F o from P. modestum) expressed in E. coli requires Atp1/ UncI from P. modestum for c-ring formation and coupled ATPase activity (Suzuki et al, 2007). Similarly, functional production of the Na + F 1 F o -ATP synthase from A. woodii in E. coli requires the A. woodii atp1/uncI gene for proper assembly (Brandt et al, 2013). In addition, c-subunit monomers and c-rings copurify together with P. modestum UncI/Atp1 (Suzuki et al, 2007), and oligomerization of P. modestum c-subunits into c 11 -rings is mediated by Atp1/ UncI in vitro (Ozaki et al, 2008).…”