Mitochondrial respiratory chain is organised into supramolecular structures that can be preserved in mild detergent solubilisates and resolved by native electrophoretic systems. Supercomplexes of respiratory complexes I, III and IV as well as multimeric forms of ATP synthase are well established. However, the involvement of complex II, linking respiratory chain with tricarboxylic acid cycle, in mitochondrial supercomplexes is questionable. Here we show that digitonin-solubilised complex II quantitatively forms high molecular weight structures (CIIhmw) that can be resolved by clear native electrophoresis. CIIhmw structures are enzymatically active and differ in electrophoretic mobility between tissues (500 - over 1000 kDa) and cultured cells (400-670 kDa). While their formation is unaffected by isolated defects in other respiratory chain complexes, they are destabilised in mtDNA-depleted, rho0 cells. Molecular interactions responsible for the assembly of CIIhmw are rather weak with the complexes being more stable in tissues than in cultured cells. While electrophoretic studies and immunoprecipitation experiments of CIIhmw do not indicate specific interactions with the respiratory chain complexes I, III or IV or enzymes of the tricarboxylic acid cycle, they point out to a specific interaction between CII and ATP synthase.

Department of Bioenergetics Institute of Physiology Academy of Sciences of the Czech Republic Prague Czech Republic

Zobrazit více v PubMed

Cecchini G (2003) Function and structure of complex II of the respiratory chain. Annu Rev Biochem 72: 77-109. doi:10.1146/annurev.biochem.72.121801.161700. PubMed: 14527321. PubMed DOI

Sun F, Huo X, Zhai YJ, Wang AJ, Xu JX et al. (2005) Crystal structure of mitochondrial respiratory membrane protein complex II. Cell 121: 1043-1057. doi:10.1016/j.cell.2005.05.025. PubMed: 15989954. PubMed DOI

Brière JJ, Favier J, El Ghouzzi V, Djouadi F, Bénit P et al. (2005) Succinate dehydrogenase deficiency in human. Cell Mol Life Sci 62: 2317-2324. doi:10.1007/s00018-005-5237-6. PubMed: 16143825. PubMed DOI PMC

Bardella C, Pollard PJ, Tomlinson I (2011) SDH mutations in cancer. Biochim Biophys Acta 1807: 1432-1443. doi:10.1016/j.bbabio.2011.07.003. PubMed: 21771581. PubMed DOI

Burnichon N, Brière JJ, Libé R, Vescovo L, Rivière J et al. (2010) SDHA is a tumor suppressor gene causing paraganglioma. Hum Mol Genet 19: 3011-3020. doi:10.1093/hmg/ddq206. PubMed: 20484225. PubMed DOI PMC

Perry CG, Young WF Jr, McWhinney SR, Bei T, Stergiopoulos S et al. (2006) Functioning paraganglioma and gastrointestinal stromal tumor of the jejunum in three women: syndrome or coincidence. Am J Surg Pathol 30: 42-49. doi:10.1097/01.pas.0000178087.69394.9f. PubMed: 16330941. PubMed DOI

Ricketts C, Woodward ER, Killick P, Morris MR, Astuti D et al. (2008) Germline SDHB mutations and familial renal cell carcinoma. J Natl Cancer Inst 100: 1260-1262. doi:10.1093/jnci/djn254. PubMed: 18728283. PubMed DOI

Kluckova K, Bezawork-Geleta A, Rohlena J, Dong L, Neuzil J (2013) Mitochondrial complex II, a novel target for anti-cancer agents. Biochim Biophys Acta 1827: 552-564. doi:10.1016/j.bbabio.2012.10.015. PubMed: 23142170. PubMed DOI

Ghezzi D, Goffrini P, Uziel G, Horvath R, Klopstock T et al. (2009) SDHAF1, encoding a LYR complex-II specific assembly factor, is mutated in SDH-defective infantile leukoencephalopathy. Nat Genet 41: 654-656. doi:10.1038/ng.378. PubMed: 19465911. PubMed DOI

Shi YB, Ghosh MC, Tong WH, Rouault TA (2009) Human ISD11 is essential for both iron-sulfur cluster assembly and maintenance of normal cellular iron homeostasis. Hum Mol Genet 18: 3014-3025. doi:10.1093/hmg/ddp239. PubMed: 19454487. PubMed DOI PMC

Hao HX, Khalimonchuk O, Schraders M, Dephoure N, Bayley JP et al. (2009) SDH5, a gene required for flavination of succinate dehydrogenase, is mutated in paraganglioma. Science 325: 1139-1142. doi:10.1126/science.1175689. PubMed: 19628817. PubMed DOI PMC

Bianchi C, Genova ML, Parenti Castelli G, Lenaz G (2004) The mitochondrial respiratory chain is partially organized in a supercomplex assembly: kinetic evidence using flux control analysis. J Biol Chem 279: 36562-36569. doi:10.1074/jbc.M405135200. PubMed: 15205457. PubMed DOI

Shibata N, Kobayashi M (2008) The role for oxidative stress in neurodegenerative diseases. Brain Nerve 60: 157-170. PubMed: 18306664. PubMed

Strauss M, Hofhaus G, Schröder RR, Kühlbrandt W (2008) Dimer ribbons of ATP synthase shape the inner mitochondrial membrane. EMBO J 27: 1154-1160. doi:10.1038/emboj.2008.35. PubMed: 18323778. PubMed DOI PMC

Schägger H, Pfeiffer K (2000) Supercomplexes in the respiratory chains of yeast and mammalian mitochondria. EMBO J 19: 1777-1783. doi:10.1093/emboj/19.8.1777. PubMed: 10775262. PubMed DOI PMC

Wittig I, Schägger H (2009) Native electrophoretic techniques to identify protein-protein interactions. Proteomics 9: 5214-5223. doi:10.1002/pmic.200900151. PubMed: 19834896. PubMed DOI

Acín-Pérez R, Fernández-Silva P, Peleato ML, Pérez-Martos A, Enriquez JA (2008) Respiratory active mitochondrial supercomplexes. Mol Cell 32: 529-539. doi:10.1016/j.molcel.2008.10.021. PubMed: 19026783. PubMed DOI

Ardehali H, Chen Z, Ko Y, Mejía-Alvarez R, Marbán E (2004) Multiprotein complex containing succinate dehydrogenase confers mitochondrial ATP-sensitive K+ channel activity. Proc Natl Acad Sci U S A 101: 11880-11885. doi:10.1073/pnas.0401703101. PubMed: 15284438. PubMed DOI PMC

Meyer FM, Gerwig J, Hammer E, Herzberg C, Commichau FM et al. (2011) Physical interactions between tricarboxylic acid cycle enzymes in Bacillus subtilis: evidence for a metabolon. Metab Eng 13: 18-27. doi:10.1016/j.ymben.2010.10.001. PubMed: 20933603. PubMed DOI

Vélot C, Mixon MB, Teige M, Srere PA (1997) Model of a quinary structure between Krebs TCA cycle enzymes: a model for the metabolon. Biochemistry 36: 14271-14276. doi:10.1021/bi972011j. PubMed: 9400365. PubMed DOI

Kovářová N, Cížková Vrbacká A, Pecina P, Stránecký V, Pronicka E et al. (2012) Adaptation of respiratory chain biogenesis to cytochrome c oxidase deficiency caused by SURF1 gene mutations. Biochim Biophys Acta 1822: 1114-1124. doi:10.1016/j.bbadis.2012.03.007. PubMed: 22465034. PubMed DOI

Piekutowska-Abramczuk D, Magner M, Popowska E, Pronicki M, Karczmarewicz E et al. (2009) SURF1 missense mutations promote a mild Leigh phenotype. Clin Genet 76: 195-204. doi:10.1111/j.1399-0004.2009.01195.x. PubMed: 19780766. PubMed DOI

Honzík T, Tesarová M, Mayr JA, Hansíková H, Jesina P et al. (2010) Mitochondrial encephalocardio-myopathy with early neonatal onset due to TMEM70 mutation. Arch Dis Child 95: 296-301. doi:10.1136/adc.2009.168096. PubMed: 20335238. PubMed DOI

King MP, Attardi G (1996) Isolation of human cell lines lacking mitochondrial DNA. Methods Enzymol 264: 304-313. doi:10.1016/S0076-6879(96)64029-4. PubMed: 8965704. PubMed DOI

Bentlage HA, Wendel U, Schägger H, ter Laak HJ, Janssen AJ et al. (1996) Lethal infantile mitochondrial disease with isolated complex I deficiency in fibroblasts but with combined complex I and IV deficiencies in muscle. Neurology 47: 243-248. doi:10.1212/WNL.47.1.243. PubMed: 8710086. PubMed DOI

Wittig I, Braun HP, Schägger H (2006) Blue native PAGE. Nat Protoc 1: 418-428. doi:10.1038/nprot.2006.62. PubMed: 17406264. PubMed DOI

Mrácek T, Pecinová A, Vrbacký M, Drahota Z, Houstek J (2009) High efficiency of ROS production by glycerophosphate dehydrogenase in mammalian mitochondria. Arch Biochem Biophys 481: 30-36. doi:10.1016/j.abb.2008.10.011. PubMed: 18952046. PubMed DOI

Wittig I, Karas M, Schägger H (2007) High resolution clear native electrophoresis for in-gel functional assays and fluorescence studies of membrane protein complexes. Mol Cell Proteomics 6: 1215-1225. doi:10.1074/mcp.M700076-MCP200. PubMed: 17426019. PubMed DOI

Schägger H, von Jagow G (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166: 368-379. doi:10.1016/0003-2697(87)90587-2. PubMed: 2449095. PubMed DOI

Moradi-Ameli M, Godinot C (1983) Characterization of monoclonal antibodies against mitochondrial F1-ATPase. Proc Natl Acad Sci U S A 80: 6167-6171. doi:10.1073/pnas.80.20.6167. PubMed: 6194526. PubMed DOI PMC

Wittig I, Schägger H (2005) Advantages and limitations of clear-native PAGE. Proteomics 5: 4338-4346. doi:10.1002/pmic.200500081. PubMed: 16220535. PubMed DOI

Ko YH, Delannoy M, Hullihen J, Chiu W, Pedersen PL (2003) Mitochondrial ATP synthasome. Cristae-enriched membranes and a multiwell detergent screening assay yield dispersed single complexes containing the ATP synthase and carriers for Pi and ADP/ATP. J Biol Chem 278: 12305-12309. doi:10.1074/jbc.C200703200. PubMed: 12560333. PubMed DOI

Lenaz G, Genova ML (2012) Supramolecular organisation of the mitochondrial respiratory chain: a new challenge for the mechanism and control of oxidative phosphorylation. Adv Exp Med Biol 748: 107-144. doi:10.1007/978-1-4614-3573-0_5. PubMed: 22729856. PubMed DOI

Rosca M, Minkler P, Hoppel CL (2011) Cardiac mitochondria in heart failure: normal cardiolipin profile and increased threonine phosphorylation of complex IV. Biochim Biophys Acta 1807: 1373-1382. doi:10.1016/j.bbabio.2011.02.003. PubMed: 21320465. PubMed DOI

Benard G, Faustin B, Passerieux E, Galinier A, Rocher C et al. (2006) Physiological diversity of mitochondrial oxidative phosphorylation. Am J Physiol Cell Physiol 291: C1172-C1182. doi:10.1152/ajpcell.00195.2006. PubMed: 16807301. PubMed DOI

Dudkina NV, Eubel H, Keegstra W, Boekema EJ, Braun HP (2005) Structure of a mitochondrial supercomplex formed by respiratory-chain complexes I and III. Proc Natl Acad Sci U S A 102: 3225-3229. doi:10.1073/pnas.0408870102. PubMed: 15713802. PubMed DOI PMC

Schäfer E, Seelert H, Reifschneider NH, Krause F, Dencher NA et al. (2006) Architecture of active mammalian respiratory chain supercomplexes. J Biol Chem 281: 15370-15375. doi:10.1074/jbc.M513525200. PubMed: 16551638. PubMed DOI

Moreno-Lastres D, Fontanesi F, García-Consuegra I, Martín MA, Arenas J et al. (2012) Mitochondrial complex I plays an essential role in human respirasome assembly. Cell Metab 15: 324-335. doi:10.1016/j.cmet.2012.01.015. PubMed: 22342700. PubMed DOI PMC

Chevallet M, Lescuyer P, Diemer H, van Dorsselaer A, Leize-Wagner E et al. (2006) Alterations of the mitochondrial proteome caused by the absence of mitochondrial DNA: A proteomic view. Electrophoresis 27: 1574-1583. doi:10.1002/elps.200500704. PubMed: 16548050. PubMed DOI PMC

Mueller EE, Mayr JA, Zimmermann FA, Feichtinger RG, Stanger O et al. (2012) Reduction of nuclear encoded enzymes of mitochondrial energy metabolism in cells devoid of mitochondrial DNA. Biochem Biophys Res Commun 417: 1052-1057. doi:10.1016/j.bbrc.2011.12.093. PubMed: 22222373. PubMed DOI

Mercy L, Pauw Ad, Payen L, Tejerina S, Houbion A et al. (2005) Mitochondrial biogenesis in mtDNA-depleted cells involves a Ca2+-dependent pathway and a reduced mitochondrial protein import. FEBS J 272: 5031-5055. doi:10.1111/j.1742-4658.2005.04913.x. PubMed: 16176275. PubMed DOI

Wojtovich AP, Smith CO, Haynes CM, Nehrke KW, Brookes PS (2013) Physiological consequences of complex II inhibition for aging, disease, and the mKATP channel. Biochim Biophys Acta 1827: 598-611. doi:10.1016/j.bbabio.2012.12.007. PubMed: 23291191. PubMed DOI PMC

Wojtovich AP, Nehrke KW, Brookes PS (2010) The mitochondrial complex II and ATP-sensitive potassium channel interaction: quantitation of the channel in heart mitochondria. Acta Biochim Pol 57: 431-434. PubMed: 21103454. PubMed PMC

Carrozzo R, Wittig I, Santorelli FM, Bertini E, Hofmann S et al. (2006) Subcomplexes of human ATP synthase mark mitochondrial biosynthesis disorders. Ann Neurol 59: 265-275. doi:10.1002/ana.20729. PubMed: 16365880. PubMed DOI

Buchet K, Godinot C (1998) Functional F1-ATPase essential in maintaining growth and membrane potential of human mitochondrial DNA-depleted rho degrees cells. J Biol Chem 273: 22983-22989. doi:10.1074/jbc.273.36.22983. PubMed: 9722521. PubMed DOI

García JJ, Ogilvie I, Robinson BH, Capaldi RA (2000) Structure, functioning, and assembly of the ATP synthase in cells from patients with the T8993G mitochondrial DNA mutation. Comparison with the enzyme in Rho(0) cells completely lacking mtdna. J Biol Chem 275: 11075-11081. doi:10.1074/jbc.275.15.11075. PubMed: 10753912. PubMed DOI

Robinson JB Jr, Inman L, Sumegi B, Srere PA (1987) Further characterization of the Krebs tricarboxylic acid cycle metabolon. J Biol Chem 262: 1786-1790. PubMed: 2433288. PubMed

Haggie PM, Verkman AS (2002) Diffusion of tricarboxylic acid cycle enzymes in the mitochondrial matrix in vivo. Evidence for restricted mobility of a multienzyme complex. J Biol Chem 277: 40782-40788. doi:10.1074/jbc.M207456200. PubMed: 12198136. PubMed DOI

Gebert N, Gebert M, Oeljeklaus S, von der Malsburg K, Stroud DA et al. (2011) Dual function of Sdh3 in the respiratory chain and TIM22 protein translocase of the mitochondrial inner membrane. Mol Cell 44: 811-818. doi:10.1016/j.molcel.2011.09.025. PubMed: 22152483. PubMed DOI

Alternative assembly of respiratory complex II connects energy stress to metabolic checkpoints

Trypanocidal action of bisphosphonium salts through a mitochondrial target in bloodstream form Trypanosoma brucei