Cross‐Linking of Chloroplast F<sub>0</sub>F<sub>1</sub>‐ATPase Subunit ɛ to γ Without Effect on Activity ɛ and γ are Parts of the Rotor

Schulenberg, Birte; Wellmer, Frank; Lill, Holger; Junge, Wolfgang; Engelbrecht, Siegfried

doi:10.1111/j.1432-1033.1997.t01-1-00134.x

Cited by 47 publications

(28 citation statements)

References 71 publications

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“…Concurrent with tryptic cleavage of ␥ at these sites is the complete loss of inhibition by the ⑀ subunit (8,9). A close physical proximity between the ⑀ and ␥ subunits has also been shown (8,10), consistent with the current view that these two subunits form part of a rotating spindle (11,12). Rotation of the ␥ subunit of CF 1 has been directly observed (13,14).…”

supporting

confidence: 66%

Structural Analysis of the Regulatory Dithiol-containing Domain of the Chloroplast ATP Synthase γ Subunit

Samra

Gao

et al. 2006

Journal of Biological Chemistry

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The ␥ subunit of the F 1 portion of the chloroplast ATP synthase contains a critically placed dithiol that provides a redox switch converting the enzyme from a latent to an active ATPase. The switch prevents depletion of intracellular ATP pools in the dark when photophosphorylation is inactive. The dithiol is located in a special regulatory segment of about 40 amino acids that is absent from the ␥ subunits of the eubacterial and mitochondrial enzymes. Site-directed mutagenesis was used to probe the relationship between the structure of the ␥ regulatory segment and its function in ATPase regulation via its interaction with the inhibitory ⑀ subunit. Mutations were designed using a homology model of the chloroplast ␥ subunit based on the analogous structures of the bacterial and mitochondrial homo-

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supporting

confidence: 66%

Structural Analysis of the Regulatory Dithiol-containing Domain of the Chloroplast ATP Synthase γ Subunit

Samra

Gao

et al. 2006

Journal of Biological Chemistry

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“…4C, the formation of ␥-bcЈ proceeded fast and was almost saturated (43%) in the first 5 min, reflecting high reactivity of both cysteines. 5 Maximal crosslink (60%) was obtained after 2 h. Consistently, the amount of ␥ and bcЈ in nonreducing SDS-PAGE decreased as the incubation time increased. It is noteworthy that the yield of ␥-bcЈ was much higher than that of bcЈ-bcЈ (ϳ15%) despite the fact that two bcЈ are associated (noncovalently) each other as a dimer in the complex.…”

Section: ␥-B Cross-link In Atp Synthasesupporting

confidence: 56%

“…F 1 is easily and reversibly detached from F o by removal of Mg 2ϩ in a low ionic strength solution. F 1 is by itself a rotary motor fueled by ATP hydrolysis (3), in which ␥ and ⑀ subunits rotate relative to ␣ 3 ␤ 3 hexamer (4,5). In the current view of ATP synthase, F o is also thought to be a rotary motor that utilizes the energy of proton movement down the electrochemical potential (6).…”

mentioning

confidence: 99%

Second Stalk of ATP Synthase

Suzuki

Mitome

et al. 2000

Journal of Biological Chemistry

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ATP synthase consists of two portions, F 1 and F o , connected by two stalks: a central rotor stalk containing ␥ and ⑀ subunits and a peripheral, second stalk formed by ␦ and two copies of F o b subunits. The second stalk is expected to keep the stator subunits from spinning along with the rotor. We isolated a TF 1 -b 2 complex (␣ 3 ␤ 3 ␥␦⑀b 2 ) of a thermophilic Bacillus PS3, in which b was a truncated cytoplasmic fragment of F o b subunit, and introduced a cysteine at its N terminus (bc). Association of b 2 or bc 2 with TF 1 did not have significant effect on ATPase activity. A disulfide bond between the introduced cysteine of bc and cysteine 109 of ␥ subunit was readily formed, and this cross-link caused inactivation of ATPase. This implies that F o b subunit bound to stator subunits of F 1 with enough strength to resist rotation of ␥ subunit and to prevent catalysis. Contrary to this apparent tight binding, some detergents such as lauryldodecylamine oxide tend to cause release of b 2 from TF 1 .ATP synthases catalyze ATP synthesis/hydrolysis coupled with a transmembrane proton transport in bacteria, chloroplasts, and mitochondria (1, 2). The enzyme is composed of two portions, a water-soluble F 1 , which has catalytic sites for ATP synthesis/hydrolysis, and a membrane-integrated F o , which mediates proton movement. The bacterial enzyme has the simplest subunit structure, ␣ 3 ␤ 3 ␥ 1 ␦ 1 ⑀ 1 for F 1 and a 1 b 2 c 9 -12 for F o . F 1 is easily and reversibly detached from F o by removal of Mg 2ϩ in a low ionic strength solution. F 1 is by itself a rotary motor fueled by ATP hydrolysis (3), in which ␥ and ⑀ subunits rotate relative to ␣ 3 ␤ 3 hexamer (4, 5). In the current view of ATP synthase, F o is also thought to be a rotary motor that utilizes the energy of proton movement down the electrochemical potential (6). Thus, proton movement through F o drives rotation of the central stalk of ␥⑀, which then enforces each catalytic site in F 1 to synthesize ATP (7). As a reverse reaction, ATP hydrolysis in F 1 drives reverse rotation of the ␥⑀ stalk, which causes proton transport in F o . The concept that ATP synthase consists of two motors connected by a common rotor shaft necessitates the second stalk, which connects and fixes stator subunits of both F 1 and F o together to hold the ␣ 3 ␤ 3 hexamer stationary when the ␥⑀ rotor stalk rotates (8). Recent electron microscopic studies visualized the second, peripheral stalk beside the central stalk (9 -11). Two copies of F o b subunit and a single copy of ␦ subunit of F 1 are the components of the second stalk (12). The ␦ subunit appears to be located near the top of F 1 complex, farthest from the membrane (13-15). F o b, composed of 156 (Escherichia coli) or 167 (thermophilic Bacillus PS3) residues, has a single membrane-spanning helix at its N-terminal region (16), and its C-terminal region interacts with ␦ (17-20). Therefore, the hydrophilic remainder of F o b must span a distance 130 Å from the membrane to the top of F 1 . The hydrophilic domain of F o b is pred...

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“…3). Because the cross-linking between the ␥ and ⑀ subunits has little effect on ATPase activity (33)(34)(35), the ␥ and ⑀ subunits are supposed to rotate together at the same angular velocity. The fact that rotational rates of the ␥ subunit in ␣ 3 ␤ 3 ␥ SA ⑀ and in ␣ 3 ␤ 3 ␥ SA were the same with each other (Fig.…”

Section: Resultsmentioning

confidence: 99%

Direct Observation of the Rotation of ε Subunit in F1-ATPase

Kato-Yamada¹,

Noji²,

Yasuda³

et al. 1998

Journal of Biological Chemistry

182

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Cross‐Linking of Chloroplast F₀F₁‐ATPase Subunit ɛ to γ Without Effect on Activity ɛ and γ are Parts of the Rotor

Cited by 47 publications

References 71 publications

Structural Analysis of the Regulatory Dithiol-containing Domain of the Chloroplast ATP Synthase γ Subunit

Structural Analysis of the Regulatory Dithiol-containing Domain of the Chloroplast ATP Synthase γ Subunit

Second Stalk of ATP Synthase

Direct Observation of the Rotation of ε Subunit in F1-ATPase

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Cross‐Linking of Chloroplast F0F1‐ATPase Subunit ɛ to γ Without Effect on Activity ɛ and γ are Parts of the Rotor

Cited by 47 publications

References 71 publications

Structural Analysis of the Regulatory Dithiol-containing Domain of the Chloroplast ATP Synthase γ Subunit

Structural Analysis of the Regulatory Dithiol-containing Domain of the Chloroplast ATP Synthase γ Subunit

Second Stalk of ATP Synthase

Direct Observation of the Rotation of ε Subunit in F1-ATPase

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Cross‐Linking of Chloroplast F₀F₁‐ATPase Subunit ɛ to γ Without Effect on Activity ɛ and γ are Parts of the Rotor