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1.
A homodimer of b subunits constitutes the peripheral stalk linking the F1 and F0 sectors of the Escherichia coli ATP synthase. Each b subunit has a single-membrane domain. The constraints on the membrane domain have been studied by systematic mutagenesis. Replacement of a segment proximal to the cytoplasmic side of the membrane had minimal impact on F1F0 ATP synthase. However, multiple substitutions on the periplasmic side resulted in defects in assembly of the enzyme complex. These mutants had insufficient oxidative phosphorylation to support growth, and biochemical studies showed little F1F0 ATPase and no detectable ATP-driven proton pumping activity. Expression of the b
N2A,T6A,Q10A
subunit was also oxidative phosphorylation deficient, but the b
N2A,T6A,Q10A
protein was incorporated into an F1F0 complex. Single amino acid substitutions had minimal reductions in F1F0 ATP synthase function. The evidence suggests that the b subunit membrane domain has several sites of interaction contributing to assembly of F0, and that these interactions are strongest on the periplasmic side of the bilayer. 相似文献
2.
Bhatt D Cole SP Grabar TB Claggett SB Cain BD 《Journal of bioenergetics and biomembranes》2005,37(2):67-74
The peripheral stalk of F1F0 ATP synthase is composed of a parallel homodimer of b subunits that extends across the cytoplasmic membrane in F0 to the top of the F1 sector. The stalk serves as the stator necessary for holding F1 against movement of the rotor. A series of insertions and deletions have been engineered into the hydrophilic domain that interacts with F1. Only the hydrophobic segment from {val-121} to {ala-132} and the extreme carboxyl terminus proved to be highly sensitive to mutation. Deletions in either site apparently abolished enzyme function as a result of defects is assembly of the F1F0 complex. Other mutations manipulating the length of the sequence between these two areas had only limited effects on enzyme function. Expression of a b subunit with insertions with as few as two amino acids into the hydrophobic segment also resulted in loss of F1F0 ATP synthase. However, a fully defective b subunit with seven additional amino acids could be stabilized in a heterodimeric peripheral stalk within a functional F1F0 complex by a normal b subunit. 相似文献
3.
Robert H. Fillingame 《Journal of bioenergetics and biomembranes》1992,24(5):485-491
The F0 sector of the ATP synthase complex facilitates proton translocation through the membrane, and via interaction with the F1 sector, couples proton transport to ATP synthesis. The molecular mechanism of function is being probed by a combination of mutant analysis and structural biochemistry, and recent progress on theEscherichia coli F0 sector is reviewed here. TheE. coli F0 is composed of three types of subunits (a, b, andc) and current information on their folding and organization in F0 is reviewed. The structure of purified subunitc in chloroform-methanol-H2O resembles that in native F0, and progress in determining the structure by NMR methods is reviewed. Genetic experiments suggest that the two helices of subunitc must interact as a functional unit around an essential carboxyl group as protons are transported. In addition, a unique class of suppressor mutations identify a transmembrane helix of subunita that is proposed to interact with the bihelical unit of subunitc during proton transport. The role of multiple units of subunitc in coupling proton translocation to ATP synthesis is considered. The special roles of Asp61 of subunitc and Arg210 of subunita in proton translocation are also discussed. 相似文献
4.
Ferruccio Guerrieri Martin Kalous Edmondo Adorisio Nicola Turturro Zdenek Drahota Palmiro Cantatore Giuseppe Santoro 《Journal of bioenergetics and biomembranes》1998,30(3):269-276
In liver mitochondria isolated from hypothyroid rats, the rate of ATP synthesis is lower than in mitochondria from normal rats. Oligomycin-sensitive ATP hydrolase activity and passive proton permeability were significantly lower in submitochondrial particles from hypothyroid rats compared to those isolated from normal rats. In mitochondria from hypothyroid rats, the changes in catalytic activities of F0F1-ATP synthase are accompanied by a decrease in the amount of immunodetected -F1, F01-PVP, and OSCP subunits of the complex. Northern blot hybridization shows a decrease in the relative cytosolic content of mRNA for -F1 subunit in liver of hypothyroid rats. Administration of 3,5,3-triodo-L-thyronine to the hypothyroid rats tends to remedy the functional and structural defects of F0F1-ATP synthase observed in the hypothyroid rats. The results obtained indicate that hypothyroidism leads to a decreased expression of F0F1-ATP synthase complex in liver mitochondria and this contributes to the decrease of the efficiency of oxidative phosphorylation. 相似文献
5.
Richter ML Samra HS He F Giessel AJ Kuczera KK 《Journal of bioenergetics and biomembranes》2005,37(6):467-473
The chloroplast F0F1-ATP synthase-ATPase is a tiny rotary motor responsible for coupling ATP synthesis and hydrolysis to the light-driven electrochemical
proton gradient. Reversible oxidation/reduction of a dithiol, located within a special regulatory domain of the γ subunit
of the chloroplast F1 enzyme, switches the enzyme between an inactive and an active state. This regulatory mechanism is unique to the ATP synthases
of higher plants and its physiological significance lies in preventing nonproductive depletion of essential ATP pools in the
dark. The three-dimensional structure of the chloroplast F1 gamma subunit has not yet been solved. To examine the mechanism of dithiol regulation, a model of the chloroplast gamma subunit
was obtained through segmental homology modeling based on the known structures of the mitochondrial and bacterial γ subunits,
together with de novo construction of the unknown regulatory domain. The model has provided considerable insight into how the dithiol might modulate
catalytic function. This has, in turn, suggested a mechanism by which rotation of subunits in F0, the transmembrane proton channel portion of the enzyme, can be coupled, via the ε subunit, to rotation of the γ subunit
of F1 to achieve the 120° (or 90°+30°) stepping action that is characteristic of F1 γ subunit rotation. 相似文献
6.
Jörg-Christian Greie Gabriele Deckers-Hebestreit Karlheinz Altendorf 《Journal of bioenergetics and biomembranes》2000,32(4):357-364
Membrane-bound ATP synthases (F1F0) catalyze the synthesis of ATP via a rotary catalyticmechanism utilizing the energy of an electrochemical ion gradient. The transmembrane potentialis supposed to propel rotation of a subunit c ring of F0 together with subunits and of F1,hereby forming the rotor part of the enzyme, whereas the remainder of the F1F0 complexfunctions as a stator for compensation of the torque generated during rotation. This reviewfocuses on our recent work on the stator part of the F0 complex, e.g., subunits a and b. Usingepitope insertion and antibody binding, subunit a was shown to comprise six transmembranehelixes with both the N- and C-terminus oriented toward the cytoplasm. By use of circulardichroism (CD) spectroscopy, the secondary structure of subunit b incorporated intoproteoliposomes was determined to be 80% -helical together with 14% turn conformation, providingflexibility to the second stalk. Reconstituted subunit b together with isolated ac subcomplexwas shown to be active in proton translocation and functional F1 binding revealing the nativeconformation of the polypeptide chain. Chemical crosslinking in everted membrane vesiclesled to the formation of subunit b homodimers around residues bQ37 to bL65, whereas bA32Ccould be crosslinked to subunit a, indicating a close proximity of subunits a and b near themembrane. Further evidence for the proposed direct interaction between subunits a and b wasobtained by purification of a stable ab
2 subcomplex via affinity chromatography using Histags fused to subunit a or b. This ab
2 subcomplex was shown to be active in proton translocationand F1 binding, when coreconstituted with subunit c. Consequences of crosslink formationand subunit interaction within the F1F0 complex are discussed. 相似文献
7.
Boyle GM Roucou X Nagley P Devenish RJ Prescott M 《Journal of bioenergetics and biomembranes》2000,32(6):595-607
We have sought to elucidate how the oligomycin sensitivity-conferring protein (OSCP) of the mitochondrial F1F0-ATP synthase (mtATPase) can influence proton channel function. Variants of OSCP, from the yeast Saccharomyces cerevisiae, having amino acid substitutions at a strictly conserved residue (Gly166) were expressed in place of normal OSCP. Cells expressing the OSCP variants were able to grow on nonfermentable substrates, albeit with some increase in generation time. Moreover, these strains exhibited increased sensitivity to oligomycin, suggestive of modification in functional interactions between the F1 and F0 sectors mediated by OSCP. Bioenergetic analysis of mitochondria from cells expressing OSCP variants indicated an increased respiratory rate under conditions of no net ATP synthesis. Using specific inhibitors of mtATPase, in conjunction with measurement of changes in mitochondrial transmembrane potential, it was revealed that this increased respiratory rate was a result of increased proton flux through the F0 sector. This proton conductance, which is not coupled to phosphorylation, is exquisitely sensitive to inhibition by oligomycin. Nevertheless, the oxidative phosphorylation capacity of these mitochondria from cells expressing OSCP variants was no different to that of the control. These results suggest that the incorporation of OSCP variants into functional ATP synthase complexes can display effects in the control of proton flux through the F0 sector, most likely mediated through altered protein—protein contacts within the enzyme complex. This conclusion is supported by data indicating impaired stability of solubilized mtATPase complexes that is not, however, reflected in the assembly of functional enzyme complexes in vivo. Given a location for OSCP atop the F1-33 hexamer that is distant from the proton channel, then the modulation of proton flux by OSCP must occur at a distance. We consider how subtle conformational changes in OSCP may be transmitted to F0. 相似文献
8.
Summary The atp operon from the extreme alkaliphile Bacillus firmus OF4 was cloned and sequenced, and shown to contain genes for the eight structural subunits of the ATP synthase, preceded by a ninth gene predicted to encode a 14 kDa hydrophobic protein. The arrangement of genes is identical to that of the atp operons from Escherichia coli, Bacillus megaterium, and thermophilic Bacillus PS3. The deduced amino acid sequences of the subunits of the enzyme are also similar to their homologs in other ATP synthases, except for several unusual substitutions, particularly in the a and c subunits. These substitutions are in domains that have been implicated in the mechanism of proton translocation through F0-ATPase, and therefore could contribute to the gating properties of the alkaliphile ATP synthase or its capacity for proton capture. 相似文献
9.
In Escherichia coli, the F1FO ATP synthase b subunits house a conserved arginine in the tether domain at position 36 where the subunit emerges from the membrane. Previous
experiments showed that substitution of isoleucine or glutamate result in a loss of enzyme activity. Double mutants have been
constructed in an attempt to achieve an intragenic suppressor of the b
arg36→ile and the b
arg36→glu mutations. The b
arg36→ile mutation could not be suppressed. In contrast, the phenotypic defect resulting from the b
arg36→glu mutation was largely suppressed in the b
arg36→glu,glu39→arg double mutant. E. coli expressing the b
arg36→glu,glu39→arg subunit grew well on succinate-based medium. F1FO ATP synthase complexes were more efficiently assembled and ATP driven proton pumping activity was improved. The evidence
suggests that efficient coupling in F1FO ATP synthase is dependent upon a basic amino acid located at the base of the peripheral stalk. 相似文献
10.
Marcus V.J. Rocha Melissa S. Caetano Elaine F.F. da Cunha 《Journal of biomolecular structure & dynamics》2013,31(10):1175-1181
Organotin compounds are the active components of some fungicides, which are potential inhibitors of the F1F0-ATP synthase. The studies about the reaction mechanism might indicate a pathway to understand how these compounds work in biological systems, however, has not been clarified so far. In this line, molecular modeling studies and density functional theory calculations were performed in order to understand the molecular behavior of those compounds when they interact with the active site of the enzyme. Our findings indicate that a strong interaction with His132 can favor a chemical reaction with organotin compounds due to π–π stacking interactions with aromatic rings of organotin compounds. Furthermore, dependence on molecule size is related to possibility of reaction with the amino acid residue His132. Thus, it can also be noticed, for organotin compounds, that substituents with four carbons work by blocking the subunit a, in view of the high energy transition found characterized by steric hindrance. 相似文献
11.
Hase Y Tatsuno M Nishi T Kataoka K Kabe Y Yamaguchi Y Ozawa N Natori M Handa H Watanabe H 《Biochemical and biophysical research communications》2008,366(1):66-72
Atrazine is a widely used triazine herbicide. Although controversy still exists, a number of recent studies have described its adverse effects on various animals including humans. Of particular interest is its effects on reproductive capacity. In this study, we investigated the mechanisms underlying the adverse effects of atrazine, with a focus on its effects on sperm. Here we show evidence that mitochondrial F1F0-ATP synthase is a molecular target of atrazine. A series of experiments with sperm and isolated mitochondria suggest that atrazine inhibits mitochondrial function through F1F0-ATP synthase. Moreover, affinity purification using atrazine as a ligand demonstrates that F1F0-ATP synthase is a major atrazine-binding protein in cells. The inhibitory activity against mitochondria and F1F0-ATP synthase is not limited to atrazine but is likely to be applicable to other triazine-based compounds. Thus, our findings may have wide relevance to pharmacology and toxicology. 相似文献
12.
ATP synthases are motor complexes comprised of F0 and F1 parts that couple the proton gradient across the membrane to the synthesis of ATP by rotary catalysis. Although a great deal of information has been accumulated regarding the structure and function of ATP synthases, their motor functions are not fully understood. For this reason, we performed the alignments and analyses of the protein sequences comprising the core of the ATP synthase motor complex, and examined carefully the locations of the conserved residues in the subunit structures of ATP synthases. A summary of the findings from this bioinformatic study is as follows. First, we found that four conserved regions in the sequence of subunit are clustered into three patches in its structure. The interactions of these conserved patches with the and subunits are likely to be critical for energy coupling and catalytic activity of the ATP synthase. Second, we located a four-residue cluster at the N-terminal domain of mitochondrial OSCP or bacterial (or chloroplast) subunit which may be critical for the binding of these subunits to F1. Third, from the localizations of conserved residues in the subunits comprising the rotors of ATP synthases, we suggest that the conserved interaction site at the interface of subunit c and (mitochondria) or (bacteria and chloroplasts) may be important for connecting the rotor of F1 to the rotor of F0. Finally, we found the sequence of mitochondrial subunit b to be highly conserved, significantly longer than bacterial subunit b, and to contain a shorter dimerization domain than that of the bacterial protein. It is suggested that the different properties of mitochondrial subunit b may be necessary for interaction with other proteins, e.g., the supernumerary subunits. 相似文献
13.
Susan M. Howitt Andrew J. W. Rodgers Lyndall P. Hatch Frank Gibson Graeme B. Cox 《Journal of bioenergetics and biomembranes》1996,28(5):415-420
F0F1-ATPase structural information gained from X-ray crystallography and electron microscopy has activated interest in a rotational mechanism for the F0F1-ATPase. Because of the subunit stoichiometry and the involvement of both thea- andc-subunits in the mechanism of proton movement, it is argued that relative movement must occur between the subunits. Various options for the arrangement and structure of the subunits involved are discussed and a mechanism proposed. 相似文献
14.
The insertion of inner membrane proteins in Escherichia coli occurs almost exclusively via the SecYEG pathway, while some membrane proteins require the membrane protein insertase YidC. In vitro analysis demonstrates that subunit a of the F1F0 ATP synthase (F0a) is strictly dependent on Ffh, SecYEG and YidC for its membrane insertion but independent of the proton motive force. The insertion of the first transmembrane segment of F0a also depends on Ffh and SecYEG but not on YidC, whereas the insertion is strongly dependent on the proton motive force, unlike the full-length F0a protein. These data demonstrate an extensive role of YidC in the assembly of the F0 sector of the F1F0 ATP synthase. 相似文献
15.
Asha?Manikkoth Balakrishna Holger Seelert Sven-Hendric Marx Norbert?A. Dencher Gerhard Grüber 《Bioscience reports》2014,34(2)
In eukaryotic and prokaryotic cells, F-ATP synthases provide energy through the synthesis of ATP. The chloroplast F-ATP synthase (CF1FO-ATP synthase) of plants is integrated into the thylakoid membrane via its FO-domain subunits a, b, b’ and c. Subunit c with a stoichiometry of 14 and subunit a form the gate for H+-pumping, enabling the coupling of electrochemical energy with ATP synthesis in the F1 sector.Here we report the crystallization and structure determination of the c14-ring of subunit c of the CF1FO-ATP synthase from spinach chloroplasts. The crystals belonged to space group C2, with unit-cell parameters a=144.420, b=99.295, c=123.51 Å, and β=104.34° and diffracted to 4.5 Å resolution. Each c-ring contains 14 monomers in the asymmetric unit. The length of the c-ring is 60.32 Å, with an outer ring diameter 52.30 Å and an inner ring width of 40 Å. 相似文献
16.
S. Kauffer R. Schmid K. Steffens G. Deckers-Hebestreit K. Altendorf 《Archives of microbiology》1987,148(3):187-192
The ATP synthase complex of Klebsiella pneumoniae (KF1F0) has been purified and characterized. SDS-gel electrophoresis of the purified F1F0 complexes revealed an identical subunit pattern for E. coli (EF1F0) and K. pneumoniae. Antibodies raised against EF1 complex and purified EF0 subunits recognized the corresponding polypeptides of EF1F0 and KF1F0 in immunoblot analysis. Protease digestion of the individual subunits generated an identical cleavage pattern for subunits , , , , a, and c of both enzymes. Only for subunit different cleavage products were obtained. The isolated subunit c of both organisms showed only a slight deviation in the amino acid composition. These data suggest that extensive homologies exist in primary and secondary structure of both ATP synthase complexes reflecting a close phylogenetic relationship between the two enterobacteric tribes.Abbreviations ACMA
9-amino-6-chloro-2-methoxyacridine
- DCCD
N,N-dicyclohexylcarbodiimide
- FITC
fluorescein isothiocyanate
- SDS
sodium dodecyl sulfate
- TTFB
4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole 相似文献
17.
18.
Araceli Cano-Estrada Alexa Villavicencio-Queijeiro Héctor Miranda-Astudillo Julio A. Mignaco Pierre Cardol Claire Remacle Diego González-Halphen 《BBA》2010,1797(8):1439-35484
Mitochondrial F1F0-ATP synthase of chlorophycean algae is a dimeric complex of 1600 kDa constituted by 17 different subunits with varying stoichiometries, 8 of them conserved in all eukaryotes and 9 that seem to be unique to the algal lineage (subunits ASA1-9). Two different models proposing the topological assemblage of the nine ASA subunits in the ATP synthase of the colorless alga Polytomella sp. have been put forward. Here, we readdressed the overall topology of the enzyme with different experimental approaches: detection of close vicinities between subunits based on cross-linking experiments and dissociation of the enzyme into subcomplexes, inference of subunit stoichiometry based on cysteine residue labelling, and general three-dimensional structural features of the complex as obtained from small-angle X-ray scattering and electron microscopy image reconstruction. Based on the available data, we refine the topological arrangement of the subunits that constitute the mitochondrial ATP synthase of Polytomella sp. 相似文献
19.
Extracellular ATP formation from ADP and inorganic phosphate, attributed to the activity of a cell surface ATP synthase, has so far only been reported in cultures of some proliferating and tumoral cell lines. We now provide evidence showing the presence of a functionally active ecto-FoF1-ATP synthase on the plasma membrane of normal tissue cells, i.e. isolated rat hepatocytes. Both confocal microscopy and flow cytometry analysis show the presence of subunits of F1 (α/β and γ) and Fo (FoI-PVP(b) and OSCP) moieties of ATP synthase at the surface of rat hepatocytes. This finding is confirmed by immunoblotting analysis of the hepatocyte plasma membrane fraction. The presence of the inhibitor protein IF1 is also detected on the hepatocyte surface. Activity assays show that the ectopic-ATP synthase can work both in the direction of ATP synthesis and hydrolysis. A proton translocation assay shows that both these mechanisms are accompanied by a transient flux of H+ and are inhibited by F1 and Fo-targeting inhibitors. We hypothesise that ecto-FoF1-ATP synthase may control the extracellular ADP/ATP ratio, thus contributing to intracellular pH homeostasis. 相似文献
20.
ATPases with unusual membrane-embedded rotor subunits were found in both F1F0 and A1A0 ATP synthases. The rotor subunit c of A1A0 ATPases is, in most cases, similar to subunit c from F0. Surprisingly, multiplied c subunits with four, six, or even 26 transmembrane spans have been found in some archaea and these multiplication events were
sometimes accompanied by loss of the ion-translocating group. Nevertheless, these enzymes are still active as ATP synthases.
A duplicated c subunit with only one ion-translocating group was found along with “normal” F0 c subunits in the Na+ F1F0 ATP synthase of the bacterium Acetobacterium woodii. These extraordinary features and exceptional structural and functional variability in the rotor of ATP synthases may have
arisen as an adaptation to different cellular needs and the extreme physicochemical conditions in the early history of life. 相似文献