Cross-linking and labeling of the Escherichia coli F1F0-ATP synthase reveal a compact hydrophilic portion of F0 close to an F1 catalytic subunit

The subunit arrangement of the F0 sector of the Escherichia coli ATP synthase is examined using hydrophilic and hydrophobic (cleavable) cross-linking reagents and the water-soluble labeling reagent [35S] diazoniumbenzenesulfonate ( [35S]DABS). Cross-linking is performed on purified ATP synthase and inverted minicell membranes. ATP synthase incorporated into liposomes is labeled with [35S]DABS. Three cross-linked products involving the F0 subunits (a, b, and c) are observed with the purified ATP synthase in solution: a-b, b2, and c2 dimers. A cross-link between the F0 and F1 is detected and occurs between the a and beta subunits. A cross-linker independent association between the b and beta subunits is also evident, suggesting that the two subunits are close enough to form a disulfide bridge. A cross-linking reagent stable to reducing agents produces a b-beta dimer, as detected by immunoblotting with anti-beta serum. The c subunit does not cross-link with any F1 polypeptide. Minicell membranes containing ATP synthase polypeptides radioactively labeled in vivo similarly show b2 and c2 dimers after cross-linking. [35S]DABS labels the a and b, but not c, subunits, showing that the a and b, but not c, subunits possess hydrophilic domains. Thus, certain domains of subunits a and b extend from the membrane and are in close proximity to one another and the F1 catalytic subunit beta.     

Probing conformations of the beta subunit of F0F1-ATP synthase in catalysis

A subcomplex of F0F1-ATP synthase (F0F1), alpha3beta3gamma, was shown to undergo the conformation(s) during ATP hydrolysis in which two of the three beta subunits have the "Closed" conformation simultaneously (CC conformation) [S.P. Tsunoda, E. Muneyuki, T. Amano, M. Yoshida, H. Noji, Cross-linking of two beta subunits in the closed conformation in F1-ATPase, J. Biol. Chem. 274 (1999) 5701-5706]. This was examined by the inter-subunit disulfide cross-linking between two mutant beta(I386C)s that was formed readily only when the enzyme was in the CC conformation. Here, we adopted the same method for the holoenzyme F0F1 from Bacillus PS3 and found that the CC conformation was generated during ATP hydrolysis but barely during ATP synthesis. The experiments using F0F1 with the epsilon subunit lacking C-terminal helices further suggest that this difference is related to dynamic nature of the epsilon subunit and that ATP synthesis is accelerated when it takes the pathway involving the CC conformation.     

In the absence of the first membrane-spanning segment of subunit 4(b), the yeast ATP synthase is functional but does not dimerize or oligomerize.

The N-terminal portion of the mitochondrial b-subunit is anchored in the inner mitochondrial membrane by two hydrophobic segments. We investigated the role of the first membrane-spanning segment, which is absent in prokaryotic and chloroplastic enzymes. In the absence of the first membrane-spanning segment of the yeast subunit (subunit 4), a strong decrease in the amount of subunit g was found. The mutant ATP synthase did not dimerize or oligomerize, and mutant cells displayed anomalous mitochondrial morphologies with onion-like structures. This phenotype is similar to that of the null mutant in the ATP20 gene that encodes subunit g, a component involved in the dimerization/oligomerization of ATP synthase. Our data indicate that the first membrane-spanning segment of the mitochondrial b-subunit is not essential for the function of the enzyme since its removal did not directly alter the oxidative phosphorylation. It is proposed that the unique membrane-spanning segment of subunit g and the first membrane-spanning segment of subunit 4 interact, as shown by cross-linking experiments. We hypothesize that in eukaryotic cells the b-subunit has evolved to accommodate the interaction with the g-subunit, an associated ATP synthase component only present in the mitochondrial enzyme.     

Organisation of the yeast ATP synthase F(0):a study based on cysteine mutants, thiol modification and cross-linking reagents

A topological study of the yeast ATP synthase membranous domain was undertaken by means of chemical modifications and cross-linking experiments on the wild-type complex and on mutated enzymes obtained by site-directed mutagenesis of genes encoding ATP synthase subunits. The modification by non-permeant maleimide reagents of the Cys-54 of mutated subunit 4 (subunit b), of the Cys-23 in the N-terminus of subunit 6 (subunit a) and of the Cys-91 in the C-terminus of mutated subunit f demonstrated their location in the mitochondrial intermembrane space. Near-neighbour relationships between subunits of the complex were demonstrated by means of homobifunctional and heterobifunctional reagents. Our data suggest interactions between the first transmembranous alpha-helix of subunit 6, the two hydrophobic segments of subunit 4 and the unique membrane-spanning segments of subunits i and f. The amino acid residue 174 of subunit 4 is close to both oscp and the beta-subunit, and the residue 209 is close to oscp. The dimerisation of subunit 4 in the membrane revealed that this component is located in the periphery of the enzyme and interacts with other ATP synthase complexes.     

Is there a relationship between the supramolecular organization of the mitochondrial ATP synthase and the formation of cristae?

Blue native polyacrylamide gel electrophoresis (BN-PAGE) analyses of detergent mitochondrial extracts have provided evidence that the yeast ATP synthase could form dimers. Cross-linking experiments performed on a modified version of the i-subunit of this enzyme indicate the existence of such ATP synthase dimers in the yeast inner mitochondrial membrane. We also show that the first transmembrane segment of the eukaryotic b-subunit (bTM1), like the two supernumerary subunits e and g, is required for dimerization/oligomerization of ATP synthases. Unlike mitochondria of wild-type cells that display a well-developed cristae network, mitochondria of yeast cells devoid of subunits e, g, or bTM1 present morphological alterations with an abnormal proliferation of the inner mitochondrial membrane. From these observations, we postulate that an anomalous organization of the inner mitochondrial membrane occurs due to the absence of ATP synthase dimers/oligomers. We provide a model in which the mitochondrial ATP synthase is a key element in cristae morphogenesis.     

The gammaepsilon-c subunit interface in the ATP synthase of Escherichia coli. cross-linking of the epsilon subunit to the c subunit ring does not impair enzyme function, that of gamma to c subunits leads to uncoupling

Mutants with a cysteine residue in the gamma subunit at position 207 and the epsilon subunit at position 31 were expressed in combination with a c-dimer construct, which contains a single cysteine at position 42 of the second c subunit. These mutants are called gammaY207C/cc'Q42C and epsilonE31C/cc'Q42C, respectively. Cross-linking of epsilon to the c subunit ring was obtained almost to completion without significant effect on any enzyme function, i.e. ATP hydrolysis, ATP synthesis, and ATP hydrolysis-driven proton translocation were all close to that of wild type. The gamma subunit could also be linked to the c subunit ring in more than 90% yield, but this affected coupling. Thus, ATP hydrolysis was increased 2. 5-fold, ATP synthesis was dramatically decreased, and ATP hydrolysis-driven proton translocation was abolished, as measured by the 9-amino-6-chloro-2-methoxyacridinequenching method. These results for epsilonE31C/cc'Q42C indicate that the c subunit ring rotates with the central stalk element. That the gamma-epsilon cross-linked enzyme retains ATPase activity also argues for a gammaepsilon-c subunit rotor. However, the uncoupling induced by cross-linking of gamma to the c subunit ring points to important conformational changes taking place in the gammaepsilon-c subunit interface during this. Blocking these structural changes by cross-linking leads to a proton leak within the F(0).     

The overexpression, purification and complete amino acid sequence of chorismate synthase from Escherichia coli K12 and its comparison with the enzyme from Neurospora crassa.

The enzyme chorismate synthase was purified in milligram quantities from an overproducing strain of Escherichia coli. The amino acid sequence was deduced from the nucleotide sequence of the aroC gene and confirmed by determining the N-terminal amino acid sequence of the purified enzyme. The complete polypeptide chain consists of 357 amino acid residues and has a calculated subunit Mr of 38,183. Cross-linking and gel-filtration experiments show that the enzyme is tetrameric. An improved purification of chorismate synthase from Neurospora crassa is also described. Cross-linking and gel-filtration experiments on the N. crassa enzyme show that it is also tetrameric with a subunit Mr of 50,000. It is proposed that the subunits of the N. crassa enzyme are larger because they contain a diaphorase domain that is absent from the E. coli enzyme.     

Insights into the rotary catalytic mechanism of F0F1 ATP synthase from the cross-linking of subunits b and c in the Escherichia coli enzyme

The transmembrane sector of the F(0)F(1) rotary ATP synthase is proposed to organize with an oligomeric ring of c subunits, which function as a rotor, interacting with two b subunits at the periphery of the ring, the b subunits functioning as a stator. In this study, cysteines were introduced into the C-terminal region of subunit c and the N-terminal region of subunit b. Cys of N2C subunit b was cross-linked with Cys at positions 74, 75, and 78 of subunit c. In each case, a maximum of 50% of the b subunit could be cross-linked to subunit c, which suggests that either only one of the two b subunits lie adjacent to the c-ring or that both b subunits interact with a single subunit c. The results support a topological arrangement of these subunits, in which the respective N- and C-terminal ends of subunits b and c extend to the periplasmic surface of the membrane and cAsp-61 lies at the center of the membrane. The cross-linking of Cys between bN2C and cV78C was shown to inhibit ATP-driven proton pumping, as would be predicted from a rotary model for ATP synthase function, but unexpectedly, cross-linking did not lead to inhibition of ATPase activity. ATP hydrolysis and proton pumping are therefore uncoupled in the cross-linked enzyme. The c subunit lying adjacent to subunit b was shown to be mobile and to exchange with c subunits that initially occupied non-neighboring positions. The movement or exchange of subunits at the position adjacent to subunit b was blocked by dicyclohexylcarbodiimide. These experiments provide a biochemical verification that the oligomeric c-ring can move with respect to the b-stator and provide further support for a rotary catalytic mechanism in the ATP synthase.     

The intermembrane space loop of subunit b (4) is a major determinant of the stability of yeast oligomeric ATP synthases

The involvement of the b-subunit, subunit 4 in yeast, a component of the peripheral stalk of the ATP synthase, in the dimerization/oligomerization process of this enzyme was investigated. Increasing deletions were introduced by site-directed mutagenesis in the loop located in the mitochondrial intermembrane space and linking the two transmembrane (TM) segments of subunit 4. The resulting strains were still able to grow on nonfermentable media, but defects were observed in ATP synthase dimerization/oligomerization along with concomitant mitochondrial morphology alterations. Surprisingly, such defects, already depicted in the absence of the so-called dimer-specific subunits e and g, were found in a mutant harboring a full amount of subunit g associated to the monomeric form of the ATP synthase. Deletion of the intermembrane space loop of subunit 4 modified the profile of cross-linking products involving cysteine residues belonging to subunits 4, g, 6, and e. This suggests that this loop of subunit 4 participates in the organization of surrounding hydrophobic membranous components (including the two TM domains of subunit 4) and thus is involved in the stability of supramolecular species of yeast ATP synthase in the mitochondrial membrane.     

Site-directed cross-linking of b to the alpha, beta, and a subunits of the Escherichia coli ATP synthase

The b subunit dimer of the Escherichia coli ATP synthase, along with the delta subunit, is thought to act as a stator to hold the alpha(3)beta(3) hexamer stationary relative to the a subunit as the gammaepsilonc(9-12) complex rotates. Despite their essential nature, the contacts between b and the alpha, beta, 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 F(1)F(0) was attempted. Cross-linking in both the full-length and truncated forms of b was obtained at positions 92 (to alpha and beta), and 109 and 110 (to alpha only). Mass spectrometric analysis of peptide fragments derived from the b(24-156)A92C cross-link revealed that cross-linking took place within the region of alpha between Ile-464 and Met-483. This result indicates that the b dimer interacts with the alpha subunit near a non-catalytic alpha/beta interface. A cysteine residue introduced in place of the highly conserved arginine at position 36 of the b subunit could be cross-linked to the a subunit of F(0) in membrane-bound ATP synthase, implying that at least 10 residues of the polar domain of b are adjacent to residues of a. Sites of cross-linking between b(24-156)A92C and beta as well as b(24-156)I109C and alpha are proposed based on the mass spectrometric data, and these sites are discussed in terms of the structure of b and its interactions with the rest of the complex.     

The epsilon subunit of the F(1)F(0) complex of Escherichia coli. cross-linking studies show the same structure in situ as when isolated.

Four double mutants in the epsilon subunit were generated, each containing two cysteines, which, based on the NMR structure of this subunit, should form internal disulfide bonds. Two of these were designed to generate interdomain cross-links that lock the C-terminal alpha-helical domain against the beta-sandwich (epsilonM49C/A126C and epsilonF61C/V130C). The second set should give cross-linking between the two C-terminal alpha-helices (epsilonA94C/L128C and epsilonA101C/L121C). All four mutants cross-linked with 90-100% efficiency upon CuCl(2) treatment in isolated Escherichia coli ATP synthase. This shows that the structure obtained for isolated epsilon is essentially the same as in the assembled complex. Functional studies revealed increased ATP hydrolysis after cross-linking between the two domains of the subunit but not after cross-linking between the C-terminal alpha-helices. None of the cross-links had any effect on proton pumping-coupled ATP hydrolysis, on DCCD sensitivity of this activity, or on ATP synthesis rates. Therefore, big conformational changes within epsilon can be ruled out as a part of the enzyme function. Protease digestion studies, however, showed that subtle changes do occur, since the epsilon subunit could be locked in an ADP or 5'-adenylyl-beta,gamma-imidodiphosphate conformation by the cross-linking with resulting differences in cleavage rates.     

Evidence of the proximity of ATP synthase subunits 6 (a) in the inner mitochondrial membrane and in the supramolecular forms of Saccharomyces cerevisiae ATP synthase

The involvement of subunit 6 (a) in the interface between yeast ATP synthase monomers has been highlighted. Based on the formation of a disulfide bond and using the unique cysteine 23 as target, we show that two subunits 6 are close in the inner mitochondrial membrane and in the solubilized supramolecular forms of the yeast ATP synthase. In a null mutant devoid of supernumerary subunits e and g that are involved in the stabilization of ATP synthase dimers, ATP synthase monomers are close enough in the inner mitochondrial membrane to make a disulfide bridge between their subunits 6, and this proximity is maintained in detergent extract containing this enzyme. The cross-linking of cysteine 23 located in the N-terminal part of the first transmembrane helix of subunit 6 suggests that this membrane-spanning segment is in contact with its counterpart belonging to the ATP synthase monomer that faces it and participates in the monomer-monomer interface.     

Phosphorylation of heart glycogen synthase by cAMP-dependent protein kinase. Regulatory effects of ATP

Glycogen synthase I, purified from bovine heart, had a specific activity of 33 units/mg and gave a single band on sodium dodecyl sulfate gel electrophoresis with a subunit molecular weight of 86,000. The enzyme was phosphorylated with cAMP-dependent protein kinase catalytic subunit, also isolated from heart. With 10 microM ATP, only one phosphate group was incorporated per subunit of glycogen synthase. The phosphorylation decreased the per cent of glycogen synthase I from 0.95 to 0.50 when activity was determined by assays with Na2SO4 and glucose 6-phosphate. Glycogen synthase containing one phosphate per subunit was designated GS-1. One additional phosphate was incorporated per synthase subunit when ATP was increased to 0.5 mM and the percent glycogen synthase I decreased from 0.50 to < 0.05. This enzyme form was designated GS-1,2. Conversion of GS-1 to Gs-1,2 gave cooperative kinetics with ATP concentration and a half-maximal stimulation at approximately 40 microM. Phosphorylation of GS-1 could also be achieved by adding other non-substrate nucleotide triphosphates such as ITP and UTP along with 10 microM ATP. Glucose-6-P and Na2SO4 were without effect on this phosphorylation reaction. Two separate peptides were obtained after CNBr cleavage of 32P-labeled GS-1,2 and only one from GS-1. Both enzyme forms contained a single phosphorylated peptide in common. Thus, heart glycogen synthase may be phosphorylated specifically in either of two different sites using appropriate concentrations of ATP. ATP acts as a substrate for the protein kinase and also affects the availability of a second site to phosphorylation by cAMP-dependent protein kinase.     

The fully-active and structurally-stable form of the mitochondrial ATP synthase of Polytomella sp. is dimeric

Mitochondrial F₁F_O-ATP synthase of chlorophycean algae is a stable dimeric complex of 1,600 kDa. It lacks the classic subunits that constitute the peripheral stator-stalk and the orthodox polypeptides involved in the dimerization of the complex. Instead, it contains nine polypeptides of unknown evolutionary origin named ASA1 to ASA9. The isolated enzyme exhibited a very low ATPase activity (0.03 Units/mg), that increased upon heat treatment, due to the release of the F₁ sector. Oligomycin was found to stabilize the dimeric structure of the enzyme, providing partial resistance to heat dissociation. Incubation in the presence of low concentrations of several non-ionic detergents increased the oligomycin-sensitive ATPase activity up to 7.0–9.0 Units/mg. Incubation with 3% (w/v) taurodeoxycholate monomerized the enzyme. The monomeric form of the enzyme exhibited diminished activity in the presence of detergents and diminished oligomycin sensitivity. Cross-linking experiments carried out with the dimeric and monomeric forms of the ATP synthase suggested the participation of the ASA6 subunit in the dimerization of the enzyme. The dimeric enzyme was more resistant to heat treatment, high hydrostatic pressures, and protease digestion than the monomeric enzyme, which was readily disrupted by these treatments. We conclude that the fully-active algal mitochondrial ATP synthase is a stable catalytically active dimer; the monomeric form is less active and less stable. Monomer-monomer interactions could be mediated by the membrane-bound subunits ASA6 and ASA9, and may be further stabilized by other polypeptides such as ASA1 and ASA5. Alexa Villavicencio-Queijeiro and Miriam Vázquez-Acevedo have contributed equally to this work.     

The second stalk of the yeast ATP synthase complex: identification of subunits showing cross-links with known positions of subunit 4 (subunit b).

A component of the stator of the yeast ATP synthase (subunit 4 or b) showed many cross-linked products with the homobifunctional reagent dithiobis[succinimidyl propionate], which reacts with the amino group of lysine residues. The positions in subunit 4 that were involved in the cross-linkings were determined by using cysteine-generated mutants constructed by site-directed mutagenesis of ATP4. Cross-linking experiments with the heterobifunctional reagent p-azidophenacyl bromide, which has a spacer arm of 9 A, were performed with mitochondria and crude Triton X-100 extracts containing the solubilized enzyme. Substitution of lysine residues by cysteine residues in the hydrophilic C-terminal part of subunit 4 allowed cross-links with subunit h from C98 and with subunit d from C141, C143, and C151. OSCP was cross-linked from C174 and C209. A cross-linked product, 4+beta, was also obtained from C174. It is concluded that the C-terminus of subunit 4 is distant from the membrane surface and close to F(1) and OSCP. The N-terminal part of subunit 4 is close to subunit g, as demonstrated by the identification of a cross-linked product involving subunit g and the cysteine residues 7 or 14 of subunit 4.     

The peripheral stalk participates in the yeast ATP synthase dimerization independently of e and g subunits

It is now clearly established that dimerization of the F(1)F(o) ATP synthase takes place in the mitochondrial inner membrane. Interestingly, oligomerization of this enzyme seems to be involved in cristae morphogenesis. As they were able to form homodimers, subunits 4, e, and g have been proposed as potential ATP synthase dimerization subunits. In this paper, we provide evidence that subunit h, a peripheral stalk component, is located either at or near the ATP synthase dimerization interface. Subunit h homodimers were formed in mitochondria and were found to be associated to ATP synthase dimers. Moreover, homodimerization of subunit h and of subunit i turned out to be independent of subunits e and g, confirming the existence of an ATP synthase dimer in the mitochondrial inner membrane in the absence of subunits e and g. For the first time, this dimer has been observed by BN-PAGE. Finally, from these results we are now able to update our model for the supramolecular organization of the ATP synthase in the membrane and propose a role for subunits e and g, which stabilize the ATP synthase dimers and are involved in the oligomerization of the complex.     

A "petite obligate" mutant of Saccharomyces cerevisiae: functional mtDNA is lethal in cells lacking the delta subunit of mitochondrial F1-ATPase

Within the mitochondrial F(1)F(0)-ATP synthase, the nucleus-encoded delta-F(1) subunit plays a critical role in coupling the enzyme proton translocating and ATP synthesis activities. In Saccharomyces cerevisiae, deletion of the delta subunit gene (Deltadelta) was shown to result in a massive destabilization of the mitochondrial genome (mitochondrial DNA; mtDNA) in the form of 100% rho(-)/rho degrees petites (i.e. cells missing a large portion (>50%) of the mtDNA (rho(-)) or totally devoid of mtDNA (rho degrees )). Previous work has suggested that the absence of complete mtDNA (rho(+)) in Deltadelta yeast is a consequence of an uncoupling of the ATP synthase in the form of a passive proton transport through the enzyme (i.e. not coupled to ATP synthesis). However, it was unclear why or how this ATP synthase defect destabilized the mtDNA. We investigated this question using a nonrespiratory gene (ARG8(m)) inserted into the mtDNA. We first show that retention of functional mtDNA is lethal to Deltadelta yeast. We further show that combined with a nuclear mutation (Deltaatp4) preventing the ATP synthase proton channel assembly, a lack of delta subunit fails to destabilize the mtDNA, and rho(+) Deltadelta cells become viable. We conclude that Deltadelta yeast cannot survive when it has the ability to synthesize the ATP synthase proton channel. Accordingly, the rho(-)/rho degrees mutation can be viewed as a rescuing event, because this mutation prevents the synthesis of the two mtDNA-encoded subunits (Atp6p and Atp9p) forming the core of this channel. This is the first report of what we have called a "petite obligate" mutant of S. cerevisiae.     

Thiol modulation of the chloroplast ATP synthase is dependent on the energization of thylakoid membranes

Thiol modulation of the chloroplast ATP synthase γ subunit has been recognized as an important regulatory system for the activation of ATP hydrolysis activity, although the physiological significance of this regulation system remains poorly characterized. Since the membrane potential required by this enzyme to initiate ATP synthesis for the reduced enzyme is lower than that needed for the oxidized form, reduction of this enzyme was interpreted as effective regulation for efficient photophosphorylation. However, no concrete evidence has been obtained to date relating to the timing and mode of chloroplast ATP synthase reduction and oxidation in green plants. In this study, thorough analysis of the redox state of regulatory cysteines of the chloroplast ATP synthase γ subunit in intact chloroplasts and leaves shows that thiol modulation of this enzyme is pivotal in prohibiting futile ATP hydrolysis activity in the dark. However, the physiological importance of efficient ATP synthesis driven by the reduced enzyme in the light could not be demonstrated. In addition, we investigated the significance of the electrochemical proton gradient in reducing the γ subunit by the reduced form of thioredoxin in chloroplasts, providing strong insights into the molecular mechanisms underlying the formation and reduction of the disulfide bond on the γ subunit in vivo.     

F0F1-ATPase/synthase is geared to the synthesis mode by conformational rearrangement of epsilon subunit in response to proton motive force and ADP/ATP balance

The epsilon subunit in F0F1-ATPase/synthase undergoes drastic conformational rearrangement, which involves the transition of two C-terminal helices between a hairpin "down"-state and an extended "up"-state, and the enzyme with the up-fixed epsilon cannot catalyze ATP hydrolysis but can catalyze ATP synthesis (Tsunoda, S. P., Rodgers, A. J. W., Aggeler, R., Wilce, M. C. J., Yoshida, M., and Capaldi, R. A. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 6560-6564). Here, using cross-linking between introduced cysteine residues as a probe, we have investigated the causes of the transition. Our findings are as follows. (i) In the up-state, the two helices of epsilon are fully extended to insert the C terminus into a deeper position in the central cavity of F1 than was thought previously. (ii) Without a nucleotide, epsilon is in the up-state. ATP induces the transition to the down-state, and ADP counteracts the action of ATP. (iii) Conversely, the enzyme with the down-state epsilon can bind an ATP analogue, 2',3'-O-(2,4,6-trinitrophenyl)-ATP, much faster than the enzyme with the up-state epsilon. (iv) Proton motive force stabilizes the up-state. Thus, responding to the increase of proton motive force and ADP, F0F1-ATPase/synthase would transform the epsilon subunit into the up-state conformation and change gear to the mode for ATP synthesis.