Abstract:Bacterial operons for F1Fo-ATP synthase typically include an uncI gene that encodes a function-unknown small hydrophobic protein.When we expressed a hybrid F 1Fo (F1 from thermophilic Bacillus PS3 and Na ؉ -translocating Fo from Propionigenium modestum) in Escherchia coli cells, we found that uncI derived from P. modestum was indispensable to produce active enzyme; without uncI, csubunits in F 1Fo existed as monomers but not as functional c11-ring. When uncI was expressed from another plasmid at the same time,… Show more
“…Spectroscopic data indeed show that the central stalk (subunits γ and ε) of the I. tartaricus ATP synthase is able to interact with c n rings of variable diameter [at least from n = 11-15 (5.5 and 6.5 nm in diameter, respectively)] with wild type-like affinity. Our findings are consistent with the observation that a given F 1 domain derived from either E. coli or Bacillus PS3 can dock either with its native c 10 ring or with c 11 rings from Propionigenium modestum, forming chimeric F 1 F o ATP synthases (39,40). Taken together, these studies suggest a high degree of structural compliance in the assembly of the rotor subcomplex (c n γε).…”
ATP synthase membrane rotors consist of a ring of c-subunits whose stoichiometry is constant for a given species but variable across different ones. We investigated the importance of c/c-subunit contacts by site-directed mutagenesis of a conserved stretch of glycines (GxGxGxGxG) in a bacterial c 11 ring. Structural and biochemical studies show a direct, specific influence on the c-subunit stoichiometry, revealing c <11 , c 12 , c 13 , c 14 , and c >14 rings. Molecular dynamics simulations rationalize this effect in terms of the energetics and geometry of the c-subunit interfaces. Quantitative data from a spectroscopic interaction study demonstrate that the complex assembly is independent of the c-ring size. Real-time ATP synthesis experiments in proteoliposomes show the mutant enzyme, harboring the larger c 12 instead of c 11 , is functional at lower ion motive force. The high degree of compliance in the architecture of the ATP synthase rotor offers a rationale for the natural diversity of c-ring stoichiometries, which likely reflect adaptations to specific bioenergetic demands. These results provide the basis for bioengineering ATP synthases with customized ion-to-ATP ratios, by sequence modifications.alpha helix packing | F 1 F o ATP synthase | membrane protein | rotary motor stoichiometry | bioenergetics
“…Spectroscopic data indeed show that the central stalk (subunits γ and ε) of the I. tartaricus ATP synthase is able to interact with c n rings of variable diameter [at least from n = 11-15 (5.5 and 6.5 nm in diameter, respectively)] with wild type-like affinity. Our findings are consistent with the observation that a given F 1 domain derived from either E. coli or Bacillus PS3 can dock either with its native c 10 ring or with c 11 rings from Propionigenium modestum, forming chimeric F 1 F o ATP synthases (39,40). Taken together, these studies suggest a high degree of structural compliance in the assembly of the rotor subcomplex (c n γε).…”
ATP synthase membrane rotors consist of a ring of c-subunits whose stoichiometry is constant for a given species but variable across different ones. We investigated the importance of c/c-subunit contacts by site-directed mutagenesis of a conserved stretch of glycines (GxGxGxGxG) in a bacterial c 11 ring. Structural and biochemical studies show a direct, specific influence on the c-subunit stoichiometry, revealing c <11 , c 12 , c 13 , c 14 , and c >14 rings. Molecular dynamics simulations rationalize this effect in terms of the energetics and geometry of the c-subunit interfaces. Quantitative data from a spectroscopic interaction study demonstrate that the complex assembly is independent of the c-ring size. Real-time ATP synthesis experiments in proteoliposomes show the mutant enzyme, harboring the larger c 12 instead of c 11 , is functional at lower ion motive force. The high degree of compliance in the architecture of the ATP synthase rotor offers a rationale for the natural diversity of c-ring stoichiometries, which likely reflect adaptations to specific bioenergetic demands. These results provide the basis for bioengineering ATP synthases with customized ion-to-ATP ratios, by sequence modifications.alpha helix packing | F 1 F o ATP synthase | membrane protein | rotary motor stoichiometry | bioenergetics
“…Purified subunit c (Atp9p) of E. coli (46) and thermophilic Bacillus PS3 (47) have been reported to spontaneously oligomerize into rings from detergent solutions. Other studies, however, indicate that conversion of bacterial subunit c to the ring is mediated by the uncI gene product of E. coli (48,49). Ring formation in S. cerevisiae has also been shown to require a protein encoded by the N-terminal half of the yeast nuclear ATP25 gene (50).…”
Background:The Atp9p rotor is an assembly module of mitochondrial ATP synthase. Results: Newly translated Atp9p and Cox6p, a subunit of yeast cytochrome oxidase (COX), are present in large complexes. Conclusion: Atp9p-Cox6p complexes serve as a source of Atp9p for rotor formation. Significance: We propose that Cox6p enhances the efficiency of Atp9p ring formation and may also coordinate balanced expression of COX and ATP synthase.
“…However, instead of additional regulatory proteins, unique polar interactions at the rotorstator interface of the F 1 subunits allow almost exclusively unidirectional rotation in the ATP synthase direction for this enzyme [31]. To our knowledge, only two other bacterial proteins have been found encoded in the atp operon in addition to the eight core subunits of bacterial F 1 F 0 (α, β, γ, δ, ε, a, b, c); these two proteins are encoded by the unc-I and urf-6 genes that correspond, respectively, to an assembly factor of the c-ring [32] and to majastridin, a cytosolic protein nonassociated with the Rhodospirillum blasticus ATP synthase [33]. In contrast, the gene encoding the 11-kDa protein of P. denitrificans is located upstream to both atp operons (one for F 0 and another for F 1 subunits) already sequenced on chromosome II of P. denitrificans (see Morales-Ríos et al 2008, submitted, and the following link: http://genome.jgi-psf.org/finished_microbes/parde/parde.home.htm).…”
Section: The Central Stalk Is Part Of the Atp Synthase Rotormentioning
The F 1 F 0 -adenosine triphosphate (ATP) synthase rotational motor synthesizes most of the ATP required for living from adenosine diphosphate, Pi, and a proton electrochemical gradient across energy-transducing membranes of bacteria, chloroplasts, and mitochondria. However, as a reversible nanomotor, it also hydrolyzes ATP during de-energized conditions in all energy-transducing systems. Thus, different subunits and mechanisms have emerged in nature to control the intrinsic rotation of the enzyme to favor the ATP synthase activity over its opposite and commonly wasteful ATPase turnover. Recent advances in the structural analysis of the bacterial and mitochondrial ATP synthases are summarized to review the distribution and mechanism of the subunits that are part of the central rotor and regulate its gyration. In eubacteria, the ε subunit works as a ratchet to favor the rotation of the central stalk in the ATP synthase direction by extending and contracting two α-helixes of its C-terminal side and also by binding ATP with low affinity in thermophilic bacteria. On the other hand, in bovine heart mitochondria, the so-called inhibitor protein (IF 1 ) interferes with the intrinsic rotational mechanism of the central γ subunit and with the opening and closing of the catalytic β-subunits to inhibit its ATPase activity. Besides its inhibitory role, the IF 1 protein also promotes the dimerization of the bovine and rat mitochondrial enzymes, albeit it is not essential for dimerization of the yeast F 1 F 0 mitochondrial complex. High-resolution electron microscopy of the dimeric enzyme in its bovine and yeast forms shows a conical shape that is compatible with the role of the ATP synthase dimer in the formation of tubular the cristae membrane of mitochondria after further oligomerization. Dimerization of the mitochondrial ATP synthase diminishes the rotational drag of the central rotor that would decrease the coupling efficiency between rotation of the central stalk and ATP synthesis taking place at the F 1 portion. In addition, F 1 F 0 dimerization and its further oligomerization also increase the stability of the enzyme to natural or experimentally induced destabilizing conditions.
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