ATP synthase: subunit-subunit interactions in the stator stalk

In ATP synthase, proton translocation through the Fo subcomplex and ATP synthesis/hydrolysis in the F1 subcomplex are coupled by subunit rotation. The static, non-rotating portions of F1 and Fo are attached to each other via the peripheral "stator stalk", which has to withstand elastic strain during subunit rotation. In Escherichia coli, the stator stalk consists of subunits b2delta; in other organisms, it has three or four different subunits. Recent advances in this area include affinity measurements between individual components of the stator stalk as well as a detailed analysis of the interaction between subunit delta (or its mitochondrial counterpart, the oligomycin-sensitivity conferring protein, OSCP) and F1. The current status of our knowledge of the structure of the stator stalk and of the interactions between its subunits will be discussed in this review.     

The Oligomycin Axis of Mitochondrial ATP Synthase: OSCP and the Proton Channel

Oligomycin has long been known as an inhibitor of mitochondrial ATP synthase, putatively binding the F_o subunits 9 and 6 that contribute to proton channel function of the complex. As its name implies, OSCP is the oligomycin sensitivity-conferring protein necessary for the intact enzyme complex to display sensitivity to oligomycin. Recent advances concerning the structure and mechanism of mitochondrial ATP synthase have led to OSCP now being considered a component of the peripheral stator stalk rather than a central stalk component. How OSCP confers oligomycin sensitivity on the enzyme is unknown, but probably reflects important protein–protein interactions made within the assembled complex and transmitted down the stator stalk, thereby influencing proton channel function. We review here our studies directed toward establishing the stoichiometry, assembly, and function of OSCP in the context of knowledge of the organization of the stator stalk and the proton channel.     

Modulation at a Distance of Proton Conductance through the Saccharomyces cerevisiae Mitochondrial F1F0-ATP Synthase by Variants of the Oligomycin Sensitivity-Conferring Protein Containing Substitutions near the C-Terminus

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.     

ATP synthase: Subunit–subunit interactions in the stator stalk

In ATP synthase, proton translocation through the F_o subcomplex and ATP synthesis/hydrolysis in the F₁ subcomplex are coupled by subunit rotation. The static, non-rotating portions of F₁ and F_o are attached to each other via the peripheral “stator stalk”, which has to withstand elastic strain during subunit rotation. In Escherichia coli, the stator stalk consists of subunits b₂δ; in other organisms, it has three or four different subunits. Recent advances in this area include affinity measurements between individual components of the stator stalk as well as a detailed analysis of the interaction between subunit δ (or its mitochondrial counterpart, the oligomycin-sensitivity conferring protein, OSCP) and F₁. The current status of our knowledge of the structure of the stator stalk and of the interactions between its subunits will be discussed in this review.     

Second stalk of ATP synthase. Cross-linking of gamma subunit in F1 to truncated Fob subunit prevents ATP hydrolysis

ATP synthase consists of two portions, F(1) and F(o), connected by two stalks: a central rotor stalk containing gamma and epsilon subunits and a peripheral, second stalk formed by delta 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 (alpha(3)beta(3)gammadeltaepsilonb'(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 gamma 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 gamma 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).     

Manipulations in the peripheral stalk of the Saccharomyces cerevisiae F1F0-ATP synthase

The Saccharomyces cerevisiae F(1)F(0)-ATP synthase peripheral stalk is composed of the OSCP, h, d, and b subunits. The b subunit has two membrane-spanning domains and a large hydrophilic domain that extends along one side of the enzyme to the top of F(1). In contrast, the Escherichia coli peripheral stalk has two identical b subunits, and subunits with substantially altered lengths can be incorporated into a functional F(1)F(0)-ATP synthase. The differences in subunit structure between the eukaryotic and prokaryotic peripheral stalks raised a question about whether the two stalks have similar physical and functional properties. In the present work, the length of the S. cerevisiae b subunit has been manipulated to determine whether the F(1)F(0)-ATP synthase exhibited the same tolerances as in the bacterial enzyme. Plasmid shuffling was used for ectopic expression of altered b subunits in a strain carrying a chromosomal disruption of the ATP4 gene. Wild type growth phenotypes were observed for insertions of up to 11 and a deletion of four amino acids on a nonfermentable carbon source. In mitochondria-enriched fractions, abundant ATP hydrolysis activity was seen for the insertion mutants. ATPase activity was largely oligomycin-insensitive in these mitochondrial fractions. In addition, very poor complementation was seen in a mutant with an insertion of 14 amino acids. Lengthier deletions yielded a defective enzyme. The results suggest that although the eukaryotic peripheral stalk is near its minimum length, the b subunit can be extended a considerable distance.     

Proton-powered subunit rotation in single membrane-bound F0F1-ATP synthase

Synthesis of ATP from ADP and phosphate, catalyzed by F(0)F(1)-ATP synthases, is the most abundant physiological reaction in almost any cell. F(0)F(1)-ATP synthases are membrane-bound enzymes that use the energy derived from an electrochemical proton gradient for ATP formation. We incorporated double-labeled F(0)F(1)-ATP synthases from Escherichia coli into liposomes and measured single-molecule fluorescence resonance energy transfer (FRET) during ATP synthesis and hydrolysis. The gamma subunit rotates stepwise during proton transport-powered ATP synthesis, showing three distinct distances to the b subunits in repeating sequences. The average durations of these steps correspond to catalytic turnover times upon ATP synthesis as well as ATP hydrolysis. The direction of rotation during ATP synthesis is opposite to that of ATP hydrolysis.     

Characterization of domain interfaces in monomeric and dimeric ATP synthase

We disassembled monomeric and dimeric yeast ATP synthase under mild conditions to identify labile proteins and transiently stable subcomplexes that had not been observed before. Specific removal of subunits alpha, beta, oligomycin sensitivity conferring protein (OSCP), and h disrupted the ATP synthase at the gamma-alpha(3)beta(3) rotor-stator interface. Loss of two F(1)-parts from dimeric ATP synthase led to the isolation of a dimeric subcomplex containing membrane and peripheral stalk proteins thus identifying the membrane/peripheral stalk sectors immediately as the dimerizing parts of ATP synthase. Almost all subunit a was found associated with a ring of 10 c-subunits in two-dimensional blue native/SDS gels. We therefore postulate that c10a1-complex is a stable structure in resting ATP synthase until the entry of protons induces a breaking of interactions and stepwise rotation of the c-ring relative to the a-subunit in the catalytic mechanism. Dimeric subunit a was identified in SDS gels in association with two c10-rings suggesting that a c10a2c10-complex may constitute an important part of the monomer-monomer interface in dimeric ATP synthase that seems to be further tightened by subunits b, i, e, g, and h. In contrast to the monomer-monomer interface, the interface between dimers in higher oligomeric structures remains largely unknown. However, we could show that the natural inhibitor protein Inh1 is not required for oligomerization.     

Movements of the epsilon-subunit during catalysis and activation in single membrane-bound H(+)-ATP synthase

F0F1-ATP synthases catalyze proton transport-coupled ATP synthesis in bacteria, chloroplasts, and mitochondria. In these complexes, the epsilon-subunit is involved in the catalytic reaction and the activation of the enzyme. Fluorescence-labeled F0F1 from Escherichia coli was incorporated into liposomes. Single-molecule fluorescence resonance energy transfer (FRET) revealed that the epsilon-subunit rotates stepwise showing three distinct distances to the b-subunits in the peripheral stalk. Rotation occurred in opposite directions during ATP synthesis and hydrolysis. Analysis of the dwell times of each FRET state revealed different reactivities of the three catalytic sites that depended on the relative orientation of epsilon during rotation. Proton transport through the enzyme in the absence of nucleotides led to conformational changes of epsilon. When the enzyme was inactive (i.e. in the absence of substrates or without membrane energization), three distances were found again, which differed from those of the active enzyme. The three states of the inactive enzyme were unequally populated. We conclude that the active-inactive transition was associated with a conformational change of epsilon within the central stalk.     

Novel features in the structure of bovine ATP synthase.

The F1F0-ATP synthase from bovine heart mitochondria catalyses the synthesis of ATP from ADP and inorganic phosphate by using the energy of an electrochemical proton gradient derived from electron transport. The enzyme consists of three major domains: the globular F1catalytic domain of known atomic structure lies outside the lipid bilayer and is attached by a central stalk to the intrinsic membrane domain, F0, which transports protons through the membrane. Proton transport through F0evokes structural changes that are probably transmitted by rotation of the stalk to the catalytic sites in F1. In an alpha3beta3gamma1subcomplex, the rotation of the central gamma subunit driven by ATP hydrolysis has been visualised by optical microscopy. In order to prevent the alpha3beta3structure from following the rotation of the central gamma subunit, it has been proposed that the enzyme might have a stator connecting static parts in F0to alpha3beta3,thereby keeping it fixed relative to the rotating parts. Here we present electron microscopy images that reveal three new features in bovine F1F0-ATPase, one of which could be a stator. The second feature is a collar structure above the membrane domain and the third feature is some additional density on top of the F1domain.     

Genetic fusions of globular proteins to the epsilon subunit of the Escherichia coli ATP synthase: Implications for in vivo rotational catalysis and epsilon subunit function

The rotational mechanism of ATP synthase was investigated by fusing three proteins from Escherichia coli, the 12-kDa soluble cytochrome b(562), the 20-kDa flavodoxin, and the 28-kDa flavodoxin reductase, to the C terminus of the epsilon subunit of the enzyme. According to the concept of rotational catalysis, because epsilon is part of the rotor a large domain added at this site should sterically clash with the second stalk, blocking rotation and fully inhibiting the enzyme. E. coli cells expressing the cytochrome b(562) fusion in place of wild-type epsilon grew using acetate as the energy source, indicating their capacity for oxidative phosphorylation. Cells expressing the larger flavodoxin or flavodoxin reductase fusions failed to grow on acetate. Immunoblot analysis showed that the fusion proteins were stable in the cells and that they had no effect on enzyme assembly. These results provide initial evidence supporting rotational catalysis in vivo. In membrane vesicles, the cytochrome b(562) fusion caused an increase in the apparent ATPase activity but a minor decrease in proton pumping. Vesicles bearing ATP synthase containing the larger fusion proteins showed reduced but significant levels of ATPase activity that was sensitive to inhibition by dicyclohexylcarbodiimide (DCCD) but no proton pumping. Thus, all fusions to epsilon generated an uncoupled component of ATPase activity. These results imply that a function of the C terminus of epsilon in F(1)F(0) is to increase the efficiency of the enzyme by specifically preventing the uncoupled hydrolysis of ATP. Given the sensitivity to DCCD, this uncoupled ATP hydrolysis may arise from rotational steps of gammaepsilon in the inappropriate direction after ATP is bound at the catalytic site. It is proposed that the C-terminal domain of epsilon functions to ensure that rotation occurs only in the direction of ATP synthesis when ADP is bound and only in the direction of hydrolysis when ATP is bound.     

Single copies of subunits d, oligomycin-sensitivity conferring protein, and b are present in the Saccharomyces cerevisiae mitochondrial ATP synthase

In the mitochondrial ATP synthase (mtATPase) of the yeast Saccharomyces cerevisiae, the stoichiometry of subunits d, oligomycin-sensitivity conferring protein (OSCP), and b is poorly defined. We have investigated the stoichiometry of these subunits by the application of hexahistidine affinity purification technology. We have previously demonstrated that intact mtATPase complexes incorporating a Hex6-tagged subunit can be isolated via Ni2+-nitrilotriacetic acid affinity chromatography (Bateson, M., Devenish, R. J., Nagley, P., and Prescott, M. (1996) Anal. Biochem. 238, 14-18). Strains were constructed in which Hex6-tagged versions of subunits d, OSCP, and b were coexpressed with the corresponding wild-type subunit. This coexpression resulted in a mixed population of mtATPase complexes containing untagged wild-type and Hex6-tagged subunits. The stoichiometry of each subunit was then assessed by determining whether or not the untagged wild-type subunit could be recovered from Ni2+-nitrilotriacetic acid purifications as an integral component of those complexes absorbed by virtue of the Hex6-tagged subunit. As only the Hex6-tagged subunit was recovered from such purifications, we demonstrate that the stoichiometry of subunits d, OSCP, and b in yeast is 1 in each case.     

The peripheral stalk of the mitochondrial ATP synthase

The peripheral stalk of F-ATPases is an essential component of these enzymes. It extends from the membrane distal point of the F1 catalytic domain along the surface of the F1 domain with subunit a in the membrane domain. Then, it reaches down some 45 A to the membrane surface, and traverses the membrane, where it is associated with the a-subunit. Its role is to act as a stator to hold the catalytic alpha3beta3 subcomplex and the a-subunit static relative to the rotary element of the enzyme, which consists of the c-ring in the membrane and the attached central stalk. The central stalk extends up about 45 A from the membrane surface and then penetrates into the alpha3beta3 subcomplex along its central axis. The mitochondrial peripheral stalk is an assembly of single copies of the oligomycin sensitivity conferral protein (the OSCP) and subunits b, d and F6. In the F-ATPase in Escherichia coli, its composition is simpler, and it consists of a single copy of the delta-subunit with two copies of subunit b. In some bacteria and in chloroplasts, the two copies of subunit b are replaced by single copies of the related proteins b and b' (known as subunits I and II in chloroplasts). As summarized in this review, considerable progress has been made towards establishing the structure and biophysical properties of the peripheral stalk in both the mitochondrial and bacterial enzymes. However, key issues are unresolved, and so our understanding of the role of the peripheral stalk and the mechanism of synthesis of ATP are incomplete.     

F1 and F0 connections in the bovine mitochondrial ATP synthase: the role of the of alpha subunit N-terminus, oligomycin-sensitivity conferring protein (OCSP) and subunit d.

We have studied the functional effect of limited proteolysis by trypsin of the constituent subunits in the native and reconstituted F1F0 complex and isolated F1 of the bovine heart mitochondrial ATP synthase (EC 3.6.1.34). Chemical cross-linking of oligomycin-sensitivity conferring protein (OSCP) with other subunits of the ATP synthase and the consequent functional effects were also investigated. The results obtained show that the alpha subunit N-terminus is essential for the correct, functional connection of F1 to F0. The alpha-subunit N-terminus contacts OSCP which, in turn, contacts the F0I-PVP(b) and the F0-d subunits. The N-terminus of subunit alpha, OSCP, a segment of subunit d and the C-terminal and central region of F0I-PVP(b) subunits are peripherally located with respect to subunits gamma and delta which are completely shielded in the F1F0 complex against trypsin digestion. This qualifies the N-terminus of subunit alpha, OSCP, subunit d and F0I-PVP(b) as components of the lateral element of the stalk. These subunits, rather than being confined at one side of the complex which would leave most of the central part of the gamma subunit uncovered, surround the gamma and the delta subunits located in the central stalk.     

Location of subunit d in the peripheral stalk of the ATP synthase from Saccharomyces cerevisiae

ATP synthase from Saccharomyces cerevisiae is an approximately 600 kDa membrane protein complex. The enzyme couples the proton motive force across the mitochondrial inner membrane to the synthesis of ATP from ADP and inorganic phosphate. The peripheral stalk subcomplex acts as a stator, preventing the rotation of the soluble F 1 region relative to the membrane-bound F O region during ATP synthesis. Component subunits of the peripheral stalk are Atp5p (OSCP), Atp4p (subunit b), Atp7p (subunit d), and Atp14p (subunit h). X-ray crystallography has defined the structure of a large fragment of the bovine peripheral stalk, including 75% of subunit d (residues 3-123). Docking the peripheral stalk structure into a cryo-EM map of intact yeast ATP synthase showed that residue 123 of subunit d lies close to the bottom edge of F 1. The 37 missing C-terminal residues are predicted to either fold back toward the apex of F 1 or extend toward the membrane. To locate the C terminus of subunit d within the peripheral stalk of ATP synthase from S. cerevisiae, a biotinylation signal was fused to the protein. The biotin acceptor domain became biotinylated in vivo and was subsequently labeled with avidin in vitro. Electron microscopy of the avidin-labeled complex showed the label tethered close to the membrane surface. We propose that the C-terminal region of subunit d spans the gap from F 1 to F O, reinforcing this section of the peripheral stalk.     

Topology and proximity relationships of yeast mitochondrial ATP synthase subunit 8 determined by unique introduced cysteine residues.

We have used site-directed chemical labelling to demonstrate the membrane topology and to identify neighbouring subunits of subunit 8 (Y8) in yeast mitochondrial ATP synthase (mtATPase). Unique cysteine residues were introduced at the N or C-terminus of Y8 by site-directed mutagenesis. Expression and targeting to mitochondria in vivo of each of these variants in a yeast Y8 null mutant was able to restore activity to an otherwise nonfunctional ATP synthase complex. The position of each introduced cysteine relative to the inner mitochondrial membrane was probed with thiol-specific nonpermeant and permeant reagents in both intact and lysed mitochondria. The data indicate that the N-terminus of Y8 is located in the intermembrane space of mitochondria whereas the C-terminus is located within the mitochondrial matrix. The proximity of Y8 to other proteins of mtATPase was tested using heterobifunctional cross-linking reagents, each with one thiol-specific reactive group and one nonspecific, photoactivatible reactive group. These experiments revealed the proximity of the C-terminal domain of Y8 to subunits d and f, and that of the N-terminal domain to subunit f. It is concluded that Y8 possesses a single transmembrane domain which extends across the inner membrane of intact mitochondria. As subunit d is a likely component of the stator stalk of mitochondrial ATP synthase, we propose, on the basis of the observed cross-links, that Y8 may also be part of the stator stalk.     

The proton-translocating a subunit of F0F1-ATP synthase is allocated asymmetrically to the peripheral stalk

The position of the a subunit of the membrane-integral F0 sector of Escherichia coli ATP synthase was investigated by single molecule fluorescence resonance energy transfer studies utilizing a fusion of enhanced green fluorescent protein to the C terminus of the a subunit and fluorescent labels attached to specific positions of the epsilon or gamma subunits. Three fluorescence resonance energy transfer levels were observed during rotation driven by ATP hydrolysis corresponding to the three resting positions of the rotor subunits, gamma or epsilon, relative to the a subunit of the stator. Comparison of these positions of the rotor sites with those previously determined relative to the b subunit dimer indicates the position of a as adjacent to the b dimer on its counterclockwise side when the enzyme is viewed from the cytoplasm. This relationship provides stability to the membrane interface between a and b2, allowing it to withstand the torque imparted by the rotor during ATP synthesis as well as ATP hydrolysis.     

Regulation of the F0F1-ATP synthase: the conformation of subunit epsilon might be determined by directionality of subunit gamma rotation

F(0)F(1)-ATP synthase couples ATP synthesis/hydrolysis with transmembrane proton transport. The catalytic mechanism involves rotation of the gamma epsilon c(approximately 10)-subunits complex relative to the rest of the enzyme. In the absence of protonmotive force the enzyme is inactivated by the tight binding of MgADP. Subunit epsilon also modulates the activity: its conformation can change from a contracted to extended form with C-terminus stretched towards F(1). The latter form inhibits ATP hydrolysis (but not synthesis). We propose that the directionality of the coiled-coil subunit gamma rotation determines whether subunit epsilon is in contracted or extended form. Block of rotation by MgADP presumably induces the extended conformation of subunit epsilon. This conformation might serve as a safety lock, stabilizing the ADP-inhibited state upon de-energization and preventing spontaneous re-activation and wasteful ATP hydrolysis. The hypothesis merges the known regulatory effects of ADP, protonmotive force and conformational changes of subunit epsilon into a consistent picture.     

The Na(+)-translocating F(1)F(0) ATP synthase of Propionigenium modestum: mechanochemical insights into the F(0) motor that drives ATP synthesis

The ATP synthase of Propionigenium modestum encloses a rotary motor involved in the production of ATP from ADP and inorganic phosphate utilizing the free energy of an electrochemical Na(+) ion gradient. This enzyme clearly belongs to the family of F(1)F(0) ATP synthases and uses exclusively Na(+) ions as the physiological coupling ion. The motor domain, F(0), comprises subunit a and the b subunit dimer which are part of the stator and the subunit c oligomer acting as part of the rotor. During ATP synthesis, Na(+) translocation through F(0) proceeds from the periplasm via the stator channel (subunit a) onto a Na(+) binding site of the rotor (subunit c). Upon rotation of the subunit c oligomer versus subunit a, the occupied rotor site leaves the interface with the stator and the Na(+) ion can freely dissociate into the cytoplasm. Recent experiments demonstrate that the membrane potential is crucial for ATP synthesis under physiological conditions. These findings support the view that voltage generates torque in F(0), which drives the rotation of the gamma subunit thus liberating tightly bound ATP from the catalytic sites in F(1). We suggest a mechanochemical model for the transduction of transmembrane Na(+)-motive force into rotary torque by the F(0) motor that can account quantitatively for the experimental data.     

Torque Transmission Mechanism via DELSEED Loop of F1-ATPase

F₁-ATPase (F₁) is an ATP-driven rotary motor in which the three catalytic β subunits in the stator ring sequentially induce the unidirectional rotation of the rotary γ subunit. Many lines of evidence have revealed open-to-closed conformational transitions in the β subunit that swing the C-terminal domain inward. This conformational transition causes a C-terminal protruding loop with conserved sequence DELSEED to push the γ subunit. Previous work, where all residues of DELSEED were substituted with glycine to disrupt the specific interaction with γ and introduce conformational flexibility, showed that F₁ still rotated, but that the torque was halved, indicating a remarkable impact on torque transmission. In this study, we conducted a stall-and-release experiment on F₁ with a glycine-substituted DELSEED loop to investigate the impact of the glycine substitution on torque transmission upon ATP binding and ATP hydrolysis. The mutant F₁ showed a significantly reduced angle-dependent change in ATP affinity, whereas there was no change in the equilibrium for ATP hydrolysis. These findings indicate that the DELSEED loop is predominantly responsible for torque transmission upon ATP binding but not for that upon ATP hydrolysis.