Detection and localization of the<i>i</i> protein in<i>Escherichia coli</i> cells using antibodies

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.

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

Brandt

Müller

Hoffmann

et al. 2012

J Bioenerg Biomembr

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Effects of the uncI gene on expression of uncB, the gene coding for the a subunit of the F₁F₀ ATPase of Escherichia coli

Hsu

Brusilow

1995

FEBS Letters

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The eight genes coding for the subunits of the E. coil F1F o ATPase are preceded by a gene, designated uncI. A homologous gene, or a gene coding for an analagous protein, is found preceding the ATPase genes of several microorganisms. No function for the I gene has been described. Using lac fusions to measure gene expression in vivo, we tested the effects of deleting uncI on the expression of the adjacent gene uncB, which codes for the a subunit of the Fo sector of the ATPase. Deleting uncI reduced the expression of three uncB'-'lacZ fusion genes in vivo, but had no effect on the expression of two uncB'-'lacZ fusion genes containing a relatively smaller amount of the uncB coding region. The uncI deletion also reduced the relative synthesis of the a subunit in vitro. The I gene therefore appears to specifically affect the expression of uncB or the synthesis of the a subunit at some step after translational initiation of uncB.

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Modulation of proton pumping efficiency in bacterial ATP synthases

Turina

Rebecchi

D'Alessandro

et al. 2006

Biochimica et Biophysica Acta (BBA) - Bioenergetics

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The ATP synthase in chromatophores of Rhodobacter caspulatus can effectively generate a transmembrane pH difference coupled to the hydrolysis of ATP. The rate of hydrolysis was rather insensitive to the depletion of ADP in the assay medium by an ATP regenerating system (phospho-enol-pyruvate (PEP) and pyruvate kinase (PK)). The steady state values of DeltapH were however drastically reduced as a consequence of ADP depletion. The clamped concentrations of ADP obtained using different PK activities in the assay medium could be calculated and an apparent Kd approximately 0.5 microM was estimated. The extent of proton uptake was also strongly dependent on the addition of phosphate to the assay medium. The Kd for this effect was about 70 microM. Analogous experiments were performed in membrane fragment from Escherichia coli. In this case, however, the hydrolysis rate was strongly inhibited by Pi, added up to 3 mM. Inhibition by Pi was nearly completely suppressed following depletion of ADP. The Kd's for the ADP and Pi were in the micromolar range and submillimolar range, respectively, and were mutually dependent from the concentration of the other ligand. Contrary to hydrolysis, the pumping of protons was rather insensitive to changes in the concentrations of the two ligands. At intermediate concentrations, proton pumping was actually stimulated, while the hydrolysis was inhibited. It is concluded that, in these two bacterial organisms, ADP and phosphate induce a functional state of the ATP synthase competent for a tightly coupled proton pumping, while the depletion of either one of these two ligands favors an inefficient (slipping) functional state. The switch between these states can probably be related to a structural change in the C-terminal alpha-helical hairpin of the epsilon-subunit, from an extended conformation, in which ATP hydrolysis is tightly coupled to proton pumping, to a retracted one, in which ATP hydrolysis and proton pumping are loosely coupled.

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Detection and localization of thei protein inEscherichia coli cells using antibodies

Cited by 16 publications

References 27 publications

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

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

Effects of the uncI gene on expression of uncB, the gene coding for the a subunit of the F₁F₀ ATPase of Escherichia coli

Modulation of proton pumping efficiency in bacterial ATP synthases

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Detection and localization of thei protein inEscherichia coli cells using antibodies

Cited by 16 publications

References 27 publications

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

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

Effects of the uncI gene on expression of uncB, the gene coding for the a subunit of the F1F0 ATPase of Escherichia coli

Modulation of proton pumping efficiency in bacterial ATP synthases

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Product

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Effects of the uncI gene on expression of uncB, the gene coding for the a subunit of the F₁F₀ ATPase of Escherichia coli