Characterization of Domain Interfaces in Monomeric and Dimeric ATP Synthase
2008; Elsevier BV; Volume: 7; Issue: 5 Linguagem: Inglês
10.1074/mcp.m700465-mcp200
ISSN1535-9484
AutoresIlka Wittig, Jean Velours, Rosemary A. Stuart, Hermann Schägger,
Tópico(s)Photosynthetic Processes and Mechanisms
ResumoWe 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 α, β, oligomycin sensitivity conferring protein (OSCP), and h disrupted the ATP synthase at the γ-α3β3 rotor-stator interface. Loss of two F1-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. 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 α, β, oligomycin sensitivity conferring protein (OSCP), and h disrupted the ATP synthase at the γ-α3β3 rotor-stator interface. Loss of two F1-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. The possibility to remove a stable hydrophilic F1-subcomplex, containing subunits α, β, γ, δ, and ε with a α3β 3γ1δ1ε1 stoichiometry, from mitochondrial ATP synthase historically led to the term F1F0-ATP synthase for the holoenzyme (1Ernster L. Hundal T. Sandri G. Resolution and reconstitution of F0F1-ATPase in beef heart submitochondrial particles.Methods Enzymol. 1986; 126: 428-433Crossref PubMed Scopus (11) Google Scholar, 2Hatefi Y. The mitochondrial electron transport and oxidative phosphorylation system.Annu. Rev. Biochem. 1985; 54: 1015-1069Crossref PubMed Google Scholar). Today the discrimination in F1- and F0-sectors is less stringent especially because F1-c subcomplex, an association of F1-complex and a ring of 10 hydrophobic c-subunits, has been isolated from yeast and crystallized (3Stock D. Leslie A.G. Walker J.E. Molecular architecture of the rotary motor in ATP synthase.Science. 1999; 286: 1700-1705Crossref PubMed Scopus (1079) Google Scholar). A similar F1-c complex but with bound natural inhibitor protein IF1 has also been identified in human mitochondria as an assembly intermediate or dead end product in the biosynthesis of ATP synthase (4Carrozzo R. Wittig I. Santorelli F.M. Bertini E. Hofmann S. Brandt U. Schägger H. Subcomplexes of human ATP synthase mark mitochondrial biosynthesis disorders.Ann. Neurol. 2006; 59: 265-275Crossref PubMed Scopus (72) Google Scholar). Focusing on functional and mechanistic aspects of ATP synthase, this rotary engine should rather be subdivided in the rotor part, which is an oligomeric ring of c-subunits connected to the central stalk (subunits γ, δ, and ε), and the residual stator part (5Walker J.E. Dickson V.K. The peripheral stalk of the mitochondrial ATP synthase.Biochim. Biophys. Acta. 2006; 1757: 286-296Crossref PubMed Scopus (151) Google Scholar). For the yeast enzyme, the stator commonly is further subdivided into the catalytic headpiece α3β3 of the stator that immediately interacts with the rotor subunit γ and also with subunits OSCP and h of the peripheral stalk (6Dickson V.K. Silvester J.A. Fearnley I.M. Leslie A.G. Walker J.E. On the structure of the stator of the mitochondrial ATP synthase.EMBO J. 2006; 25: 2911-2918Crossref PubMed Scopus (155) Google Scholar, 7Rubinstein J.L. Dickson V.K. Runswick M.J. Walker J.E. ATP synthase from Saccharomyces cerevisiae: location of subunit h in the peripheral stalk region.J. Mol. Biol. 2005; 345: 513-520Crossref PubMed Scopus (28) Google Scholar). Two further subunits, the hydrophilic subunit d and the hydrophobic subunit b comprising two transmembrane helices, are also assigned to the peripheral stalk (5Walker J.E. Dickson V.K. The peripheral stalk of the mitochondrial ATP synthase.Biochim. Biophys. Acta. 2006; 1757: 286-296Crossref PubMed Scopus (151) Google Scholar). Subunits i, f, e, and g, all containing one transmembrane helix, and possibly subunit k may be regarded as accessory peripheral stalk proteins that reach into the membrane where they presumably stabilize the most hydrophobic subunit, a (or subunit 6 or ATP6), and therefore assist with its stator function. The a-subunit must transiently bind to the oligomeric c-ring, which is an essential part of the rotor. Cross-linking experiments in Escherichia coli have shown a close neighborship of subunits c and a (8Jiang W. Fillingame R.H. Interacting helical faces of subunits a and c in the F1F0 ATP synthase of Escherichia coli defined by disulfide cross-linking.Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6607-6612Crossref PubMed Scopus (150) Google Scholar) but associates of subunits c and a without using cross-linkers could not be verified experimentally so far. One major aim of the present work was to isolate transiently stable subcomplexes that had not been experimentally verified before to obtain further structural information on ATP synthase.Mitochondrial F1F0-ATP synthase from yeast and mammals is commonly isolated as a catalytically functional monomeric complex (9Arnold I. Pfeiffer K. Neupert W. Stuart R.A. Schägger H. ATP synthase of yeast mitochondria. Isolation of subunit j and disruption of the ATP18 gene.J. Biol. Chem. 1999; 274: 36-40Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 10Arselin G. Vaillier J. Graves P.V. Velours J. ATP synthase of yeast mitochondria. Isolation of the subunit h and disruption of the ATP14 gene.J. Biol. Chem. 1996; 271: 20284-20290Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 11Belogrudov G.I. Tomich J.M. Hatefi Y. Membrane topography and near-neighbor relationships of the mitochondrial ATP synthase subunits e, f, and g.J. Biol. Chem. 1996; 271: 20340-20345Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 12Rubinstein J.L. Walker J.E. Henderson R. Structure of the mitochondrial ATP synthase by electron cryomicroscopy.EMBO J. 2003; 22: 6182-6192Crossref PubMed Scopus (184) Google Scholar), but several lines of evidence suggested that this complex is dimeric in the membrane (13Arnold I. Pfeiffer K. Neupert W. Stuart R.A. Schägger H. Yeast mitochondrial F1F0-ATP synthase exists as a dimer: identification of three dimer-specific subunits.EMBO J. 1998; 17: 7170-7178Crossref PubMed Scopus (361) Google Scholar, 14Pfeiffer K. Gohil V. Stuart R.A. Hunte C. Brandt U. Greenberg M.L. Schägger H. Cardiolipin stabilizes respiratory chain supercomplexes.J. Biol. Chem. 2003; 278: 52873-52880Abstract Full Text Full Text PDF PubMed Scopus (623) Google Scholar) or even oligomeric (15Arselin G. Giraud M.F. Dautant A. Vaillier J. Brethes D. Coulary-Salin B. Schaeffer J. Velours J. The GxxxG motif of the transmembrane domain of subunit e is involved in the dimerization/oligomerization of the yeast ATP synthase complex in the mitochondrial membrane.Eur. J. Biochem. 2003; 270: 1875-18784Crossref PubMed Scopus (111) Google Scholar, 16Krause F. Reifschneider N.H. Goto S. Dencher N.A. Active oligomeric ATP synthases in mammalian mitochondria.Biochem. Biophys. Res. Commun. 2005; 329: 583-590Crossref PubMed Scopus (97) Google Scholar, 17Wittig I. Schägger H. Advantages and limitations of clear native polyacrylamide gel electrophoresis.Proteomics. 2005; 5: 4338-4346Crossref PubMed Scopus (205) Google Scholar). Two specific proteins, the so-called dimer-specific subunits e and g, have been identified as promotors of dimerization of ATP synthase (13Arnold I. Pfeiffer K. Neupert W. Stuart R.A. Schägger H. Yeast mitochondrial F1F0-ATP synthase exists as a dimer: identification of three dimer-specific subunits.EMBO J. 1998; 17: 7170-7178Crossref PubMed Scopus (361) Google Scholar, 18Arselin G. Vaillier J. Salin B. Schaeffer J. Giraud M.F. Dautant A. Brethes D. Velours J. The modulation in subunits e and g amounts of yeast ATP synthase modifies mitochondrial cristae morphology.J. Biol. Chem. 2004; 279: 40392-40399Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 19Brunner S. Everard-Gigot V. Stuart R.A. Su e of the yeast F1F0-ATP synthase forms homodimers.J. Biol. Chem. 2002; 277: 48484-48489Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 20Everard-Gigot V. Dunn C.D. Dolan B.M. Brunner S. Jensen R.E. Stuart R.A. Functional analysis of subunit e of the F1F0-ATP synthase of the yeast Saccharomyces cerevisiae: importance of the N-terminal membrane anchor region.Eukaryot. Cell. 2005; 4: 346-355Crossref PubMed Scopus (54) Google Scholar, 21Bustos D.M. Velours J. The modification of the conserved GXXXG motif of the membrane-spanning segment of subunit g destabilizes the supramolecular species of yeast ATP synthase.J. Biol. Chem. 2005; 280: 29004-29010Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 22Saddar S. Stuart R.A. The yeast F1F0-ATP synthase. Analysis of the molecular organization of subunit g and the importance of a conserved GXXXG motif.J. Biol. Chem. 2005; 280: 24435-24442Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) and as essential components for normal mitochondrial crista morphology (23Paumard P. Vaillier J. Coulary B. Schaeffer J. Soubannier V. Mueller D.M. Brethes D. di Rago J.P. Velours J. The ATP synthase is involved in generating mitochondrial cristae morphology.EMBO J. 2002; 21: 221-230Crossref PubMed Scopus (579) Google Scholar). The term "dimer-specific subunits" originated historically from the presence of subunits e and g in dimeric yeast ATP synthase and a complete lack of these subunits in the monomeric ATP synthase isolated by blue native (BN) 1The abbreviations used are: BN, blue native; CN, clear native; complex III, ubiquinol:cytochrome c reductase; complex IV, cytochrome c oxidase; complex V, ATP synthase; DDM, dodecyl β-d-maltoside; dSDS, doubled SDS; M/P, membrane/peripheral stalk; 1-D, first dimension; 2-D, second dimension; 3-D, third dimension; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; Su, subunit; WT, wild-type; VD, dimeric complex V; VM, monomeric complex V; VINT, intermediate complex V; OSCP, oligomycin sensitivity conferring protein. 1The abbreviations used are: BN, blue native; CN, clear native; complex III, ubiquinol:cytochrome c reductase; complex IV, cytochrome c oxidase; complex V, ATP synthase; DDM, dodecyl β-d-maltoside; dSDS, doubled SDS; M/P, membrane/peripheral stalk; 1-D, first dimension; 2-D, second dimension; 3-D, third dimension; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; Su, subunit; WT, wild-type; VD, dimeric complex V; VM, monomeric complex V; VINT, intermediate complex V; OSCP, oligomycin sensitivity conferring protein.-PAGE after solubilization by low Triton X-100 concentrations. This term is potentially misleading because low amounts of subunit g and/or subunit e can be isolated with monomeric yeast ATP synthase following solubilization by digitonin and separation by BN-PAGE as described under "Results". The presence of subunits e and g favors dimerization but is not essential for dimerization, suggesting that other F0-proteins are also involved (24Gavin P.D. Prescott M. Devenish R.J. F1F0-ATP synthase complex interactions in vivo can occur in the absence of the dimer specific subunit e.J. Bioenerg. Biomembr. 2005; 37: 55-66Crossref PubMed Scopus (36) Google Scholar, 25Fronzes R. Weimann T. Vaillier J. Velours J. Brethes D. The peripheral stalk participates in the yeast ATP synthase dimerization independently of e and g subunits.Biochemistry. 2006; 45: 6715-6723Crossref PubMed Scopus (45) Google Scholar). Cross-linking evidence for the involvement of subunits h, i, and b in supporting the dimerization interface has recently been presented (25Fronzes R. Weimann T. Vaillier J. Velours J. Brethes D. The peripheral stalk participates in the yeast ATP synthase dimerization independently of e and g subunits.Biochemistry. 2006; 45: 6715-6723Crossref PubMed Scopus (45) Google Scholar, 26Paumard P. Arselin G. Vaillier J. Chaignepain S. Bathany K. Schmitter J.M. Brethes D. Velours J. Two ATP synthases can be linked through subunits i in the inner mitochondrial membrane of Saccharomyces cerevisiae..Biochemistry. 2002; 41: 10390-10396Crossref PubMed Scopus (39) Google Scholar, 27Soubannier V. Vaillier J. Paumard P. Coulary B. Schaeffer J. Velours J. 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.J. Biol. Chem. 2002; 277: 10739-10745Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). A second goal of this work was to molecularly define the interface formed by the dimerizing ATP synthase monomers. Controlled disassembly of dimeric ATP synthase under various mild conditions, as used here, was expected to confirm known components of the interface of two ATP synthase monomers and should reveal novel important protein-protein interactions.A third focus of the present work was on the higher oligomeric states of ATP synthase. In contrast to a relatively high but still incomplete knowledge on the protein-protein interactions in the monomer-monomer interface, not much is known on the interaction of dimers. These deficits may partly be due to the vanishing amounts of ATP synthase that could be isolated in higher oligomeric forms from yeast mitochondria so far (27Soubannier V. Vaillier J. Paumard P. Coulary B. Schaeffer J. Velours J. 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.J. Biol. Chem. 2002; 277: 10739-10745Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Especially interesting candidates potentially inducing oligomerization were the natural inhibitor protein Inh1 and associated proteins Stf1, Stf2, and Sfl2 that previously have been shown to be not essential for the initial dimerization (28Dienhart M. Pfeiffer K. Schägger H. Stuart R.A. Formation of the yeast F1F0-ATP synthase dimeric complex does not require the ATPase inhibitor protein Inh1.J. Biol. Chem. 2002; 277: 39289-39295Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). We can show that this inhibitor and the associated proteins are not essential for oligomerization and do not even favor higher order structures.DISCUSSIONThe rotor-stator interface is a well defined part of ATP synthase especially because the structure of the F1-ATPase has been resolved (42Abrahams J.P. Leslie A.G. Lutter R. Walker J.E. Structure at 2.8 Å resolution of F1-ATPase from bovine heart mitochondria.Nature. 1994; 370: 621-628Crossref PubMed Scopus (2736) Google Scholar), and the subunit rotation in F1-ATPase has been experimentally verified (43Sabbert D. Engelbrecht S. Junge W. Intersubunit rotation in active F-ATPase.Nature. 1996; 381: 623-625Crossref PubMed Scopus (461) Google Scholar, 44Noji H. Yasuda R. Yoshida M. Kinosita K. Direct observation of the rotation of F1-ATPase.Nature. 1997; 386: 299-302Crossref PubMed Scopus (1942) Google Scholar). Even transient loosening of interactions between the γ-subunit of the rotor part and the α3β3-headpiece of the stator part that must occur during the catalytic cycle did not lead to dissociation of the two parts. Here we show for the first time that yeast ATP synthase can dissociate at the presumed rotor-stator interface, i.e. the α3β3-headpiece dissociated from the γ-subunit that was still assembled in a very large residual complex. The concomitant removal of subunits h and OSCP with α3β3 suggested that these four subunits confer stability to the α3β3-headpiece on the one side and to interact with subunits b and d on the other side as suggested by the structure of a subcomplex crystallized from recombinantly expressed proteins (6Dickson V.K. Silvester J.A. Fearnley I.M. Leslie A.G. Walker J.E. On the structure of the stator of the mitochondrial ATP synthase.EMBO J. 2006; 25: 2911-2918Crossref PubMed Scopus (155) Google Scholar).Subunits e and g are neighboring subunits in monomeric bovine ATP synthase (11Belogrudov G.I. Tomich J.M. Hatefi Y. Membrane topography and near-neighbor relationships of the mitochondrial ATP synthase subunits e, f, and g.J. Biol. Chem. 1996; 271: 20340-20345Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). In yeast, they stabilize the dimeric form of ATP synthase so that dimers can be isolated under the conditions of BN-PAGE (13Arnold I. Pfeiffer K. Neupert W. Stuart R.A. Schägger H. Yeast mitochondrial F1F0-ATP synthase exists as a dimer: identification of three dimer-specific subunits.EMBO J. 1998; 17: 7170-7178Crossref PubMed Scopus (361) Google Scholar, 15Arselin G. Giraud M.F. Dautant A. Vaillier J. Brethes D. Coulary-Salin B. Schaeffer J. Velours J. The GxxxG motif of the transmembrane domain of subunit e is involved in the dimerization/oligomerization of the yeast ATP synthase complex in the mitochondrial membrane.Eur. J. Biochem. 2003; 270: 1875-18784Crossref PubMed Scopus (111) Google Scholar, 18Arselin G. Vaillier J. Salin B. Schaeffer J. Giraud M.F. Dautant A. Brethes D. Velours J. The modulation in subunits e and g amounts of yeast ATP synthase modifies mitochondrial cristae morphology.J. Biol. Chem. 2004; 279: 40392-40399Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 19Brunner S. Everard-Gigot V. Stuart R.A. Su e of the yeast F1F0-ATP synthase forms homodimers.J. Biol. Chem. 2002; 277: 48484-48489Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 20Everard-Gigot V. Dunn C.D. Dolan B.M. Brunner S. Jensen R.E. Stuart R.A. Functional analysis of subunit e of the F1F0-ATP synthase of the yeast Saccharomyces cerevisiae: importance of the N-terminal membrane anchor region.Eukaryot. Cell. 2005; 4: 346-355Crossref PubMed Scopus (54) Google Scholar, 21Bustos D.M. Velours J. The modification of the conserved GXXXG motif of the membrane-spanning segment of subunit g destabilizes the supramolecular species of yeast ATP synthase.J. Biol. Chem. 2005; 280: 29004-29010Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 22Saddar S. Stuart R.A. The yeast F1F0-ATP synthase. Analysis of the molecular organization of subunit g and the importance of a conserved GXXXG motif.J. Biol. Chem. 2005; 280: 24435-24442Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), but the two proteins were postulated to be not essential for dimerization in the membrane because fluorescence resonance energy transfer analyses (24Gavin P.D. Prescott M. Devenish R.J. F1F0-ATP synthase complex interactions in vivo can occur in the absence of the dimer specific subunit e.J. Bioenerg. Biomembr. 2005; 37: 55-66Crossref PubMed Scopus (36) Google Scholar) and cross-linkage of subunits h (25Fronzes R. Weimann T. Vaillier J. Velours J. Brethes D. The peripheral stalk participates in the yeast ATP synthase dimerization independently of e and g subunits.Biochemistry. 2006; 45: 6715-6723Crossref PubMed Scopus (45) Google Scholar) using subunits g and e null mutants suggested interactions of ATP synthases in the membrane also in the absence of subunits e and g. Here we identified dimeric and also minimal amounts of tetrameric ATP synthase in null mutants of subunits e and g. This supports previous postulations that subunits e and g favor supramolecular structures of ATP synthase but are not essential for the formation of dimers/oligomers (24Gavin P.D. Prescott M. Devenish R.J. F1F0-ATP synthase complex interactions in vivo can occur in the absence of the dimer specific subunit e.J. Bioenerg. Biomembr. 2005; 37: 55-66Crossref PubMed Scopus (36) Google Scholar, 25Fronzes R. Weimann T. Vaillier J. Velours J. Brethes D. The peripheral stalk participates in the yeast ATP synthase dimerization independently of e and g subunits.Biochemistry. 2006; 45: 6715-6723Crossref PubMed Scopus (45) Google Scholar) by a completely different and direct CN-PAGE approach.In the membrane-embedded ATP synthase, the c-ring rotates against the laterally orientated a-subunit that in turn is bound to subunit b in a bacterial ATP synthase (45Ono S. Sone N. Yoshida M. Suzuki T. ATP synthase that lacks F0 a-subunit. Isolation, properties, and indication of F0 b2-subunits as an anchor rail of a rotating c-ring.J. Biol. Chem. 2004; 279: 33409-33412Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar) and to further membrane/peripheral stalk subunits in the mammalian complex. The mechanism of proton translocation must involve sophisticated interactions between subunit a and the rotating c-ring to grant stability of the subunit c-a complex and at the same time to allow for almost frictionless rotation of the c-ring against subunit a (46Ueno H. Suzuki T. Kinosita Jr., K. Yoshida M. ATP-driven stepwise rotation of F0F1-ATP synthase.Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 1333-1338Crossref PubMed Scopus (133) Google Scholar, 47Dimroth P. von Ballmoos C. Meier T. Catalytic and mechanical cycles in F-ATP synthases.EMBO Rep. 2006; 7: 276-282Crossref PubMed Scopus (106) Google Scholar). This seemed to explain why subunit c-a complexes could not be isolated so far. Unexpectedly subunit c-a complexes could be isolated here by SDS-PAGE, and even more surprising the monomeric c-a complex was not isolated as a minor by-product, but both subunits (c and a) were almost quantitatively isolated in this associated form. This suggested that the mechanism of ATP synthase includes a resting position with stable subunit c-a association.We asked what is special in our SDS gels allowing isolation of the subunit c-a complex because we and others had previously separated the c-ring from the a-subunit following normal SDS incubation of isolated ATP synthase (9Arnold I. Pfeiffer K. Neupert W. Stuart R.A. Schägger H. ATP synthase of yeast mitochondria. Isolation of subunit j and disruption of the ATP18 gene.J. Biol. Chem. 1999; 274: 36-40Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Here we used 1-D native gels first to separate the native complexes. Two different protocols were then used for the transition from BN-PAGE to 2-D SDS-PAGE. Either native gel strips were wetted for 30 min with 1% SDS before starting the SDS-PAGE (Figs. 1, C and D, and 2, C and D), a common protocol for 2-D BN/SDS-PAGE previously not known to favor artificial hydrophobic interactions, or the gel strips were just wetted with water (Fig. 4, A–F). The SDS concentration in the 2-D SDS gel (in the latter case solely originating from the 0.1% SDS-containing cathode buffer) was sufficiently low to retain the subunit c-a complex in both situations. We think that we have demonstrated more than just a situation where subunits c and a are more stable in association with one another than in the surrounding medium because (i) no other proteins except subunits c and a could be identified with the monomeric c-a subcomplex, (ii) the stoichiometry was uniform (c10a1), and (iii) the recovery was almost quantitative. SDS-PAGE from BN gel strips also revealed some percentage of a larger complex with a minimal (c10)2a2 composition (further protein constituents cannot be excluded because of low protein amount) from which c10-ring and a2 dimer could be dissociated in SDS gels. Because no hints for an immediate association of two c-rings via c-c interfaces were obtained from the 2-D gels and this interface would not be compatible with rotation, we postulate that subcomplex (d) contained two c-rings linked by dimeric subunit a. Assuming that native subunit interactions existing in mitochondrial membranes are maintained through BN-PAGE and also through further and successive steps of electrophoresis, it follows that subunit a dimers can link two c10-rings in dimeric ATP synthase. Subunit a possesses more transmembrane helices than every other subunit of the complex (five and seven transmembrane helices predicted by TMpred and HMMTOP, respectively) suggesting dimeric a-subunit as the central component of a basic c-a2-c monomer-monomer interface that can be tightened by subunits e, g, b, i, and h.Natural inhibitor Inh1 and associated proteins Stf1, Stf2, and Sfl2 have been excluded here as important linkers of dimers. Other candidates that must be considered as potential linkers are the carriers for phosphate and ADP/ATP that have been described to form a supercomplex with the ATP synthase, the ATP synthasome (48Chen C. Ko Y. Delannoy M. Ludtke S.J. Chiu W. Pedersen P.L. Mitochondrial ATP synthasome. Three-dimensional structure by electron microscopy of the ATP synthase in complex formation with carriers for Pi and ADP/ATP.J. Biol. Chem. 2004; 279: 31761-31768Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar), and F0-subunits f, ATP8, and subunit a (involving transmembrane helices not engaged in monomer-monomer interactions). Based on the postulations that the metabolic state of a cell correlates with mitochondrial crista morphology (49Mannella C.A. The relevance of mitochondrial membrane topology to mitochondrial function.Biochim. Biophys. Acta. 2006; 1762: 140-147Crossref PubMed Scopus (216) Google Scholar) and that establishment of the normal crista membrane architecture requires the presence of Su e and Su g, which support the dimerization and oligomerization of ATP synthase (18Arselin G. Vaillier J. Salin B. Schaeffer J. Giraud M.F. Dautant A. Brethes D. Velours J. The modulation in subunits e and g amounts of yeast ATP synthase modifies mitochondrial cristae morphology.J. Biol. Chem. 2004; 279: 40392-40399Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 23Paumard P. Vaillier J. Coulary B. Schaeffer J. Soubannier V. Mueller D.M. Brethes D. di Rago J.P. Velours J. The ATP synthase is involved in generating mitochondrial cristae morphology.EMBO J. 2002; 21: 221-230Crossref PubMed Scopus (579) Google Scholar), the elucidation of the ATP synthase dimer-dimer interface represents an important future task. The possibility to remove a stable hydrophilic F1-subcomplex, containing subunits α, β, γ, δ, and ε with a α3β 3γ1δ1ε1 stoichiometry, from mitochondrial ATP synthase historically led to the term F1F0-ATP synthase for the holoenzyme (1Ernster L. Hundal T. Sandri G. Resolution and reconstitution of F0F1-ATPase in beef heart submitochondrial particles.Methods Enzymol. 1986; 126: 428-433Crossref PubMed Scopus (11) Google Scholar, 2Hatefi Y. The mitochondrial electron transport and oxidative phosphorylation system.Annu. Rev. Biochem. 1985; 54: 1015-1069Crossref PubMed Google Scholar). Today the discrimination in F1- and F0-sectors is less stringent especially because F1-c subcomplex, an association of F1-complex and a ring of 10 hydrophobic c-subunits, has been isolated from yeast and crystallized (3Stock D. Leslie A.G. Walker J.E. Molecular architecture of the rotary motor in ATP synthase.Science. 1999; 286: 1700-1705Crossref PubMed Scopus (1079) Google Scholar). A similar F1-c complex but with bound natural inhibitor protein IF1 has also been identified in human mitochondria as an assembly intermediate or dead end product in the biosynthesis of ATP synthase (4Carrozzo R. Wittig I. Santorelli F.M. Bertini E. Hofmann S. Brandt U. Schägger H. Subcomplexes of human ATP synthase mark mitochondrial biosynthesis disorders.Ann. Neurol. 2006; 59: 265-275Crossref PubMed Scopus (72) Google Scholar). Focusing on functional and mechanistic aspects of ATP synthase, this rotary engine should rather be subdivided in the rotor part, which is an oligomeric ring of c-subunits connected to the central stalk (subunits γ, δ, and ε), and the residual stator part (5Walker J.E. Dickson V.K. The peripheral stalk of the mitochondrial ATP synthase.Biochim. Biophys. Acta. 2006; 1757: 286-296Crossref PubMed Scopus (151) Google Scholar). For the yeast enzyme, the stator commonly is further subdivided into the catalytic headpiece α3β3 of the stator that immediately interacts with the rotor subunit γ and also with subunits OSCP and h of the peripheral stalk (6Dickson V.K. Silvester J.A. Fearnley I.M. Leslie A.G. Walker J.E. On the structure of the stator of the mitochondrial ATP synthase.EMBO J. 2006;
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