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. 2013 Sep 6;288(36):25880-94.
doi: 10.1074/jbc.M113.484675. Epub 2013 Jul 17.

Subunit δ Is the Key Player for Assembly of the H(+)-translocating Unit of Escherichia Coli F(O)F1 ATP Synthase

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Free PMC article

Subunit δ Is the Key Player for Assembly of the H(+)-translocating Unit of Escherichia Coli F(O)F1 ATP Synthase

Florian Hilbers et al. J Biol Chem. .
Free PMC article

Abstract

The ATP synthase (F(O)F1) of Escherichia coli couples the translocation of protons across the cytoplasmic membrane to the synthesis or hydrolysis of ATP. This nanomotor is composed of the rotor c10γε and the stator ab2α3β3δ. To study the assembly of this multimeric enzyme complex consisting of membrane-integral as well as peripheral hydrophilic subunits, we combined nearest neighbor analyses by intermolecular disulfide bond formation or purification of partially assembled F(O)F1 complexes by affinity chromatography with the use of mutants synthesizing different sets of F(O)F1 subunits. Together with a time-delayed in vivo assembly system, the results demonstrate that F(O)F1 is assembled in a modular way via subcomplexes, thereby preventing the formation of a functional H(+)-translocating unit as intermediate product. Surprisingly, during the biogenesis of F(O)F1, F1 subunit δ is the key player in generating stable F(O). Subunit δ serves as clamp between ab2 and c10α3β3γε and guarantees that the open H(+) channel is concomitantly assembled within coupled F(O)F1 to maintain the low membrane proton permeability essential for viability, a general prerequisite for the assembly of multimeric H(+)-translocating enzymes.

Keywords: ATP Synthase; Escherichia coli; F1Fo ATPase; H+-ATPase; Membrane Proteins; Protein Assembly; Protein Cross-linking.

Figures

FIGURE 1.
FIGURE 1.
Comparison of cross-linked subunit b or subunits b and c in membranes of cells expressing the atp operon (FOF1) and cells exclusively expressing atpF (subunit b) or atpEF (subunits b and c). A, cross-linking of subunit b. FOF1, DK8 transformed with pBWU13 derivatives (b Cys-less, pSTK3; bQ10C, pBH16; bL65C, pSTK8; bS139C, pBH17) were grown in minimal medium with 0.5% (v/v) glycerol and harvested at OD = 0.8–1.0. subunit b, cells of E. coli HB1(DE3) transformed with pET-22b derivatives (bQ10C, pET22-atpF1; bL65C, pET22-atpF3; bS139C, pET22-atpF2) were grown in LB medium with ampicillin to OD = 0.6 prior to induction of atpF gene expression with 1 mm IPTG for 1 h. Membranes (20 μg of protein/lane) containing cysteine-substituted subunit b were separated after cross-linking with CuP at pH 8.2 by non-reducing SDS-PAGE and analyzed by immunoblotting. B, cross-linking of subunits b and c. FOF1, DK8 transformed with pHB3 (bN2C/cV74C); subunits b + c, cells of HB1(DE3) transformed with pET22-atpEF1 (bN2C/cV74C) were grown and analyzed as described in A. Using the Odyssey system, the intensities of the fluorescence detected in the individually immunolabeled bands are adjusted between a minimum and a maximum for each blot membrane scanned and separately for both detection channels. Due to the extremely high intensity of the red-labeled c and c-c bands of the membranes containing only subunits b and c (right), it was not possible to verify the presence of subunits b and c in the b-c cross-link band of FOF1-containing membranes. Therefore, the left panel shows FOF1 of the same immunoblot after cropping the right part of the right panel to readjust the intensities of the fluorescence.
FIGURE 2.
FIGURE 2.
Presence of FOF1 subunits in membrane vesicles of single-subunit knock-out mutants Δa, Δb, and Δδ compared with wild type. A, detection of the different FOF1 subunits to verify their presence in membrane vesicles. DK8 transformed with pBWU13 (WT), pHB5 (Δa), pBWU13.Δb (Δb), and pBWU13.Δδ (Δδ), respectively, was grown in LB medium with ampicillin and harvested at OD = 0.8–1.0. Membranes were prepared in TMG buffer in the presence of protease inhibitor mix without EDTA and analyzed by immunoblotting. B, Cu2+-catalyzed cross-linking within the FOF1 core complex c10α3β3γϵ of single-subunit knock-out mutants Δb and Δδ compared with wild type. DK8 transformed with pBWU13 derivatives was grown, membranes were prepared, and after cross-linking with CuP at pH 7.5, proteins were separated by non-reducing SDS-PAGE and analyzed by immunoblotting. Top, formation of a cross-linked c10 ring using cysteine pair cA21C/cM65C (WT, pBWU13.NOC; Δb, pJGA5.1; Δδ, pRE3) immunolabeled with monoclonal anti-c antibody GDH 9-2A2. Due to an incomplete cross-linking, all intermediate products are visible. Bottom, formation of α-γ and β-γ via cysteine substitutions αP280C/γL262C and βD380C/γC87, respectively (α-γ: WT, pBH26.1; Δb, pJGA3; Δδ, pRE8; β-γ: WT, pBH29; Δb, pJGA4; Δδ, pRE10). Immunolabeling was performed with polyclonal mouse (anti-α, anti-β; green) and polyclonal rabbit (anti-γ; red) antibodies.
FIGURE 3.
FIGURE 3.
Cu2+-catalyzed cross-linking of subunit b to subunit α, β, or c in membranes of knock-out mutants Δa and Δδ compared with wild type. DK8 transformed with pBWU13 derivatives was grown, and membranes were prepared as described in the legend of Fig. 2. Membranes of wild type, Δa, and Δδ, containing FOF1 subunits individually substituted with cysteines, were separated after cross-linking with CuP at pH 7.5 by non-reducing SDS-PAGE and analyzed by immunoblotting. A, structural homology model of c10α3β3γϵ of E. coli ATP synthase. The model is composed of several partial structures combined with biochemical data as described in detail by Junge et al. (1) and drawn by RasMol version 2.7.2.1.1 to mark the positions of the cross-linking pairs used, with b2 illustrated as yellow rectangles. B, formation of b dimer via cysteine substitution bL65C (WT, pSTK8; Δa, pHB17; Δδ, pRE4). C, cross-linking between subunits b and c using cysteine pair bN2C/cV74C (WT, pHB3; Δa, pHB15; Δδ, pRE2). D, cross-linking between subunits b and α using cysteine pair bA92C/αR477C (WT, pKB1.αR477C; Δa, pBH55.bα; Δδ, pRE14). E, cross-linking between subunits b and β via cysteine pair bA92C/βQ351C (WT, pJPKB.βQ351C; Δa, pBH55.bβ; Δδ, pBH56.bβ). B–E, left, WT, 20 μg/lane; Δa, 20 μg/lane. B–E, right, WT, 5 μg/lane; Δδ, 35 μg/lane. unsp., unspecific.
FIGURE 4.
FIGURE 4.
Purification of partially assembled FOF1 of mutants Δa and Δδ by affinity chromatography in comparison with wild type. A and C, comparison of membranes and purified FOF1 complexes. B and D, detection of the different FOF1 subunits to verify their presence in the complexes purified. DK8 transformed with pBWU13 derivatives was grown as described in the legend of Fig. 2. Membranes were prepared and solubilized, and FOF1 complexes were purified by affinity chromatography via a His6 tag (A and B) present N-terminal to subunit β (WT, pBH7; Δa, pBH55.β-His; Δδ, pBH56.β-His) or a His12 tag (C and D) fused to the N terminus of subunit a (WT, pBH1; Δδ, pBH56.a-His12). Membranes and purified FOF1 were analyzed by immunoblotting. S, molecular mass standard; C, subunits a and b detected in membranes of DK8/pBWU13.
FIGURE 5.
FIGURE 5.
Cross-linking between subunits a and b in membranes of Δδ compared with wild type. Cell growth, membrane preparation, and cross-linking at pH 8.2 were performed as described in the legend of Fig. 3. Membranes were separated by non-reducing SDS-PAGE and analyzed by immunoblotting. A, generation of an a-b-b subcomplex by combining cross-linking pairs bA13C/aN238C and bE155C (Cys-less, pSTK3; b-b, pBH20.1; b-a, pKB7.aN238C; b-b/b-a, pBH152) using 20 μg protein/lane. B, cross-linking between subunits b and a using the cysteine pair bA13C/aN238C in mutant Δδ (WT, pKB7.aN238C; Δδ, pSP2.a238/b13). C, formation of a cross-linked a-b-b subcomplex in mutant Δδ (WT, pBH152; Δδ, pBH152.Δδ) using different amounts of protein (WT, 5 μg/lane; Δδ, 35 μg/lane). S, molecular mass standard; unsp., unspecific.
FIGURE 6.
FIGURE 6.
Design of the time-delayed in vivo assembly system and its different states during cell growth. ΔatpB-C strain DK8 was transformed with three plasmids bearing different resistance genes and different origins to gain compatibility: (i) a pBAD33 derivative (p15A ori; CmR) with structural genes of the atp operon carrying an early stop codon (δY11end) in atpH (atpBEFH*AGDC) under control of ParaBAD; (ii) a pET-22b derivative (pMB1 ori, ApR) carrying the atpH gene under control of the IPTG-inducible T7lac promoter using the weak start codon TTG for atpH; (iii) a pSC101 derivative (KanR) containing T7 gene1 under control of the IPTG-inducible T5N25lac promoter, encoding the RNA polymerase specific for promoters of phage T7. Left, cells were inoculated with OD = 0.05; ParaBAD was induced by arabinose to allow expression of the atpBEFH*AGDC genes, and the FOF1 subunits except for subunit δ (FOF1−δ) were synthesized. During this growth phase, the lac operator-controlled promoters are completely repressed. Middle, at OD = 0.3, ParaBAD is repressed by the simultaneous addition of glucose and d-fucose, and after further growth for 20 min, the atp mRNA present within the cell is completely degraded. Due to this time delay, the de novo biosynthesis of FOF1 subunits is prevented. Right, time-delayed IPTG induction of lac operator-controlled T7 and T5N25 promoters for 1 h enables the synthesis of subunit δ assembling together with the preformed FOF1 subcomplexes ab2 and c10α3β3γϵ (FOF1−δ+δ), a functional FOF1 complex. Red cross, repressed or uninduced state of promoters.
FIGURE 7.
FIGURE 7.
Stability of FOF1−δ (A) and degradation of atp mRNA (B) after repression of ParaBAD controlling expression of atpBEFH*AGDC. DK8 carrying plasmids pBAD33.Δδ, pET22.atpH-TTG and pT7POL26 (FOF1−δ) was grown as described under “Experimental Procedures.” At each time point indicated, cells were harvested for immunoblot analysis (A) and isolation of RNA (B). A, stability of FOF1−δ. After resuspension of cell lysates in sample loading buffer, cells were incubated for 5 min at 99 °C. The amount of cell extract (20 μg/lane) was calculated according to the determination of Neidhardt et al. (34) that 160 μg of protein is present per ml of growth medium at OD = 1. Proteins were separated by SDS-PAGE and detected by immunolabeling as indicated. B, degradation of atp mRNA. rt-RT-PCR was performed using primer pairs atpEF (dark gray) and atpA (light gray). The amount of atp mRNA present in the FOF1 sample grown with Ara was set to 100%. Error bars, S.E.
FIGURE 8.
FIGURE 8.
Time-delayed in vivo assembly system of subunit δ into preformed FOF1 subcomplexes yielding functional FOF1. A–D, DK8 carrying plasmids pBAD33.atp, pET-22b, and pT7POL26 (FOF1) or pBAD33.Δδ, pET22-atpH-TTG, and pT7POL26 (FOF1−δ; FOF1−δ+δ) was grown as described under “Experimental Procedures.” Seven independent cell batches were prepared in parallel containing the additives indicated. The data represent average values of three independent measurements. A, level of atp mRNA. The amount of atp mRNA was determined via rt-RT-PCR using primer pairs atpEF (dark gray) and atpA (light gray). The amount of atp mRNA present in the FOF1 sample grown with Ara was set to 100%. B, immunoblot analysis of membrane vesicles (20 μg of protein/lane) as indicated. C, ATPase activity of membrane vesicles. Gray and white portions of the bars represent DCCD-sensitive and DCCD-insensitive fractions of ATP hydrolysis, respectively. D, ATP-driven proton translocation of membrane vesicles. The relative magnitude of ACMA fluorescence quenching induced by ATP is shown. Error bars, S.E.
FIGURE 9.
FIGURE 9.
Model for the assembly of E. coli ATP synthase. The assembled c10 ring and b2 are independently present in cytoplasmic membranes. Formation of α3β3γϵ is not known in detail for E. coli. For FOF1 from yeast mitochondria, it has been proposed that two subcomplexes, α3β3 hexamer and γϵ, are assembled, which combine to form α3β3γϵ, before it subsequently binds to the membrane-bound c10 ring (58, 19). The membrane insertion of subunit a is interdependent on the presence of b and c. Integration of b2 into the complex is dependent on subunit δ, whereas binding of a as well as δ to b2 possibly has no preferred sequence.

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