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W1998375235 abstract "Energy transduction in mitochondria involves five oligomeric complexes embedded within the inner membrane. They are composed of catalytic and noncatalytic subunits, the role of these latter proteins often being difficult to assign. One of these complexes, the bc1 complex, is composed of three catalytic subunits including cytochrome b and seven or eight noncatalytic subunits. Recently, several mutations in the human cytochrome b gene have been linked to various diseases. We have studied in detail the effects of a cardiomyopathy generating mutation G252D in yeast. This mutation disturbs the biogenesis of the bc1 complex at 36 °C and decreases the steady-state level of the noncatalytic subunit Qcr9p. In addition, theG252D mutation and the deletion of QCR9 show synergetic defects that can be partially bypassed by suppressor mutations at position 252 and by a new cytochrome bmutation, P174T. Altogether, our results suggest that the supernumerary subunit Qcr9p enhances or stabilizes the interactions between the catalytic subunits, this role being essential at high temperature. Energy transduction in mitochondria involves five oligomeric complexes embedded within the inner membrane. They are composed of catalytic and noncatalytic subunits, the role of these latter proteins often being difficult to assign. One of these complexes, the bc1 complex, is composed of three catalytic subunits including cytochrome b and seven or eight noncatalytic subunits. Recently, several mutations in the human cytochrome b gene have been linked to various diseases. We have studied in detail the effects of a cardiomyopathy generating mutation G252D in yeast. This mutation disturbs the biogenesis of the bc1 complex at 36 °C and decreases the steady-state level of the noncatalytic subunit Qcr9p. In addition, theG252D mutation and the deletion of QCR9 show synergetic defects that can be partially bypassed by suppressor mutations at position 252 and by a new cytochrome bmutation, P174T. Altogether, our results suggest that the supernumerary subunit Qcr9p enhances or stabilizes the interactions between the catalytic subunits, this role being essential at high temperature. Numerous cellular functions are performed by oligomeric complexes composed of catalytic and noncatalytic subunits and the role of these latter proteins is often difficult to assign. Energy transduction in mitochondria involves five oligomeric complexes that are embedded within the inner membrane. One of these complexes, the bc1complex is composed of three catalytic and seven or eight noncatalytic subunits (for review, see Ref. 1Berry E.A. Guergova-Kuras M. Huang L.S. Crofts A.R. Annu. Rev. Biochem. 2000; 69: 1005-1075Crossref PubMed Scopus (394) Google Scholar). The three catalytic subunits, cytochrome b, cytochrome c1, and the Rieske iron-sulfur protein (Rieske) are conserved in the bacterial equivalents of this complex. However, several noncatalytic subunits are present in the mitochondrial bc1 complexes and are often referred to as supernumerary subunits although some are required for the enzymatic activity. In Saccharomyces cerevisiae the bc1complex contains seven supernumerary subunits that are conserved in the mammalian enzyme. The mammalian bc1 complex presents an additional subunit that has no equivalent in yeast. The mitochondrialbc1 complexes from bovine, chicken, and yeast have been crystallized (2Zhang Z. Huang L. Shulmeister V.M. Chi Y. Kim K.K. Hung L. Crofts A.R. Berry E.A. Kim S. Nature. 1998; 392: 677-684Crossref PubMed Scopus (927) Google Scholar, 3Xia D. Yu C.-A. Kim H. Xia J.-Z. Kachurin A.M. Zhang L. Yu L. Deinsenhofer J. Science. 1997; 277: 60-66Crossref PubMed Scopus (867) Google Scholar, 4Iwata S. Lee J.W. Okada K. Lee J.K. Iwata M. Rasmussen B. Link T.A. Ramaswamy S. Jap B.K. Science. 1998; 281: 64-71Crossref PubMed Scopus (1054) Google Scholar, 5Hunte C. Koepke J. Lange C. Rossmanith T. Michel H. Structure. 2000; 8: 669-684Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar) and an analysis of these structures reveals that the general shape, size, and topology of the complexes are similar in the three organisms.The mitochondrial respiratory chain is required for electron transport and oxidative phosphorylation. The bc1 complex transfers electrons from ubiquinol to cytochrome c and translocates protons into the intermembrane space, the resulting electrochemical gradient is utilized by the ATP synthase to produce ATP in the mitochondrial matrix. The bc1 complex exhibits two sites of ubiquinol binding, the Qo site in the intermembrane side and Qi site in the matrix side. The oxidation of ubiquinol at the Qo site releases two electrons; one is transferred to Rieske, then to cytochromec1, and finally to cytochrome c. The second electron is transferred to the low potential hemeb L, then to the high potential hemeb H and to ubiquinone at the Qi site. Genetic analyses in yeast have shown that the absence of a catalytic subunit always leads to a complete block of electron transfer, whereas the absence of noncatalytic subunits causes various defects inbc1 complex activity (for review, Ref. 6Tzagoloff A. Methods Enzymol. 1995; 260: 51-63Crossref PubMed Scopus (16) Google Scholar). For example, the absence of the supernumerary subunits Qcr7p or Qcr8p (7Schoppink P. Berden J. Grivell L. Eur. J. Biochem. 1989; 181: 475-483Crossref PubMed Scopus (28) Google Scholar, 8Maarse A. De Haan M. Schoppink P. Berden J. Grivell L. Eur. J. Biochem. 1988; 172: 179-184Crossref PubMed Scopus (41) Google Scholar) leads to a complete respiratory deficiency at any temperature, whereas Qcr6p or Qcr9p appears to be essential only at 36 °C (9Phillips J. Graham L. Trumpower B. J. Biol. Chem. 1993; 268: 11727-11736Abstract Full Text PDF PubMed Google Scholar, 10Yang M. Trumpower B.L. J. Biol. Chem. 1994; 269: 1270-1275Abstract Full Text PDF PubMed Google Scholar). The precise role of these supernumerary subunits and the nature of the functional interactions existing between them and the catalytic subunits still remain to be elucidated.Cytochrome b plays a crucial role in the activity of thebc1 complex because it harbors the two b hemes and participates in determining the shape of the two ubiquinone fixation sites. This integral membrane protein containing eight transmembrane segments is the only bc1 subunit encoded by the mitochondrial genome. The sequence alignment between yeast and bovine cytochrome b shows 51% identity and presents also a striking structural conservation between the cytochrome b of the two organisms (5Hunte C. Koepke J. Lange C. Rossmanith T. Michel H. Structure. 2000; 8: 669-684Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar).Recently several mutations in the human cytochrome b gene have been linked to diseases such as cardiomyopathy, exercise intolerance or Leber's hereditary optic neuropathy (for review, see Ref. 11Fisher N. Meunier B. Eur. J. Biochem. 2001; 268: 1155-1162Crossref PubMed Scopus (45) Google Scholar and references therein; Refs. 12Legros F. Chatzoglou E. Frachon P. Ogier De Baulny H. Laforet P. Jardel C. Godinot C. Lombes A. Eur. J. Hum. Genet. 2001; 9: 510-518Crossref PubMed Scopus (40) Google Scholar, 13Wibrand F. Ravn K. Schwartz M. Rosenberg T. Horn N. Vissing J. Ann. Neurol. 2001; 50: 540-543Crossref PubMed Scopus (67) Google Scholar, 14Lamantea E. Carrara F. Mariotti C. Morandi L. Tiranti V. Zeviani M. Neuromuscul. Disord. 2002; 12: 49-52Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 15Schuelke M. Krude H. Finckh B. Mayatepek E. Janssen A. Schmelz M. Trefz F. Trijbels F. Smeitink J. Ann. Neurol. 2002; 51: 388-392Crossref PubMed Scopus (68) Google Scholar). In particular, one mutation that substitutes a glycine by an aspartate residue at position 251 of cytochrome b (G251D) is associated with a histiocytoid cardiomyopathy (16Papadimitriou A. Neustein H.B. Dimauro S. Stanton R. Bresolin N. Pediatr. Res. 1984; 18: 1023-1028Crossref PubMed Google Scholar, 17Andreu A. Checcarelli N. Iwata S. Shanske S. DiMauro S. Pediatr. Res. 2000; 48: 311-314Crossref PubMed Scopus (127) Google Scholar). This mutation is located in the intermembrane space loop connecting the fifth and sixth transmembrane segments. Analyses on mitochondria purified from patient heart have established a defect in the succinate cytochrome coxidoreductase activity and in cytochrome b assembly, showing the importance of residue 251 for bc1 function.The fact that S. cerevisiae is a facultative aerobe, considerably facilitates the study of respiratory deficient mutants. Moreover, the rapid segregation of mitochondrial chromosomes in yeast permits the generation of homoplasmic cells containing only the mutated mitochondrial DNA. On the contrary, wild type and mutated mitochondrial DNAs often coexist within the cells of patients carrying a mitochondrial pathology. Thus, yeast mitochondrial mutants can provide invaluable help to fully understand the consequences of a mitochondrial mutation observed in a patient.In this paper, we have studied in detail the effects of the histiocytoid cardiomyopathy corresponding mutation in yeast (G252D). This mutation strongly disturbs the respiratory functions of cells grown at high temperature (36 °C). We have found that the presence of the aspartate residue at position 252 renders the noncatalytic subunit Qcr9p essential for the activity of thebc1 complex at 28 °C, and that this defect can be bypassed by a second mutation in the cytochrome b gene (P174T) located in a region close to Rieske. We propose that the supernumerary subunit Qcr9p enhances or stabilizes the interactions between cytochromes b, c1, and Rieske, particularly at high temperature.DISCUSSIONIn this study, we have shown that the intron-less mitochondrial chromosome from the yeast S. cerevisiae carries a mutation leading to the substitution of a glycine residue by an aspartate at position 252 of cytochrome b. The same mutation has been recently described in a patient with a cardiomyopathy (17Andreu A. Checcarelli N. Iwata S. Shanske S. DiMauro S. Pediatr. Res. 2000; 48: 311-314Crossref PubMed Scopus (127) Google Scholar) associated with a defect in succinate cytochrome c oxidoreductase activity (16Papadimitriou A. Neustein H.B. Dimauro S. Stanton R. Bresolin N. Pediatr. Res. 1984; 18: 1023-1028Crossref PubMed Google Scholar). This G252D mutation has no major effect on respiratory function of yeast cells grown at 28 °C, the appropriate growth temperature of yeast but leads to a 50% decrease of thebc1 complex activity at 36 °C, which is close to the temperature of the human body.A growing number of mutations in the human mitochondrial DNA were shown to be responsible for numerous pathologies (for review, see Ref. 30DiMauro S. Andreu A.L. Ann. Med. 2001; 33: 472-476Crossref PubMed Scopus (22) Google Scholar). Among a dozen mutations mapped to the human cytochrome bgene, only two have been investigated in the yeast system (11Fisher N. Meunier B. Eur. J. Biochem. 2001; 268: 1155-1162Crossref PubMed Scopus (45) Google Scholar). TheG34S mutation has been observed in a patient suffering from exercise intolerance (31Andreu A.L. Hanna M.G. Reichmann H. Bruno C. Penn A.S. Tanji K. Pallotti F. Iwata S. Bonilla E. Lach B. Morgan-Hughes J. DiMauro S. N. Engl. J. Med. 1999; 341: 1037-1044Crossref PubMed Scopus (359) Google Scholar) and the substitution of the corresponding glycine by aspartate in yeast leads to a total defect of thebc1 complex activity at 28 °C (32Coppee J.Y. Brasseur G. Brivet-Chevillotte P. Colson A.M. J. Biol. Chem. 1994; 269: 4221-4226Abstract Full Text PDF PubMed Google Scholar). The G339Emutation is responsible for a human myopathy and the same mutation totally abolishes the bc1 complex assembly in yeast at 28 °C (33Lemesle-Meunier D. Brivet-Chevillotte P. di Rago J.P. Slonimski P.P. Bruel C. Tron T. Forget N. J. Biol. Chem. 1993; 268: 15626-15632Abstract Full Text PDF PubMed Google Scholar, 34Andreu A.L. Bruno C. Shanske S. Shtilbans A. Hirano M. Krishna S. Hayward L. Systrom D.S. Brown Jr., R.H. DiMauro S. Neurology. 1998; 51: 1444-1447Crossref PubMed Scopus (74) Google Scholar). Thus, G252D is the first cytochromeb mutation corresponding to a human pathology that leads to a thermosensitive bc1 activity. This stresses the interest of testing different growth temperatures when using yeast as a model to study mutations implicated in human pathology.The glycine 252 of yeast cytochrome b is not only conserved in mammals and plants but is also present in several bacteria such asR. capsulatus. The G252D mutation leads to the substitution of a small amino acid by a large and negatively charged one. Thus, it is reasonable to postulate that the respiratory defect is because of steric and/or electrostatic conflicts within cytochromeb or between cytochrome b and another subunit. The glycine 252 is located in the E-ef loop at the positive side of the membrane, close to the ubiquinol oxidation site, Qo, according to the crystal structure (5Hunte C. Koepke J. Lange C. Rossmanith T. Michel H. Structure. 2000; 8: 669-684Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar). It was previously proposed (17Andreu A. Checcarelli N. Iwata S. Shanske S. DiMauro S. Pediatr. Res. 2000; 48: 311-314Crossref PubMed Scopus (127) Google Scholar) that the presence of Asp instead of Gly in human cytochrome b should cause charge repulsion with glutamate 271 of the Qo site (Glu-272 in yeast) and would impair hydroquinone binding. The fact that both the mutations D252N and D252Y, in an otherwise wild type background, totally restore the respiratory function at 36 °C suggests that the defect observed in the G252D mutant is rather because of the negative charge brought by the aspartate residue rather than the increased size. If this charge inhibits the Qo site function, cytochrome b can be only reduced at the Qi site. However, the cytochrome spectra of the Δqcr9 G252D double mutant, recorded with antimycin that blocks the Qi site, revealed the presence of reduced cytochrome b (data not shown). This suggests that the cytochrome b hemes can be reduced by ubiquinol at the Qo site in the G252D mutant.Our results suggest the existence of an interaction between cytochrome b residue 252 and Qcr9p, the mutation G252D leading to a decrease in the steady-state level of Qcr9p. However, according to Ref. 5Hunte C. Koepke J. Lange C. Rossmanith T. Michel H. Structure. 2000; 8: 669-684Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar, residue 252 is far from Qcr9p and cannot directly interact with Qcr9p although cytochrome c1 is close to residue 252 (Fig. 6). Thus, it is tempting to propose that the interaction between residue 252 and Qcr9p would be a long range interaction via cytochrome c1.Because, the closest amino acid in cytochrome c1 is lysine 182, the G252D mutation could create an illegitimate electrostatic interaction with this positively charged amino acid. This illegitimate interaction would affect cytochrome c1conformation and partially destabilize Qcr9p or affect the insertion of Qcr9p within the membrane. This would lead to a decrease of thebc1 complex activity at high temperature, as in theΔqcr9 mutant. Previous studies have shown that Qcr9p interacts with Rieske and cytochrome c1 (5Hunte C. Koepke J. Lange C. Rossmanith T. Michel H. Structure. 2000; 8: 669-684Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar, 9Phillips J. Graham L. Trumpower B. J. Biol. Chem. 1993; 268: 11727-11736Abstract Full Text PDF PubMed Google Scholar, 35Gonzalez-Halphen D. Lindorfer M.A. Capaldi R.A. Biochemistry. 1988; 27: 7021-7031Crossref PubMed Scopus (100) Google Scholar), therefore we propose that Qcr9p could enhance or stabilize the interactions between these two catalytic subunits. It was previously shown that in the total absence of Qcr9p, the conformation of Rieske and its iron-sulfur cluster insertion is altered (9Phillips J. Graham L. Trumpower B. J. Biol. Chem. 1993; 268: 11727-11736Abstract Full Text PDF PubMed Google Scholar). Thus, in theΔqcr9 G252D double mutant, the conformation of both cytochrome c1 and Rieske would be affected and this would completely prevent the electron transfer between Rieske and cytochrome c1 even at 28 °C. This hypothesis is consistent with our results showing a block in electron transfer between cytochrome b and cytochrome c1. However, direct evidence of this hypothetical illegitimate interaction could be provided by introducing a compensatory mutation in the partner residue,e.g. the proposed candidate lysine 182 of cytochromec1.The suppressor mutations able to restore the respiratory growth of theΔqcr9 G252D double mutant were mapped either at codon 252 or codon 174 of the cytochrome b gene. All partially compensate for the respiratory defect at 28 °C but not at 36 °C, suggesting that they restore the electron transfer between cytochromeb and cytochrome c1 but do not compensate for the absence of Qcr9p. This suggests that the cytochrome bsubstitutions, that we have isolated, cannot strengthen the interactions between Rieske and cytochrome c1 at high temperatures and thus cannot functionally substitute for Qcr9p. Whereas suppressor mutations at residue 252 could lower the illegitimate interactions occurring at this position in the original mutant, the suppression mechanism by proline 174 ought to be different. Both glycine 252 and proline 174 are located in the intermembrane space but are quite distant (Fig. 6), the proline 174 is at the vicinity of the tether domain of Rieske, which is necessary for the Rieske movement and the electron transfer between the iron-sulfur cluster and the heme of cytochrome c1 (2Zhang Z. Huang L. Shulmeister V.M. Chi Y. Kim K.K. Hung L. Crofts A.R. Berry E.A. Kim S. Nature. 1998; 392: 677-684Crossref PubMed Scopus (927) Google Scholar, 36Ghosh M. Wang Y. Ebert C.E. Vadlamuri S. Beattie D.S. Biochemistry. 2001; 40: 327-335Crossref PubMed Scopus (24) Google Scholar, 37Darrouzet E. Daldal F. J. Biol. Chem. 2002; 277: 3471-3476Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Substituting a proline by a threonine in this domain could improve the interaction between Rieske and cytochrome c1 that is deficient in the Δqcr9 G252D double mutant, and allow partial electron transfer at 28 °C.Altogether, these results show that residue 252 plays an important role in the biogenesis of the bc1 complex at 36 °C and is probably involved in long range interactions within the bc1complex and between the supernumerary subunit Qcr9p and the two other catalytic subunits, cytochrome c1 and Rieske. It has recently been shown that protein-phospholipid interactions also play a role in the structure of the bc1 complex (38Lange C. Nett J.H. Trumpower B.L. Hunte C. EMBO J. 2001; 20: 6591-6600Crossref PubMed Scopus (359) Google Scholar) and it is well known that phospholipids are highly sensitive to temperature. Thus, elucidating the relative roles of noncatalytic subunits and phospholipids constitutes the next challenge to fully understand respiratory complex assembly at high temperatures. Numerous cellular functions are performed by oligomeric complexes composed of catalytic and noncatalytic subunits and the role of these latter proteins is often difficult to assign. Energy transduction in mitochondria involves five oligomeric complexes that are embedded within the inner membrane. One of these complexes, the bc1complex is composed of three catalytic and seven or eight noncatalytic subunits (for review, see Ref. 1Berry E.A. Guergova-Kuras M. Huang L.S. Crofts A.R. Annu. Rev. Biochem. 2000; 69: 1005-1075Crossref PubMed Scopus (394) Google Scholar). The three catalytic subunits, cytochrome b, cytochrome c1, and the Rieske iron-sulfur protein (Rieske) are conserved in the bacterial equivalents of this complex. However, several noncatalytic subunits are present in the mitochondrial bc1 complexes and are often referred to as supernumerary subunits although some are required for the enzymatic activity. In Saccharomyces cerevisiae the bc1complex contains seven supernumerary subunits that are conserved in the mammalian enzyme. The mammalian bc1 complex presents an additional subunit that has no equivalent in yeast. The mitochondrialbc1 complexes from bovine, chicken, and yeast have been crystallized (2Zhang Z. Huang L. Shulmeister V.M. Chi Y. Kim K.K. Hung L. Crofts A.R. Berry E.A. Kim S. Nature. 1998; 392: 677-684Crossref PubMed Scopus (927) Google Scholar, 3Xia D. Yu C.-A. Kim H. Xia J.-Z. Kachurin A.M. Zhang L. Yu L. Deinsenhofer J. Science. 1997; 277: 60-66Crossref PubMed Scopus (867) Google Scholar, 4Iwata S. Lee J.W. Okada K. Lee J.K. Iwata M. Rasmussen B. Link T.A. Ramaswamy S. Jap B.K. Science. 1998; 281: 64-71Crossref PubMed Scopus (1054) Google Scholar, 5Hunte C. Koepke J. Lange C. Rossmanith T. Michel H. Structure. 2000; 8: 669-684Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar) and an analysis of these structures reveals that the general shape, size, and topology of the complexes are similar in the three organisms. The mitochondrial respiratory chain is required for electron transport and oxidative phosphorylation. The bc1 complex transfers electrons from ubiquinol to cytochrome c and translocates protons into the intermembrane space, the resulting electrochemical gradient is utilized by the ATP synthase to produce ATP in the mitochondrial matrix. The bc1 complex exhibits two sites of ubiquinol binding, the Qo site in the intermembrane side and Qi site in the matrix side. The oxidation of ubiquinol at the Qo site releases two electrons; one is transferred to Rieske, then to cytochromec1, and finally to cytochrome c. The second electron is transferred to the low potential hemeb L, then to the high potential hemeb H and to ubiquinone at the Qi site. Genetic analyses in yeast have shown that the absence of a catalytic subunit always leads to a complete block of electron transfer, whereas the absence of noncatalytic subunits causes various defects inbc1 complex activity (for review, Ref. 6Tzagoloff A. Methods Enzymol. 1995; 260: 51-63Crossref PubMed Scopus (16) Google Scholar). For example, the absence of the supernumerary subunits Qcr7p or Qcr8p (7Schoppink P. Berden J. Grivell L. Eur. J. Biochem. 1989; 181: 475-483Crossref PubMed Scopus (28) Google Scholar, 8Maarse A. De Haan M. Schoppink P. Berden J. Grivell L. Eur. J. Biochem. 1988; 172: 179-184Crossref PubMed Scopus (41) Google Scholar) leads to a complete respiratory deficiency at any temperature, whereas Qcr6p or Qcr9p appears to be essential only at 36 °C (9Phillips J. Graham L. Trumpower B. J. Biol. Chem. 1993; 268: 11727-11736Abstract Full Text PDF PubMed Google Scholar, 10Yang M. Trumpower B.L. J. Biol. Chem. 1994; 269: 1270-1275Abstract Full Text PDF PubMed Google Scholar). The precise role of these supernumerary subunits and the nature of the functional interactions existing between them and the catalytic subunits still remain to be elucidated. Cytochrome b plays a crucial role in the activity of thebc1 complex because it harbors the two b hemes and participates in determining the shape of the two ubiquinone fixation sites. This integral membrane protein containing eight transmembrane segments is the only bc1 subunit encoded by the mitochondrial genome. The sequence alignment between yeast and bovine cytochrome b shows 51% identity and presents also a striking structural conservation between the cytochrome b of the two organisms (5Hunte C. Koepke J. Lange C. Rossmanith T. Michel H. Structure. 2000; 8: 669-684Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar). Recently several mutations in the human cytochrome b gene have been linked to diseases such as cardiomyopathy, exercise intolerance or Leber's hereditary optic neuropathy (for review, see Ref. 11Fisher N. Meunier B. Eur. J. Biochem. 2001; 268: 1155-1162Crossref PubMed Scopus (45) Google Scholar and references therein; Refs. 12Legros F. Chatzoglou E. Frachon P. Ogier De Baulny H. Laforet P. Jardel C. Godinot C. Lombes A. Eur. J. Hum. Genet. 2001; 9: 510-518Crossref PubMed Scopus (40) Google Scholar, 13Wibrand F. Ravn K. Schwartz M. Rosenberg T. Horn N. Vissing J. Ann. Neurol. 2001; 50: 540-543Crossref PubMed Scopus (67) Google Scholar, 14Lamantea E. Carrara F. Mariotti C. Morandi L. Tiranti V. Zeviani M. Neuromuscul. Disord. 2002; 12: 49-52Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 15Schuelke M. Krude H. Finckh B. Mayatepek E. Janssen A. Schmelz M. Trefz F. Trijbels F. Smeitink J. Ann. Neurol. 2002; 51: 388-392Crossref PubMed Scopus (68) Google Scholar). In particular, one mutation that substitutes a glycine by an aspartate residue at position 251 of cytochrome b (G251D) is associated with a histiocytoid cardiomyopathy (16Papadimitriou A. Neustein H.B. Dimauro S. Stanton R. Bresolin N. Pediatr. Res. 1984; 18: 1023-1028Crossref PubMed Google Scholar, 17Andreu A. Checcarelli N. Iwata S. Shanske S. DiMauro S. Pediatr. Res. 2000; 48: 311-314Crossref PubMed Scopus (127) Google Scholar). This mutation is located in the intermembrane space loop connecting the fifth and sixth transmembrane segments. Analyses on mitochondria purified from patient heart have established a defect in the succinate cytochrome coxidoreductase activity and in cytochrome b assembly, showing the importance of residue 251 for bc1 function. The fact that S. cerevisiae is a facultative aerobe, considerably facilitates the study of respiratory deficient mutants. Moreover, the rapid segregation of mitochondrial chromosomes in yeast permits the generation of homoplasmic cells containing only the mutated mitochondrial DNA. On the contrary, wild type and mutated mitochondrial DNAs often coexist within the cells of patients carrying a mitochondrial pathology. Thus, yeast mitochondrial mutants can provide invaluable help to fully understand the consequences of a mitochondrial mutation observed in a patient. In this paper, we have studied in detail the effects of the histiocytoid cardiomyopathy corresponding mutation in yeast (G252D). This mutation strongly disturbs the respiratory functions of cells grown at high temperature (36 °C). We have found that the presence of the aspartate residue at position 252 renders the noncatalytic subunit Qcr9p essential for the activity of thebc1 complex at 28 °C, and that this defect can be bypassed by a second mutation in the cytochrome b gene (P174T) located in a region close to Rieske. We propose that the supernumerary subunit Qcr9p enhances or stabilizes the interactions between cytochromes b, c1, and Rieske, particularly at high temperature. DISCUSSIONIn this study, we have shown that the intron-less mitochondrial chromosome from the yeast S. cerevisiae carries a mutation leading to the substitution of a glycine residue by an aspartate at position 252 of cytochrome b. The same mutation has been recently described in a patient with a cardiomyopathy (17Andreu A. Checcarelli N. Iwata S. Shanske S. DiMauro S. Pediatr. Res. 2000; 48: 311-314Crossref PubMed Scopus (127) Google Scholar) associated with a defect in succinate cytochrome c oxidoreductase activity (16Papadimitriou A. Neustein H.B. Dimauro S. Stanton R. Bresolin N. Pediatr. Res. 1984; 18: 1023-1028Crossref PubMed Google Scholar). This G252D mutation has no major effect on respiratory function of yeast cells grown at 28 °C, the appropriate growth temperature of yeast but leads to a 50% decrease of thebc1 complex activity at 36 °C, which is close to the temperature of the human body.A growing number of mutations in the human mitochondrial DNA were shown to be responsible for numerous pathologies (for review, see Ref. 30DiMauro S. Andreu A.L. Ann. Med. 2001; 33: 472-476Crossref PubMed Scopus (22) Google Scholar). Among a dozen mutations mapped to the human cytochrome bgene, only two have been investigated in the yeast system (11Fisher N. Meunier B. Eur. J. Biochem. 2001; 268: 1155-1162Crossref PubMed Scopus (45) Google Scholar). TheG34S mutation has been observed in a patient suffering from exercise intolerance (31Andreu A.L. Hanna M.G. Reichmann H. Bruno C. Penn A.S. Tanji K. Pallotti F. Iwata S. Bonilla E. Lach B. Morgan-Hughes J. DiMauro S. N. Engl. J. Med. 1999; 341: 1037-1044Crossref PubMed Scopus (359) Google Scholar) and the substitution of the corresponding glycine by aspartate in yeast leads to a total defect of thebc1 complex activity at 28 °C (32Coppee J.Y. Brasseur G. Brivet-Chevillotte P. Colson A.M. J. Biol. Chem. 1994; 269: 4221-4226Abstract Full Text PDF PubMed Google Scholar). The G339Emutation is responsible for a human myopathy and the same mutation totally abolishes the bc1 complex assembly in yeast at 28 °C (33Lemesle-Meunier D. Brivet-Chevillotte P. di Rago J.P. Slonimski P.P. Bruel C. Tron T. Forget N. J. Biol. Chem. 1993; 268: 15626-15632Abstract Full Text PDF PubMed Google Scholar, 34Andreu A.L. Bruno C. Shanske S. Shtilbans A. Hirano M. Krishna S. Hayward L. Systrom D.S. Brown Jr., R.H. DiMauro S. Neurology. 1998; 51: 1444-1447Crossref PubMed Scopus (74) Google Scholar). Thus, G252D is the first cytochromeb mutation corresponding to a human pathology that leads to a thermosensitive bc1 activity. This stresses the interest of testing different growth temperatures when using yeast as a model to study mutations implicated in human pathology.The glycine 252 of yeast cytochrome b is not only conserved in mammals and plants but is also present in several bacteria such asR. capsulatus. The G252D mutation leads to the substitution of a small amino acid by a large and negatively charged one. Thus, it is reasonable to postulate that the respiratory defect is because of steric and/or electrostatic conflicts within cytochromeb or between cytochrome b and another subunit. The glycine 252 is located in the E-ef loop at the positive side of the membrane, close to the ubiquinol oxidation site, Qo, according to the crystal structure (5Hunte C. Koepke J. Lange C. Rossmanith T. Michel H. Structure. 2000; 8: 669-684Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar). It was previously proposed (17Andreu A. Checcarelli N. Iwata S. Shanske S. DiMauro S. Pediatr. Res. 2000; 48: 311-314Crossref PubMed Scopus (127) Google Scholar) that the presence of Asp instead of Gly in human cytochrome b should cause charge repulsion with glutamate 271 of the Qo site (Glu-272 in yeast) and would impair hydroquinone binding. The fact that both the mutations D252N and D252Y, in an otherwise wild type background, totally restore the respiratory function at 36 °C suggests that the defect observed in the G252D mutant is rather because of the negative charge brought by the aspartate residue rather than the increased size. If this charge inhibits the Qo site function, cytochrome b can be only reduced at the Qi site. However, the cytochrome spectra of the Δqcr9 G252D double mutant, recorded with antimycin that blocks the Qi site, revealed the presence of reduced cytochrome b (data not shown). This suggests that the cytochrome b hemes can be reduced by ubiquinol at the Qo site in the G252D mutant.Our results suggest the existence of an interaction between cytochrome b residue 252 and Qcr9p, the mutation G252D leading to a decrease in the steady-state level of Qcr9p. However, according to Ref. 5Hunte C. Koepke J. Lange C. Rossmanith T. Michel H. Structure. 2000; 8: 669-684Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar, residue 252 is far from Qcr9p and cannot directly interact with Qcr9p although cytochrome c1 is close to residue 252 (Fig. 6). Thus, it is tempting to propose that the interaction between residue 252 and Qcr9p would be a long range interaction via cytochrome c1.Because, the closest amino acid in cytochrome c1 is lysine 182, the G252D mutation could create an illegitimate electrostatic interaction with this positively charged amino acid. This illegitimate interaction would affect cytochrome c1conformation and partially destabilize Qcr9p or affect the insertion of Qcr9p within the membrane. This would lead to a decrease of thebc1 complex activity at high temperature, as in theΔqcr9 mutant. Previous studies have shown that Qcr9p interacts with Rieske and cytochrome c1 (5Hunte C. Koepke J. Lange C. Rossmanith T. Michel H. Structure. 2000; 8: 669-684Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar, 9Phillips J. Graham L. Trumpower B. J. Biol. Chem. 1993; 268: 11727-11736Abstract Full Text PDF PubMed Google Scholar, 35Gonzalez-Halphen D. Lindorfer M.A. Capaldi R.A. Biochemistry. 1988; 27: 7021-7031Crossref PubMed Scopus (100) Google Scholar), therefore we propose that Qcr9p could enhance or stabilize the interactions between these two catalytic subunits. It was previously shown that in the total absence of Qcr9p, the conformation of Rieske and its iron-sulfur cluster insertion is altered (9Phillips J. Graham L. Trumpower B. J. Biol. Chem. 1993; 268: 11727-11736Abstract Full Text PDF PubMed Google Scholar). Thus, in theΔqcr9 G252D double mutant, the conformation of both cytochrome c1 and Rieske would be affected and this would completely prevent the electron transfer between Rieske and cytochrome c1 even at 28 °C. This hypothesis is consistent with our results showing a block in electron transfer between cytochrome b and cytochrome c1. However, direct evidence of this hypothetical illegitimate interaction could be provided by introducing a compensatory mutation in the partner residue,e.g. the proposed candidate lysine 182 of cytochromec1.The suppressor mutations able to restore the respiratory growth of theΔqcr9 G252D double mutant were mapped either at codon 252 or codon 174 of the cytochrome b gene. All partially compensate for the respiratory defect at 28 °C but not at 36 °C, suggesting that they restore the electron transfer between cytochromeb and cytochrome c1 but do not compensate for the absence of Qcr9p. This suggests that the cytochrome bsubstitutions, that we have isolated, cannot strengthen the interactions between Rieske and cytochrome c1 at high temperatures and thus cannot functionally substitute for Qcr9p. Whereas suppressor mutations at residue 252 could lower the illegitimate interactions occurring at this position in the original mutant, the suppression mechanism by proline 174 ought to be different. Both glycine 252 and proline 174 are located in the intermembrane space but are quite distant (Fig. 6), the proline 174 is at the vicinity of the tether domain of Rieske, which is necessary for the Rieske movement and the electron transfer between the iron-sulfur cluster and the heme of cytochrome c1 (2Zhang Z. Huang L. Shulmeister V.M. Chi Y. Kim K.K. Hung L. Crofts A.R. Berry E.A. Kim S. Nature. 1998; 392: 677-684Crossref PubMed Scopus (927) Google Scholar, 36Ghosh M. Wang Y. Ebert C.E. Vadlamuri S. Beattie D.S. Biochemistry. 2001; 40: 327-335Crossref PubMed Scopus (24) Google Scholar, 37Darrouzet E. Daldal F. J. Biol. Chem. 2002; 277: 3471-3476Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Substituting a proline by a threonine in this domain could improve the interaction between Rieske and cytochrome c1 that is deficient in the Δqcr9 G252D double mutant, and allow partial electron transfer at 28 °C.Altogether, these results show that residue 252 plays an important role in the biogenesis of the bc1 complex at 36 °C and is probably involved in long range interactions within the bc1complex and between the supernumerary subunit Qcr9p and the two other catalytic subunits, cytochrome c1 and Rieske. It has recently been shown that protein-phospholipid interactions also play a role in the structure of the bc1 complex (38Lange C. Nett J.H. Trumpower B.L. Hunte C. EMBO J. 2001; 20: 6591-6600Crossref PubMed Scopus (359) Google Scholar) and it is well known that phospholipids are highly sensitive to temperature. Thus, elucidating the relative roles of noncatalytic subunits and phospholipids constitutes the next challenge to fully understand respiratory complex assembly at high temperatures. In this study, we have shown that the intron-less mitochondrial chromosome from the yeast S. cerevisiae carries a mutation leading to the substitution of a glycine residue by an aspartate at position 252 of cytochrome b. The same mutation has been recently described in a patient with a cardiomyopathy (17Andreu A. Checcarelli N. Iwata S. Shanske S. DiMauro S. Pediatr. Res. 2000; 48: 311-314Crossref PubMed Scopus (127) Google Scholar) associated with a defect in succinate cytochrome c oxidoreductase activity (16Papadimitriou A. Neustein H.B. Dimauro S. Stanton R. Bresolin N. Pediatr. Res. 1984; 18: 1023-1028Crossref PubMed Google Scholar). This G252D mutation has no major effect on respiratory function of yeast cells grown at 28 °C, the appropriate growth temperature of yeast but leads to a 50% decrease of thebc1 complex activity at 36 °C, which is close to the temperature of the human body. A growing number of mutations in the human mitochondrial DNA were shown to be responsible for numerous pathologies (for review, see Ref. 30DiMauro S. Andreu A.L. Ann. Med. 2001; 33: 472-476Crossref PubMed Scopus (22) Google Scholar). Among a dozen mutations mapped to the human cytochrome bgene, only two have been investigated in the yeast system (11Fisher N. Meunier B. Eur. J. Biochem. 2001; 268: 1155-1162Crossref PubMed Scopus (45) Google Scholar). TheG34S mutation has been observed in a patient suffering from exercise intolerance (31Andreu A.L. Hanna M.G. Reichmann H. Bruno C. Penn A.S. Tanji K. Pallotti F. Iwata S. Bonilla E. Lach B. Morgan-Hughes J. DiMauro S. N. Engl. J. Med. 1999; 341: 1037-1044Crossref PubMed Scopus (359) Google Scholar) and the substitution of the corresponding glycine by aspartate in yeast leads to a total defect of thebc1 complex activity at 28 °C (32Coppee J.Y. Brasseur G. Brivet-Chevillotte P. Colson A.M. J. Biol. Chem. 1994; 269: 4221-4226Abstract Full Text PDF PubMed Google Scholar). The G339Emutation is responsible for a human myopathy and the same mutation totally abolishes the bc1 complex assembly in yeast at 28 °C (33Lemesle-Meunier D. Brivet-Chevillotte P. di Rago J.P. Slonimski P.P. Bruel C. Tron T. Forget N. J. Biol. Chem. 1993; 268: 15626-15632Abstract Full Text PDF PubMed Google Scholar, 34Andreu A.L. Bruno C. Shanske S. Shtilbans A. Hirano M. Krishna S. Hayward L. Systrom D.S. Brown Jr., R.H. DiMauro S. Neurology. 1998; 51: 1444-1447Crossref PubMed Scopus (74) Google Scholar). Thus, G252D is the first cytochromeb mutation corresponding to a human pathology that leads to a thermosensitive bc1 activity. This stresses the interest of testing different growth temperatures when using yeast as a model to study mutations implicated in human pathology. The glycine 252 of yeast cytochrome b is not only conserved in mammals and plants but is also present in several bacteria such asR. capsulatus. The G252D mutation leads to the substitution of a small amino acid by a large and negatively charged one. Thus, it is reasonable to postulate that the respiratory defect is because of steric and/or electrostatic conflicts within cytochromeb or between cytochrome b and another subunit. The glycine 252 is located in the E-ef loop at the positive side of the membrane, close to the ubiquinol oxidation site, Qo, according to the crystal structure (5Hunte C. Koepke J. Lange C. Rossmanith T. Michel H. Structure. 2000; 8: 669-684Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar). It was previously proposed (17Andreu A. Checcarelli N. Iwata S. Shanske S. DiMauro S. Pediatr. Res. 2000; 48: 311-314Crossref PubMed Scopus (127) Google Scholar) that the presence of Asp instead of Gly in human cytochrome b should cause charge repulsion with glutamate 271 of the Qo site (Glu-272 in yeast) and would impair hydroquinone binding. The fact that both the mutations D252N and D252Y, in an otherwise wild type background, totally restore the respiratory function at 36 °C suggests that the defect observed in the G252D mutant is rather because of the negative charge brought by the aspartate residue rather than the increased size. If this charge inhibits the Qo site function, cytochrome b can be only reduced at the Qi site. However, the cytochrome spectra of the Δqcr9 G252D double mutant, recorded with antimycin that blocks the Qi site, revealed the presence of reduced cytochrome b (data not shown). This suggests that the cytochrome b hemes can be reduced by ubiquinol at the Qo site in the G252D mutant. Our results suggest the existence of an interaction between cytochrome b residue 252 and Qcr9p, the mutation G252D leading to a decrease in the steady-state level of Qcr9p. However, according to Ref. 5Hunte C. Koepke J. Lange C. Rossmanith T. Michel H. Structure. 2000; 8: 669-684Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar, residue 252 is far from Qcr9p and cannot directly interact with Qcr9p although cytochrome c1 is close to residue 252 (Fig. 6). Thus, it is tempting to propose that the interaction between residue 252 and Qcr9p would be a long range interaction via cytochrome c1.Because, the closest amino acid in cytochrome c1 is lysine 182, the G252D mutation could create an illegitimate electrostatic interaction with this positively charged amino acid. This illegitimate interaction would affect cytochrome c1conformation and partially destabilize Qcr9p or affect the insertion of Qcr9p within the membrane. This would lead to a decrease of thebc1 complex activity at high temperature, as in theΔqcr9 mutant. Previous studies have shown that Qcr9p interacts with Rieske and cytochrome c1 (5Hunte C. Koepke J. Lange C. Rossmanith T. Michel H. Structure. 2000; 8: 669-684Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar, 9Phillips J. Graham L. Trumpower B. J. Biol. Chem. 1993; 268: 11727-11736Abstract Full Text PDF PubMed Google Scholar, 35Gonzalez-Halphen D. Lindorfer M.A. Capaldi R.A. Biochemistry. 1988; 27: 7021-7031Crossref PubMed Scopus (100) Google Scholar), therefore we propose that Qcr9p could enhance or stabilize the interactions between these two catalytic subunits. It was previously shown that in the total absence of Qcr9p, the conformation of Rieske and its iron-sulfur cluster insertion is altered (9Phillips J. Graham L. Trumpower B. J. Biol. Chem. 1993; 268: 11727-11736Abstract Full Text PDF PubMed Google Scholar). Thus, in theΔqcr9 G252D double mutant, the conformation of both cytochrome c1 and Rieske would be affected and this would completely prevent the electron transfer between Rieske and cytochrome c1 even at 28 °C. This hypothesis is consistent with our results showing a block in electron transfer between cytochrome b and cytochrome c1. However, direct evidence of this hypothetical illegitimate interaction could be provided by introducing a compensatory mutation in the partner residue,e.g. the proposed candidate lysine 182 of cytochromec1. The suppressor mutations able to restore the respiratory growth of theΔqcr9 G252D double mutant were mapped either at codon 252 or codon 174 of the cytochrome b gene. All partially compensate for the respiratory defect at 28 °C but not at 36 °C, suggesting that they restore the electron transfer between cytochromeb and cytochrome c1 but do not compensate for the absence of Qcr9p. This suggests that the cytochrome bsubstitutions, that we have isolated, cannot strengthen the interactions between Rieske and cytochrome c1 at high temperatures and thus cannot functionally substitute for Qcr9p. Whereas suppressor mutations at residue 252 could lower the illegitimate interactions occurring at this position in the original mutant, the suppression mechanism by proline 174 ought to be different. Both glycine 252 and proline 174 are located in the intermembrane space but are quite distant (Fig. 6), the proline 174 is at the vicinity of the tether domain of Rieske, which is necessary for the Rieske movement and the electron transfer between the iron-sulfur cluster and the heme of cytochrome c1 (2Zhang Z. Huang L. Shulmeister V.M. Chi Y. Kim K.K. Hung L. Crofts A.R. Berry E.A. Kim S. Nature. 1998; 392: 677-684Crossref PubMed Scopus (927) Google Scholar, 36Ghosh M. Wang Y. Ebert C.E. Vadlamuri S. Beattie D.S. Biochemistry. 2001; 40: 327-335Crossref PubMed Scopus (24) Google Scholar, 37Darrouzet E. Daldal F. J. Biol. Chem. 2002; 277: 3471-3476Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Substituting a proline by a threonine in this domain could improve the interaction between Rieske and cytochrome c1 that is deficient in the Δqcr9 G252D double mutant, and allow partial electron transfer at 28 °C. Altogether, these results show that residue 252 plays an important role in the biogenesis of the bc1 complex at 36 °C and is probably involved in long range interactions within the bc1complex and between the supernumerary subunit Qcr9p and the two other catalytic subunits, cytochrome c1 and Rieske. It has recently been shown that protein-phospholipid interactions also play a role in the structure of the bc1 complex (38Lange C. Nett J.H. Trumpower B.L. Hunte C. EMBO J. 2001; 20: 6591-6600Crossref PubMed Scopus (359) Google Scholar) and it is well known that phospholipids are highly sensitive to temperature. Thus, elucidating the relative roles of noncatalytic subunits and phospholipids constitutes the next challenge to fully understand respiratory complex assembly at high temperatures. We are particularly grateful to N. Lachacinsky for technical assistance. We thank Drs. C. Godinot, A. Lombes, and J. M. Galan for the gift of antisera. We also thank C. Lemaire, O. Groudinsky, C. J. Herbert, and P. Hamel for the critical reading of the manuscript." @default.
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W1998375235 title "A Pathogenic Cytochrome b Mutation Reveals New Interactions between Subunits of the Mitochondrial bc1Complex" @default.
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