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 theATP20 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 mitochondrialb-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 subunitg and the first membrane-spanning segment of subunit4 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. 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 theATP20 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 mitochondrialb-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 subunitg and the first membrane-spanning segment of subunit4 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. blue native-polyacrylamide gel electrophoresis The F0F1-ATP synthase is a molecular rotary motor that is responsible for the aerobic synthesis of ATP. It exhibits a tripartite structure consisting of a headpiece (catalytic sector), basepiece (membrane sector), and two connecting stalks. The sector F1 containing the headpiece is a water-soluble unit retaining the ability to hydrolyze ATP when in a soluble form. F0 is embedded in the membrane and is mainly composed of hydrophobic subunits forming a specific proton conducting pathway. When the F1 and F0 sectors are coupled, the enzyme functions as a reversible H+-transporting ATPase or ATP synthase (1Fillingame R.H. Science. 1999; 286: 1687-1688Crossref PubMed Scopus (56) Google Scholar, 2Pedersen P.L. Ko Y.H. Hong S. J. Bioenerg. Biomembr. 2000; 32: 325-422Crossref PubMed Scopus (79) Google Scholar, 3Stock D. Gibbons C. Arechaga I. Leslie A.G.W. Walker J.E. Curr. Opin. Struct. Biol. 2000; 10: 672-679Crossref PubMed Scopus (259) Google Scholar). The enzyme displays two connecting stalks that are constituted of components from both F1 and F0. The first stalk is the rotor part of the enzyme. The second stalk is part of the stator which relays the catalytic domain F1 and hydrophobic membranous components of the enzyme. High resolution x-ray crystallographic data have been used to solve the structure of the F1 (4Abrahams J.P. Leslie A.G.W. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2734) Google Scholar, 5Bianchet M.A. Hullien J. Pedersen P.L. Amzel L.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11065-11070Crossref PubMed Scopus (224) Google Scholar, 6Hausrath A.C. Gruber G. Matthews B.W. Capaldi R.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13697-13702Crossref PubMed Scopus (82) Google Scholar, 7Gibbons C. Montgomery M.G. Leslie A.G. Walker J.E. Nat. Struct. Biol. 2000; 11: 1055-1061Google Scholar) from different sources. Recently, Stock et al. (8Stock D. Leslie A.G.W. Walker J.E. Science. 1999; 286: 1700-1705Crossref PubMed Scopus (1078) Google Scholar) reported the 3.9-Å resolution x-ray diffraction structure of yeast F1 associated with the c-ring oligomer, but a large part of the structure of F0 remains unknown. InEscherichia coli, F0 is composed of subunitsa, b, and c. The mitochondrial F0 of mammalian is composed of 10 different subunits (9Collinson I.R. Runswick M.J. Buchanan S.K. Fearnley I.M. Skehel J.M. van Raaij M.J. Griffiths D.E. Walker J.E. Biochemistry. 1994; 33: 7971-7978Crossref PubMed Scopus (162) Google Scholar). The same 10 components have been identified in the Saccharomyces cerevisiae enzyme (10Velours J. Arselin G. J. Bioenerg. Biomembr. 2000; 32: 383-390Crossref PubMed Scopus (91) Google Scholar). However, two of them (subunitse and g) have been classified as associated proteins since they are not essential in the structure or for the activity of the yeast complex (11Arnold I. Bauer M.F. Brunner M. Neupert W. Stuart R.A. FEBS Lett. 1997; 411: 195-200Crossref PubMed Scopus (69) Google Scholar, 12Arnold I. Pfieffer K. Neupert W. Stuart R.A. Schägger H. EMBO J. 1998; 17: 7170-7178Crossref PubMed Scopus (361) Google Scholar, 13Boyle G.M. Roucou X. Nagley P. Devenish R.J. Prescott M. Eur. J. Biochem. 1999; 262: 315-323Crossref PubMed Scopus (47) Google Scholar). In addition, two new components named subunits i/j and k have recently been identified in the yeast F0 as associated proteins (14Arnold I. Pfeiffer K. Neupert W. Stuart R.A. Schägger H. J. Biol. Chem. 1999; 274: 36-40Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar,15Vaillier J. Arselin G. Graves P.V. Camougrand N. Velours J. J. Biol. Chem. 1999; 274: 543-548Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). It has been reported that subunits e and gare involved in the dimerization of the yeast enzyme (12Arnold I. Pfieffer K. Neupert W. Stuart R.A. Schägger H. EMBO J. 1998; 17: 7170-7178Crossref PubMed Scopus (361) Google Scholar). We have recently reported that null mutants in either TIM11 orATP20 genes encoding subunits e and g, respectively, led to anomalous mitochondrial morphology with proliferation of the inner mitochondrial membrane, in turn leading to onion-like structures enclosed inside a continuous envelope of outer mitochondrial membrane (16Paumard P. Vaillier J. Coulary B. Schaeffer J. Soubannier V. Mueller D.M. Brèthes D. diRago J.P. Velours J. EMBO J. 2002; 21: 221-230Crossref PubMed Scopus (579) Google Scholar). It was hypothesized that ATP synthase itself participates in the mitochondrial morphogenesis through a oligomerization process mediated at least by subunits e, g,and 4.Subunit 4, which is homologous to the mammalianb-subunit, is a major component of the stator. It shows interactions with other components of F0 such as subunitsd, F6, OSCP, and g (17Collinson I.R. van Raaij M.J. Runswick M.J. Fearnley I.M. Skehel J.M. Orriss G.L. Miroux B. Walker J.E. J. Mol. Biol. 1994; 242: 408-421PubMed Google Scholar, 18Soubannier V. Rusconi F. Vaillier J. Arselin G. Chaignepain S. Graves P.V. Schmitter J.M. Zhang J.L. Mueller D. Velours J. Biochemistry. 1999; 38: 15017-15024Crossref PubMed Scopus (34) Google Scholar). There is only one copy of subunit b per mitochondrial ATP synthase molecule (17Collinson I.R. van Raaij M.J. Runswick M.J. Fearnley I.M. Skehel J.M. Orriss G.L. Miroux B. Walker J.E. J. Mol. Biol. 1994; 242: 408-421PubMed Google Scholar, 19Collinson I.R. Skehel J.M. Fearnley I.M. Runswick M.J. Walker J.E. Biochemistry. 1996; 35: 12640-12646Crossref PubMed Scopus (72) Google Scholar, 20Spannagel C. Vaillier J. Arselin G. Graves P.V. Grandier-Vazeille X. Velours J. Biochim. Biophys. Acta. 1998; 1414: 260-264Crossref PubMed Scopus (47) Google Scholar, 21Bateson M. Devenish R.J. Nagley P. Prescott M. J. Biol. Chem. 1999; 274: 7462-7466Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar) whereas there are two in the prokaryotic and chloroplastic enzymes. Subunit 4 is also able to dimerize with another subunit 4 belonging to another complex (20Spannagel C. Vaillier J. Arselin G. Graves P.V. Grandier-Vazeille X. Velours J. Biochim. Biophys. Acta. 1998; 1414: 260-264Crossref PubMed Scopus (47) Google Scholar). Unlike the prokaryotic and chloroplastic b-subunits which have only one membrane-spanning segment in their N-terminal part, the mitochondrial b-subunit has two membrane-spanning segments. In a previous work, we have shown that the N-terminal part of subunit4 is close to subunit g (18Soubannier V. Rusconi F. Vaillier J. Arselin G. Chaignepain S. Graves P.V. Schmitter J.M. Zhang J.L. Mueller D. Velours J. Biochemistry. 1999; 38: 15017-15024Crossref PubMed Scopus (34) Google Scholar). The purpose of the present work was to investigate the role of the first additional 43 amino acid residues bearing the first membrane-spanning segment of the mitochondrial b-subunit, and the involvement of the latter in the interaction with subunit g. We now show that the first membrane-spanning segment of the b-subunit is essential for the ATP synthase dimerization/oligomerization but is not essential for the structure and function of the yeast ATP synthase. The F0F1-ATP synthase is a molecular rotary motor that is responsible for the aerobic synthesis of ATP. It exhibits a tripartite structure consisting of a headpiece (catalytic sector), basepiece (membrane sector), and two connecting stalks. The sector F1 containing the headpiece is a water-soluble unit retaining the ability to hydrolyze ATP when in a soluble form. F0 is embedded in the membrane and is mainly composed of hydrophobic subunits forming a specific proton conducting pathway. When the F1 and F0 sectors are coupled, the enzyme functions as a reversible H+-transporting ATPase or ATP synthase (1Fillingame R.H. Science. 1999; 286: 1687-1688Crossref PubMed Scopus (56) Google Scholar, 2Pedersen P.L. Ko Y.H. Hong S. J. Bioenerg. Biomembr. 2000; 32: 325-422Crossref PubMed Scopus (79) Google Scholar, 3Stock D. Gibbons C. Arechaga I. Leslie A.G.W. Walker J.E. Curr. Opin. Struct. Biol. 2000; 10: 672-679Crossref PubMed Scopus (259) Google Scholar). The enzyme displays two connecting stalks that are constituted of components from both F1 and F0. The first stalk is the rotor part of the enzyme. The second stalk is part of the stator which relays the catalytic domain F1 and hydrophobic membranous components of the enzyme. High resolution x-ray crystallographic data have been used to solve the structure of the F1 (4Abrahams J.P. Leslie A.G.W. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2734) Google Scholar, 5Bianchet M.A. Hullien J. Pedersen P.L. Amzel L.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11065-11070Crossref PubMed Scopus (224) Google Scholar, 6Hausrath A.C. Gruber G. Matthews B.W. Capaldi R.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13697-13702Crossref PubMed Scopus (82) Google Scholar, 7Gibbons C. Montgomery M.G. Leslie A.G. Walker J.E. Nat. Struct. Biol. 2000; 11: 1055-1061Google Scholar) from different sources. Recently, Stock et al. (8Stock D. Leslie A.G.W. Walker J.E. Science. 1999; 286: 1700-1705Crossref PubMed Scopus (1078) Google Scholar) reported the 3.9-Å resolution x-ray diffraction structure of yeast F1 associated with the c-ring oligomer, but a large part of the structure of F0 remains unknown. InEscherichia coli, F0 is composed of subunitsa, b, and c. The mitochondrial F0 of mammalian is composed of 10 different subunits (9Collinson I.R. Runswick M.J. Buchanan S.K. Fearnley I.M. Skehel J.M. van Raaij M.J. Griffiths D.E. Walker J.E. Biochemistry. 1994; 33: 7971-7978Crossref PubMed Scopus (162) Google Scholar). The same 10 components have been identified in the Saccharomyces cerevisiae enzyme (10Velours J. Arselin G. J. Bioenerg. Biomembr. 2000; 32: 383-390Crossref PubMed Scopus (91) Google Scholar). However, two of them (subunitse and g) have been classified as associated proteins since they are not essential in the structure or for the activity of the yeast complex (11Arnold I. Bauer M.F. Brunner M. Neupert W. Stuart R.A. FEBS Lett. 1997; 411: 195-200Crossref PubMed Scopus (69) Google Scholar, 12Arnold I. Pfieffer K. Neupert W. Stuart R.A. Schägger H. EMBO J. 1998; 17: 7170-7178Crossref PubMed Scopus (361) Google Scholar, 13Boyle G.M. Roucou X. Nagley P. Devenish R.J. Prescott M. Eur. J. Biochem. 1999; 262: 315-323Crossref PubMed Scopus (47) Google Scholar). In addition, two new components named subunits i/j and k have recently been identified in the yeast F0 as associated proteins (14Arnold I. Pfeiffer K. Neupert W. Stuart R.A. Schägger H. J. Biol. Chem. 1999; 274: 36-40Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar,15Vaillier J. Arselin G. Graves P.V. Camougrand N. Velours J. J. Biol. Chem. 1999; 274: 543-548Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). It has been reported that subunits e and gare involved in the dimerization of the yeast enzyme (12Arnold I. Pfieffer K. Neupert W. Stuart R.A. Schägger H. EMBO J. 1998; 17: 7170-7178Crossref PubMed Scopus (361) Google Scholar). We have recently reported that null mutants in either TIM11 orATP20 genes encoding subunits e and g, respectively, led to anomalous mitochondrial morphology with proliferation of the inner mitochondrial membrane, in turn leading to onion-like structures enclosed inside a continuous envelope of outer mitochondrial membrane (16Paumard P. Vaillier J. Coulary B. Schaeffer J. Soubannier V. Mueller D.M. Brèthes D. diRago J.P. Velours J. EMBO J. 2002; 21: 221-230Crossref PubMed Scopus (579) Google Scholar). It was hypothesized that ATP synthase itself participates in the mitochondrial morphogenesis through a oligomerization process mediated at least by subunits e, g,and 4. Subunit 4, which is homologous to the mammalianb-subunit, is a major component of the stator. It shows interactions with other components of F0 such as subunitsd, F6, OSCP, and g (17Collinson I.R. van Raaij M.J. Runswick M.J. Fearnley I.M. Skehel J.M. Orriss G.L. Miroux B. Walker J.E. J. Mol. Biol. 1994; 242: 408-421PubMed Google Scholar, 18Soubannier V. Rusconi F. Vaillier J. Arselin G. Chaignepain S. Graves P.V. Schmitter J.M. Zhang J.L. Mueller D. Velours J. Biochemistry. 1999; 38: 15017-15024Crossref PubMed Scopus (34) Google Scholar). There is only one copy of subunit b per mitochondrial ATP synthase molecule (17Collinson I.R. van Raaij M.J. Runswick M.J. Fearnley I.M. Skehel J.M. Orriss G.L. Miroux B. Walker J.E. J. Mol. Biol. 1994; 242: 408-421PubMed Google Scholar, 19Collinson I.R. Skehel J.M. Fearnley I.M. Runswick M.J. Walker J.E. Biochemistry. 1996; 35: 12640-12646Crossref PubMed Scopus (72) Google Scholar, 20Spannagel C. Vaillier J. Arselin G. Graves P.V. Grandier-Vazeille X. Velours J. Biochim. Biophys. Acta. 1998; 1414: 260-264Crossref PubMed Scopus (47) Google Scholar, 21Bateson M. Devenish R.J. Nagley P. Prescott M. J. Biol. Chem. 1999; 274: 7462-7466Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar) whereas there are two in the prokaryotic and chloroplastic enzymes. Subunit 4 is also able to dimerize with another subunit 4 belonging to another complex (20Spannagel C. Vaillier J. Arselin G. Graves P.V. Grandier-Vazeille X. Velours J. Biochim. Biophys. Acta. 1998; 1414: 260-264Crossref PubMed Scopus (47) Google Scholar). Unlike the prokaryotic and chloroplastic b-subunits which have only one membrane-spanning segment in their N-terminal part, the mitochondrial b-subunit has two membrane-spanning segments. In a previous work, we have shown that the N-terminal part of subunit4 is close to subunit g (18Soubannier V. Rusconi F. Vaillier J. Arselin G. Chaignepain S. Graves P.V. Schmitter J.M. Zhang J.L. Mueller D. Velours J. Biochemistry. 1999; 38: 15017-15024Crossref PubMed Scopus (34) Google Scholar). The purpose of the present work was to investigate the role of the first additional 43 amino acid residues bearing the first membrane-spanning segment of the mitochondrial b-subunit, and the involvement of the latter in the interaction with subunit g. We now show that the first membrane-spanning segment of the b-subunit is essential for the ATP synthase dimerization/oligomerization but is not essential for the structure and function of the yeast ATP synthase. We are grateful to Drs. D. Brèthes, X. Grandier-Vazeille, and C. Napias for stimulating discussions.
No takes yet. Share an insight, caveat, or question.
Soubannier et al. (2002) studied this question.
Synapse has enriched 4 closely related papers on similar clinical questions. Consider them for comparative context: