Subunit b of the membrane moiety (F0) of ATP synthase (F1F0) from Escherichia coli is indispensable for H+ translocation and binding of the water-soluble F1 moiety.

Schneider, Erwin; Altendorf, Karlheinz

doi:10.1073/pnas.81.23.7279

Cited by 70 publications

(58 citation statements)

References 29 publications

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“…Purification of Subunit a-After labeling, membrane vesicles were resuspended in 100 mM Tris-HCl (pH 8.0), 1.5% octyl glucoside, 0.1% deoxycholate, 0.5% cholate, 10 mM ␤-mercaptoethanol, 10 mM imidazole, and 1% Tween 20 (53). The samples were incubated with agitation for 1 h at 4°C and centrifuged at 14,000 rpm (16,000 ϫ g) for 10 min in a microcentrifuge (1.5 ml).…”

Section: Methodsmentioning

confidence: 99%

Membrane Topology of Subunit a of the F1F0 ATP Synthase as Determined by Labeling of Unique Cysteine Residues

Long

Wang²,

Vik

1998

Journal of Biological Chemistry

117

116

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The membrane topology of the a subunit of the F 1 F 0 ATP synthase from Escherichia coli has been probed by surface labeling using 3-(N-maleimidylpropionyl) biocytin. Subunit a has no naturally occurring cysteine residues, allowing unique cysteines to be introduced at the following positions: 8, 24, 27, 69, 89, 128, 131, 172, 176, 196, 238, 241, and 277 (following the COOH-terminal 271 and a hexahistidine tag). None of the single mutations affected the function of the enzyme, as judged by growth on succinate minimal medium. Membrane vesicles with an exposed cytoplasmic surface were prepared using a French pressure cell. Before labeling, the membranes were incubated with or without a highly charged sulfhydryl reagent, 4-acetamido-4-maleimidylstilbene-2,2-disulfonic acid. After labeling with the less polar biotin maleimide, the samples were solubilized with octyl glucoside/cholate and the subunit a was purified via the oligohistidine at its COOH terminus using immobilized nickel chromatography. The purified samples were electrophoresed and transferred to nitrocellulose for detection by avidin conjugated to alkaline phosphatase. Results indicated cytoplasmic accessibility for residues 69, 172, 176, and 277 and periplasmic accessibility for residues 8, 24, 27, and 131. On the basis of these and earlier results, a transmembrane topology for the subunit a is proposed.The F 1 F 0 ATP synthase from Escherichia coli is typical of the ATP synthases found in mitochondria, chloroplasts, and many other bacteria (for recent reviews, see Refs.

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Section: Methodsmentioning

confidence: 99%

Membrane Topology of Subunit a of the F1F0 ATP Synthase as Determined by Labeling of Unique Cysteine Residues

Long

Wang²,

Vik

1998

Journal of Biological Chemistry

117

116

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“…First, they may be important for anchoring the b subunits to a, maintaining the integrity of the stator complex. Second, given the observation that the b subunit is required for the formation of a functional proton pore upon reconstitution of F 0 from the purified subunits (47,48), the interactions between b and a in this region may have a role in assembly and organization of F 0 . This idea is further supported by evidence implicating the second stalk in the maturation of F 0 to a proton-conducting state (49,50).…”

Section: Figmentioning

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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“…Growth Polyclonal antibodies to F1F0 ATPase. The Fo ATPase was purified as described by Schneider and Altendorf (18). This preparation, which contained primarily Fo subunits but was contaminated with F1 subunits, was injected into rabbits for the purpose of raising polyclonal antibodies against Fo subunits.…”

Section: Methodsmentioning

confidence: 99%

Role of the delta subunit in enhancing proton conduction through the F0 of the Escherichia coli F1F0 ATPase

Monticello

Brusilow

1994

J Bacteriol

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We studied the effect of the 8 subunit of the Escherichia coli F1 ATPase on the proton permeability of the Fo proton channel synthesized and assembled in vivo. Membranes isolated from an unc deletion strain carrying a plasmid containing the genes for the Fo subunits and the 8 subunit were significantly more permeable to protons than membranes isolated from the same strain carrying a plasmid containing the genes for the Fo subunits alone. This increased proton permeability could be blocked by treatment with either dicyclohexylcarbodiimide or purified Fl, both of which block proton conduction through the Fo. After reconstitution with purified F1 in vitro, both membrane preparations could couple proton pumping to ATP hydrolysis. These results demonstrate that an interaction between the 8 subunit and the Fo during synthesis and assembly produces a significant change in the proton permeability of the Fo proton channel. We have proposed a model for Fo assembly in which the Fo is synthesized and assembled in a relatively proton-impermeable form (3,15). Instead of the F1 subunits catalyzing membrane insertion of certain Fo subunits, the Fo subunits are inserted spontaneously and assemble in an immature form. It is through interactions with F1 subunits that this immature "closed" channel is opened, and the flow of protons through the complex is then regulated by the activity of the F1 sector. In this study, we examined the role of the 8 subunit of the F1 sector during assembly of the Fo sector. The 8 subunit is coded for by the uncH gene, which is the first F1 gene in the operon, located immediately following the Fo genes. Previous genetic studies implicated the 8 subunit as causing the F1-dependent proton permeability of the Fo (1 MATERIALS AND METHODSStrains and plasmids. These studies used E. coli JM103 A(uncB-uncD), which is strain JM103 (11) with a deletion of seven of the nine unc genes, including all the Fo genes (10).Plasmids pEA5, pWSB30.0, and pWSB33 were described previously (13, 22). pRM1, which carries the Fo genes plus uncH (S subunit) cloned behind the lac promoter, was constructed by digesting pWSB30.0 with HindIll and EcoRI and ligating the resulting fragments with the pUC9 (23) vector, which had been digested with HindIIl and EcoRI. pRM1 was digested with AflII, which cuts at the stop codon for uncH, treated with mung bean nuclease to form blunt ends, and then ligated with a Sall adapter (5'-TGGTGTCGACACCA-3') to produce plasmid pRM6. pRM7, which carries the Fo genes and all of uncH fused in frame to a biotinylation sequence, was constructed by ligating the SalI-EcoRI fragment from the biotinylation vector YEp352-Bio7 into pRM6, which had been digested with the same enzymes. YEp352-Bio7 was a generous gift from A. Tzagoloff. Treatment with mung bean nuclease and insertion of the Sall adapter removed the stop codon for uncH and allowed the biotin attachment sequence to be cloned in frame. This region of the resultant plasmid was sequenced to verify the construction. pRM8, which encodes the uncH-biotin a...

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Subunit b of the membrane moiety (F0) of ATP synthase (F1F0) from Escherichia coli is indispensable for H+ translocation and binding of the water-soluble F1 moiety.

Cited by 70 publications

References 29 publications

Membrane Topology of Subunit a of the F1F0 ATP Synthase as Determined by Labeling of Unique Cysteine Residues

Membrane Topology of Subunit a of the F1F0 ATP Synthase as Determined by Labeling of Unique Cysteine Residues

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

Role of the delta subunit in enhancing proton conduction through the F0 of the Escherichia coli F1F0 ATPase

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