Transmembrane topology of Escherichia coli H+‐ATPase (ATP synthase) subunit a

Yamada, Hiroshi; Moriyama, Yoshinori; Maeda, Masatomo; Futai, Masamitsu

doi:10.1016/0014-5793(96)00621-7

Cited by 34 publications

(13 citation statements)

References 25 publications

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“…Yamada et al (18) reported a preliminary topographical model for subunit a based upon the binding of three antipeptide-directed antisera to ISO membrane vesicles bound to microtiter plates. The location of the COOH terminus and a cytoplasmic loop agrees with the topographical model predicted here, but the NH 2 terminus was also concluded to be cytoplasmic, leading to a six-helix model.…”

Section: Discussionmentioning

confidence: 99%

Transmembrane Topography of Subunit a in theEscherichia coli F1F0 ATP Synthase

Valiyaveetil

Fillingame

1998

Journal of Biological Chemistry

136

117

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Subunit a is the least understood of the three subunits that compose the F 0 sector in the Escherichia coli F 0 F 1 ATP synthase. In this study, we have substituted Cys into predicted extramembranous loops of the protein and used chemical modification to obtain topographical information on the folding of subunit a. The extent of labeling of the substituted Cys residues by fluorescein-5-maleimide was determined. The localization of reactive Cys residues was inferred from differences in the extent of labeling in inside out and right side out membrane vesicles. The NH 2 -terminal segment of subunit a was localized to the outside (periplasmic) surface and the COOH terminus to the cytoplasmic surface by these procedures. Loop residues in two periplasmic extramembranous loops and in two cytoplasmic extramembranous loops were also localized. The localization of two cytoplasmic Cys residues was confirmed by using 4-acetamido-4-maleimidylstilbene-2,2-disulfonic acid to block fluorescein-5-maleimide labeling. From the localization of the Cys residues, a model for the topography is proposed that consists of five transmembrane segments with the NH 2 terminus periplasmic and the COOH terminus cytoplasmic. The positions of second site suppressors, including several isolated here to the nonfunctional E219C and H245C substitutions, provide support for the topographical model proposed.

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Section: Discussionmentioning

confidence: 99%

Transmembrane Topography of Subunit a in theEscherichia coli F1F0 ATP Synthase

Valiyaveetil

Fillingame

1998

Journal of Biological Chemistry

136

117

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“…In particular, studies using the labeling of cysteine substitutions (18,19), epitope insertions (20), and peptide-directed antibodies (20,21), to identify surface accessible regions of the protein have come to significant agreement. However, the location of the N terminus remains controversial, leading to models of five-or six-transmembrane spans.…”

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confidence: 99%

“…Studies using the labeling of cysteine substitutions (18,19) concluded five-transmembrane spans, with a periplasmic N terminus. Studies using epitope insertion and peptide-directed antibodies (20,21) found cytoplasmic localization of the N terminus, leading to six-transmembrane spans. All of the studies required the preparation of oriented membrane vesicles for probing potentially accessible surface residues or regions.…”

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confidence: 99%

A Novel Labeling Approach Supports the Five-transmembrane Model of Subunit a of the Escherichia coli ATP Synthase

Wada

Long

Zhang

et al. 1999

Journal of Biological Chemistry

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Cysteine mutagenesis and surface labeling has been used to define more precisely the transmembrane spans of subunit a of the Escherichia coli ATP synthase. Regions of subunit a that are exposed to the periplasmic space have been identified by a new procedure, in which cells are incubated with polymyxin B nonapeptide (PMBN), an antibiotic derivative that partially permeabilizes the outer membrane of E. coli, along with a sulfhydryl reagent, 3-(N-maleimidylpropionyl) biocytin (MPB). This procedure permits reaction of sulfhydryl groups in the periplasmic space with MPB, but residues in the cytoplasm are not labeled. Using this procedure, residues 8, 27, 37, 127, 131, 230, 231, and 232 were labeled and so are thought to be exposed in the periplasm. Using inside-out membrane vesicles, residues near the end of transmembrane spans 1, 64, 67, 68, 69, and 70 and residues near the end of transmembrane spans 5, 260, 263, and 265 were labeled. Residues 62 and 257 were not labeled. None of these residues were labeled in PMBNpermeabilized cells. These results provide a more detailed view of the transmembrane spans of subunit a and also provide a simple and reliable technique for detection of periplasmic regions of inner membrane proteins in E. coli.The ATP synthase from Escherichia coli is typical of the ATP synthases found in mitochondria, chloroplasts, and many other bacteria (for reviews, see Refs. 1-3). It contains an F 1 sector, with subunits for nucleotide binding and catalysis, and an F 0 sector, which conducts protons across the membrane. Five different subunits are found in the E. coli F 1 : ␣, ␤, ␥, ␦, and ⑀, in a stoichiometry of 3:3:1:1:1. Three different subunits named a, b, and c form the E. coli F 0 with a stoichiometry of 1:2:12 (4) . The mechanism by which an electrochemical proton gradient across the membrane drives ATP synthesis is thought to involve a rotary mechanism. The crystallization of F 1 from bovine mitochondria (5) led to a high resolution structure of the ␣ 3 ␤ 3 hexamer, plus parts of ␥ in the central core . Subsequently, the hypothesis of rotation of ␥ and ⑀ and relative to ␣ 3 ␤ 3 has been supported by direct visualization of rotation of fluorescently labeled actin filaments covalently attached to ␥ (6) or ⑀ (7). It has been proposed that F 0 subunit c drives the rotation of ␥ and ⑀ as a rotor (8), whereas subunits a and b function as the stator. Recent theoretical work has indicated the feasibility of this proposal (9), but as of yet there is no direct evidence of rotation by F 0 subunits.Information about the tertiary and quaternary structure of F 0 subunits will be necessary for an understanding of how F 0 translocates protons, and how it might drive rotation of ␥ and ⑀ subunits in F 1 . Subunit b seems to be embedded in the membrane via a span of hydrophobic amino acids at its N terminus. A truncated, soluble form of b has been shown to be extended and dimeric (10) . Recent NMR studies of c have confirmed the ␣-helical hairpin structure of the two predicted transmembrane spans, and also ...

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“…The b subunits attach the a subunit to an ␣ subunit of the ␣ 3 ␤ 3 headpiece via the ␦ subunit (18,19). The a subunit is an integral membrane protein consisting of five or six membranespanning ␣-helices (20)(21)(22)(23). According to NMR analyses of E. coli subunit c in chloroform͞methanol͞water, the two helices pack closely to one another, extending over 40 and 30 residues, respectively, with a short interruption after D61 of the Cterminal ␣-helix (24).…”

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Energy transduction in the sodium F-ATPase of Propionigenium modestum

Dimroth

Wang

Grabe

et al. 1999

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

145

142

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The F-ATPase of the bacterium Propionigenium modestum is driven by an electrochemical sodium gradient between the cell interior and its environment. Here we present a mechanochemical model for the transduction of transmembrane sodium-motive force into rotary torque. The same mechanism is likely to operate in other F-ATPases, including the proton-driven F-ATPases of Escherichia coli.The culmination of metabolism is the generation of ATP from a transmembrane electromotive gradient. This conversion of electrochemical energy into the chemical energy of the terminal phosphoric anhydride bond of ATP is accomplished by the enzyme ATP synthase-also called F 1 F o -ATP synthase, or F 1 F o -ATPase. The latter name reflects the fact that the enzyme is reversible and can act as a proton (or Na ϩ ) -pumping ATPase. Closely related ATP synthases are found in the bacterial cytoplasmic membrane, the inner membrane of mitochondria, and the thylakoid membrane of chloroplasts. These enzymes consist of a membrane-embedded portion, F o , which contains the ion channel, and the soluble F 1 moiety, which harbors the nucleotide-binding sites in which ATP is synthesized or hydrolyzed (for reviews see refs. 1-6). Fig. 1 shows a schematic diagram of ATP synthase that summarizes a great deal of experimental work. Isolated F 1 (also called F 1 -ATPase) hydrolyzes ATP and is composed of subunits ␣ 3 ␤ 3 ␥␦ (subscripts refer to the subunit stoichiometry). The majority of the ␣ 3 ␤ 3 ␥ structure has been solved by x-ray crystallography (7). The structure shows a cylinder formed by the three ␣ and three ␤ subunits alternating around a central cavity through which the asymmetrically bent shaft of the ␥ subunit extends and protrudes. The ␥ subunit asymmetry correlates with the different conformations assumed by each of the three catalytic ␤s, in accordance to Boyer's ''binding change mechanism '' (8). This strongly supports the view that the binding changes of the ␤ subunits are coupled to the rotation of the ␥ subunit. This rotational coupling is confirmed by several recent studies which demonstrate unequivocally that ATP hydrolysis drives rotation of the ␥ subunit within the ␣ 3 ␤ 3 ␥ subcomplex of F 1 (9-12), and that subunit rotates with ␥ as a unit (13,14). The rotation is reversed during ATP synthesis, driven by the downhill movement of the coupling ions through the F o sector.The F o portion of ATP synthase has been most intensely studied with the enzymes from the bacteria Escherichia coli and Propionigenium modestum. The subunit composition of F o is ab 2 c 12 . The c subunit consists of twin membrane-spanning ␣-helices that are connected by a cytoplasmic loop. Evidence from electron and atomic force microscopy indicates that the c subunits are assembled into a 12-unit ring that is flanked at the periphery by the a and the two b subunits (15-17). The b subunits attach the a subunit to an ␣ subunit of the ␣ 3 ␤ 3 headpiece via the ␦ subunit (18, 19). The a subunit is an integral membrane protein consisting of five or six membranes...

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Transmembrane topology of Escherichia coli H⁺‐ATPase (ATP synthase) subunit a

Cited by 34 publications

References 25 publications

Transmembrane Topography of Subunit a in theEscherichia coli F1F0 ATP Synthase

Transmembrane Topography of Subunit a in theEscherichia coli F1F0 ATP Synthase

A Novel Labeling Approach Supports the Five-transmembrane Model of Subunit a of the Escherichia coli ATP Synthase

Energy transduction in the sodium F-ATPase of Propionigenium modestum

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