Transmembrane Topography of Subunit a in theEscherichia coli F1F0 ATP Synthase

Valiyaveetil, Francis I.; Fillingame, Robert H.

doi:10.1074/jbc.273.26.16241

Cited by 136 publications

(126 citation statements)

References 35 publications

(40 reference statements)

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“…The a-subunit of the bovine enzyme and the equivalent subunit of the T. thermophilus enzyme are expected to have dramatically different topologies. The F-type ATP synthase a-subunit has been proposed to contain five transmembrane helices (34,35), whereas the equivalent T. thermophilus subunit is thought to contain eight transmembrane α-helices (29,36). Despite this difference, both subunits appear to form structures that present the midmembrane ends of both proton-conducting half-channels to the c-ring with a small contact area.…”

Section: Discussionmentioning

confidence: 99%

Arrangement of subunits in intact mammalian mitochondrial ATP synthase determined by cryo-EM

Baker

Watt

Runswick

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

115

109

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Mitochondrial ATP synthase is responsible for the synthesis of ATP, a universal energy currency in cells. Whereas X-ray crystallography has revealed the structure of the soluble region of the complex and the membrane-intrinsic c-subunits, little is known about the structure of the six other proteins (a, b, f, A6L, e, and g) that comprise the membrane-bound region of the complex in animal mitochondria. Here, we present the structure of intact bovine mitochondrial ATP synthase at ∼18 Å resolution by electron cryomicroscopy of single particles in amorphous ice. The map reveals that the a-subunit and c 8 -ring of the complex interact with a small contact area and that the b-subunit spans the membrane without contacting the c 8 -ring. The e-and g-subunits extend from the a-subunit density distal to the c 8 -ring. The map was calculated from images of a preparation of the enzyme solubilized with the detergent dodecyl maltoside, which is visible in electron cryomicroscopy maps. The structure shows that the micelle surrounding the complex is curved. The observed bend in the micelle of the detergent-solubilized complex is consistent with previous electron tomography experiments and suggests that monomers of ATP synthase are sufficient to produce curvature in lipid bilayers.A TP synthases are responsible for the synthesis of ATP from ADP and inorganic phosphate. In mammalian mitochondria, the enzyme is an ∼600-kDa membrane protein complex comprised of a catalytic F 1 region and a membrane-bound F O region. The F 1 region consists of subunits α 3 β 3 γδε (1, 2) and the F O region consists of subunits abc 8 defg(A6L)F 6 (3). The F 1 and F O regions are connected by a central stalk, comprised of the γ-, δ-, and ε-subunits from the F 1 region, and a peripheral stalk, comprised of the oligomycin sensitivity conferral protein (OSCP) and subunits b, d, and F 6 from the F O region (4, 5). Previously, electron cryomicroscopy (cryo-EM) of intact detergent-solubilized bovine ATP synthase particles embedded in a thin layer of amorphous ice revealed the overall shape of the complex at 32 Å resolution (6), and subsequent analysis of the Saccharomyces cerevisiae enzyme produced a similar map at 24 Å resolution (7). X-ray crystallography of subcomplexes of the bovine enzyme has defined the arrangement of subunits in the F 1 region (8) and the peripheral stalk (9, 10) and showed the presence of a ring of eight c-subunits in the F O region (11). The structure and arrangement of the remaining subunits in the membrane-bound region of the enzyme are not known.The ATP synthase functions by a rotary catalytic mechanism. Proton translocation through the F O region requires the a-, b-, and c-subunits (12-14) and induces rotation of the membranebound c 8 -ring (15). The structure of the c 8 -ring is thought to be stabilized by binding of cardiolipin to a lysine residue conserved throughout animalia that has been shown to be trimethylated at the ε-amino group in all animal ATP synthases tested (11,16,17). The c 8 -ring is attached to the central st...

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Section: Discussionmentioning

confidence: 99%

Arrangement of subunits in intact mammalian mitochondrial ATP synthase determined by cryo-EM

Baker

Watt

Runswick

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

115

109

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“…Chemical cross-linking of purified ATP synthase (22) or F 0 (23) indicates that a and b are proximal, and analysis of second-site revertants to a bG9D mutation identified residue 240 of the a subunit, within the fifth putative transmembrane helix (24,25), as a site that may be close to b within the membrane (26). The propensity of cysteines introduced within residues 2-18 of b to form disulfides suggests that the two b subunits are adjacent to one another in the membrane (27).…”

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confidence: 99%

Site-directed Cross-linking of b to the α, β, anda Subunits of the Escherichia coli ATP Synthase

McLachlin¹,

Coveny

Clark

et al. 2000

Journal of Biological Chemistry

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The b subunit dimer of the Escherichia coli ATP synthase, along with the ␦ subunit, is thought to act as a stator to hold the ␣ 3 ␤ 3 hexamer stationary relative to the a subunit as the ␥⑀c 9 -12 complex rotates. Despite their essential nature, the contacts between b and the ␣, ␤, and a subunits remain largely undefined. We have introduced cysteine residues individually at various positions within the wild type membrane-bound b subunit, or within b 24 -156 , a truncated, soluble version consisting only of the hydrophilic C-terminal domain. The introduced cysteine residues were modified with a photoactivatable cross-linking agent, and cross-linking to subunits of the F 1 sector or to complete ATP synthase, or F 1 F 0 -ATPase, utilizes a transmembrane proton gradient to synthesize ATP and is responsible for the final step in oxidative phosphorylation and photophosphorylation. The enzyme (reviewed in Refs. 1-3) is composed of two sectors. The membrane-integral F 0 sector is a proton pore, and in Escherichia coli has a subunit composition of ab 2 c 9 -12 . The membrane-peripheral F 1 sector has a subunit stoichiometry of ␣ 3 ␤ 3 ␥␦⑀. A key feature of the F 1 sector, as seen in the bovine heart mitochondrial crystal structure (4), is that the ␣ and ␤ subunits alternate in a ring around a lengthy pair of ␣-helices of ␥. Each ␤ subunit bears one catalytic nucleotide-binding site, while non-catalytic nucleotide-binding sites are found on the ␣ subunits. These nucleotide-binding sites are located close to the interfaces between ␣ and ␤ subunits, with one site near each of the six interfaces.Subunits from each sector contribute to the formation of two stalks that join F 1 and F 0 . The ␥ and ⑀ subunits form the central stalk, rotation of which is believed to be caused by translocation of protons across the membrane by the a and c subunits. This rotation is thought to cause conformational changes in the catalytic sites, driving synthesis of ATP (1). It is believed that the ␣ and ␤ subunits are prevented from rotating by a peripheral stalk consisting of ␦ and the two b subunits (5, 6) that joins the ␣ 3 ␤ 3 complex to the a subunit.In recent years the interaction of the b dimer and ␦ has been well established by a variety of evidence (7-10). The ␦ subunit appears to be located near the crown of the F 1 complex, the part of F 1 furthest from the membrane (11-15). Because b has a single membrane-spanning region at its N terminus, the remainder of the subunit must span a distance of over 100 Å to come in contact with ␦. Consistent with this proposed arrangement, the region of interaction between b and ␦ has been localized to the C terminus of b (10, 16). The hydrophilic domain of b by itself is mostly ␣-helical as measured by circular dichroism (17), and an isolated complex composed of ␦ with the hydrophilic domain of b was demonstrated by sedimentation velocity ultracentrifugation to be highly extended (9). The dimerization domain of b, encompassing residues 53-122, was also shown to be highly extended (18). Therefore the b 2...

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“…Subunit a consists of five transmembrane helices, four of which likely interact as a four-helix bundle (31)(32)(33)(34). Subunit a lies on the periphery of the c-ring with TMHs 4 and 5 from subunit a and TMH2 from subunit c forming the a-c interface (35,36).…”

Section: F 0 Reconstituted Into Liposomes the Results Suggest That Tmentioning

confidence: 99%

Residues in the Polar Loop of Subunit c in Escherichia coli ATP Synthase Function in Gating Proton Transport to the Cytoplasm

Steed

Fillingame

2014

Journal of Biological Chemistry

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Transmembrane Topography of Subunit a in theEscherichia coli F1F0 ATP Synthase

Cited by 136 publications

References 35 publications

Arrangement of subunits in intact mammalian mitochondrial ATP synthase determined by cryo-EM

Arrangement of subunits in intact mammalian mitochondrial ATP synthase determined by cryo-EM

Site-directed Cross-linking of b to the α, β, anda Subunits of the Escherichia coli ATP Synthase

Residues in the Polar Loop of Subunit c in Escherichia coli ATP Synthase Function in Gating Proton Transport to the Cytoplasm

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