Analysis of Mitochondrial Respiratory Chain Supercomplexes Using Blue Native Polyacrylamide Gel Electrophoresis (BN‐PAGE)

Pooja Jha1, Xu Wang1, Johan Auwerx1

1 Laboratory for Integrative and Systems Physiology, École Polytechnique Fédérale de Lausanne, Lausanne
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/9780470942390.mo150182
Online Posting Date:  March, 2016
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Abstract

Mitochondria are cellular organelles that harvest energy in the form of ATP through a process termed oxidative phosphorylation (OXPHOS), which occurs via the protein complexes of the electron transport chain (ETC). In recent years it has become unequivocally clear that mitochondrial complexes of the ETC are not static entities in the inner mitochondrial membrane. These complexes are dynamic and in mammals they aggregate in different stoichiometric combinations to form supercomplexes (SCs) or respirasomes. It has been proposed that the net respiration is more efficient via SCs than via isolated complexes. However, it still needs to be determined whether the activity of a particular SC is associated with a disease etiology. Here we describe a simplified method to visualize and assess in‐gel activity of SCs and the individual complexes with good resolution using blue native polyacrylamide gel electrophoresis (BN‐PAGE). © 2016 by John Wiley & Sons, Inc.

Keywords: supercomplex; in‐gel activity; mitochondria; oxidative phosphorylation; Cox7a2l; SCAFI

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Analysis of Respiratory Chain Supercomplexes
  • Alternate Protocol 1: CN‐Page and In‐Gel Activity of Complexes I, IV, IV + I, II, V, and III
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Analysis of Respiratory Chain Supercomplexes

  Materials
  • Mouse tissue, ∼30 mg liver; ∼15 mg from organs with high mitochondrial content, e.g., brain and heart (e.g., The Jackson Laboratory, Charles River, Taconic)
  • Isolation buffer (IB; see recipe)
  • Bovine serum albumin (BSA; Sigma‐Aldrich, cat. no. A7030)
  • Bradford protein assay (Bio‐Rad, cat. no. 500‐0006)
  • 5% digitonin (Thermo Fisher Scientific, cat. no. BN2006)
  • 4× NativePAGE sample buffer (Thermo Fisher Scientific, cat. no. BN20032)
  • NativePAGE 5% G‐250 sample additive (Thermo Fisher Scientific, cat. no. BN2004)
  • 20× NativePAGE running buffer (Thermo Fisher Scientific, cat. no. BN2001)
  • Coomassie Brilliant Blue G‐250 (SERVA, cat. no. 17524)
  • NativePAGE Novex 3% to 12% Bis‐Tris protein gels (1.0 mm, 15 well, Thermo Fisher Scientific, cat. no. BN2012BX10 or 1.0 mm, 10 well, Thermo Fisher Scientific, cat. no. BN2011BX10)
  • NativeMark Unstained Protein Standard (Thermo Fisher Scientific, cat. no. LC0725)
  • Colloidal blue staining kit (Thermo Fisher Scientific, cat. no. LC6025)
  • 20× NuPAGE transfer buffer (Thermo Fisher Scientific, cat. no. NP0006‐1)
  • Methanol (Fisher Scientific, cat. no. M/4000/17)
  • 8% acetic acid
  • WesternBreeze chromogenic kit, anti‐mouse (Thermo Fisher Scientific, cat. no. WB7103; use only for mouse; for rabbit use Thermo Fisher Scientific, cat. no. WB7105 and for goat Thermo Fisher Scientific, cat. no. WB7107, where only the secondary antibody is different; all kits contain blocker/diluent part A, blocker/diluent part B, 16× antibody wash solution, secondary antibody solution, chromogenic substrate)
  • NativePAGE anode buffer (see recipe)
  • Dark blue cathode buffer (see recipe)
  • Light blue cathode buffer (see recipe)
  • Blocking solution (see recipe)
  • OxPhos complex kit antibody cocktail (Thermo Fisher Scientific, cat. no. 45‐7999)
  • Anti‐MTCO1 antibody (Abcam, cat. no. ab14705)
  • Primary antibody solution (contains OxPhos antibody cocktail and anti‐MTCO1 antibody; see recipe)
  • Antibody wash solution (see recipe)
  • Substrates for complex I, II, IV, and V activity (see reciperecipes)
  • XCell SureLock Mini‐Cell (Thermo Fisher Scientific, cat. no. EI0001)
  • Prot/Elec tips (Bio‐Rad, cat. no. 223‐9915)
  • iBlot gel transfer device (Invitrogen, cat. no. IB1001EU)
  • iBlot gel transfer stacks, PVDF, mini (Thermo Fisher Scientific, cat. no. IB4010‐02)
  • POLYMIX PX‐SR 50 E stirrer
  • Wheaton Glass 2‐ml Potter‐Elvehjem tissue grinder set (Wheaton, cat. no. 358029)
  • 15‐ml polypropylene Falcon tubes
  • 20P pipet tips
  • Centrifuge
  • Additional reagents and equipment for anesthesia (Adams and Pacharinsak, ) and Bradford protein assay (Simonian and Smith, )
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Figures

Videos

Literature Cited

Literature Cited
  Acin‐Perez, R., Fernandez‐Silva, P., Peleato, M.L., Perez‐Martos, A., and Enriquez, J.A. 2008. Respiratory active mitochondrial supercomplexes. Mol. Cell 32:529‐539. doi: 10.1016/j.molcel.2008.10.021
  Adams, S. and Pacharinsak, C. 2015. Mouse anesthesia and analgesia. Curr. Protoc. Mouse Biol. 5:51‐63. doi: 10.1002/9780470942390.mo140179
  Barrientos, A. and Ugalde, C. 2013. I function, therefore I am: Overcoming skepticism about mitochondrial supercomplexes. Cell Metab. 18:147‐149. doi: 10.1016/j.cmet.2013.07.010
  Budde, S.M., van den Heuvel, L.P., Janssen, A.J., Smeets, R.J., Buskens, C.A., DeMeirleir, L., Van Coster, R., Baethmann, M., Voit, T., Trijbels, J.M., and Smeitink, J.A. 2000. Combined enzymatic complex I and III deficiency associated with mutations in the nuclear encoded NDUFS4 gene. Biochem. Biophys. Res. Commun. 275:63‐68. doi: 10.1006/bbrc.2000.3257
  Chaban, Y., Boekema, E.J., and Dudkina, N.V. 2014. Structures of mitochondrial oxidative phosphorylation supercomplexes and mechanisms for their stabilisation. Biochim. Biophys. Acta 1837:418‐426. doi: 10.1016/j.bbabio.2013.10.004
  Champy, M.F., Selloum, M., Zeitler, V., Caradec, C., Jung, B., Rousseau, S., Pouilly, L., Sorg, T., and Auwerx, J. 2008. Genetic background determines metabolic phenotypes in the mouse. Mamm. Genome 19:318‐331. doi: 10.1007/s00335-008-9107-z
  D'Aurelio, M., Gajewski, C.D., Lenaz, G., and Manfredi, G. 2006. Respiratory chain supercomplexes set the threshold for respiration defects in human mtDNA mutant cybrids. Hum. Mol. Genet. 15:2157‐2169. doi: 10.1093/hmg/ddl141
  Gomez, L.A. and Hagen, T.M. 2012. Age‐related decline in mitochondrial bioenergetics: Does supercomplex destabilization determine lower oxidative capacity and higher superoxide production? Semin. Cell Dev. Biol. 23:758‐767. doi: 10.1016/j.semcdb.2012.04.002
  Lamantea, E., Carrara, F., Mariotti, C., Morandi, L., Tiranti, V., and Zeviani, M. 2002. A novel nonsense mutation (Q352X) in the mitochondrial cytochrome b gene associated with a combined deficiency of complexes I and III. Neuromuscul. Disord. 12:49‐52. doi: 10.1016/S0960-8966(01)00244-9
  Lapuente‐Brun, E., Moreno‐Loshuertos, R., Acin‐Perez, R., Latorre‐Pellicer, A., Colas, C., Balsa, E., Perales‐Clemente, E., Quiros, P.M., Calvo, E., Rodriguez‐Hernandez, M.A., Navas, P., Cruz, R., Carracedo, A., Lopez‐Otin, C., Perez‐Martos, A., Fernandez‐Silva, P., Fernandez‐Vizarra, E., and Enriquez, J.A. 2013. Supercomplex assembly determines electron flux in the mitochondrial electron transport chain. Science 340:1567‐1570. doi: 10.1126/science.1230381
  Lenaz, G. and Genova, M.L. 2010. Structure and organization of mitochondrial respiratory complexes: A new understanding of an old subject. Antioxid. Redox Signal. 12:961‐1008. doi: 10.1089/ars.2009.2704
  McKenzie, M., Lazarou, M., Thorburn, D.R., and Ryan, M.T. 2006. Mitochondrial respiratory chain supercomplexes are destabilized in Barth Syndrome patients. J. Mol. Biol. 361:462‐469. doi: 10.1016/j.jmb.2006.06.057
  Moreno‐Lastres, D., Fontanesi, F., Garcia‐Consuegra, I., Martin, M.A., Arenas, J., Barrientos, A., and Ugalde, C. 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
  Mourier, A., Matic, S., Ruzzenente, B., Larsson, N.G., and Milenkovic, D. 2014. The respiratory chain supercomplex organization is independent of COX7a2l isoforms. Cell Metab. 20:1069‐1075. doi: 10.1016/j.cmet.2014.11.005
  Paigen, B., Morrow, A., Brandon, C., Mitchell, D., and Holmes, P. 1985. Variation in susceptibility to atherosclerosis among inbred strains of mice. Atherosclerosis 57:65‐73. doi: 10.1016/0021-9150(85)90138-8
  Saada, A., Edvardson, S., Shaag, A., Chung, W.K., Segel, R., Miller, C., Jalas, C., and Elpeleg, O. 2012. Combined OXPHOS complex I and IV defect, due to mutated complex I assembly factor C20ORF7. J. Inherit. Metab. Dis. 35:125‐131. doi: 10.1007/s10545-011-9348-y
  Schagger, H. and von Jagow, G. 1991. Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal. Biochem. 199:223‐231. doi: 10.1016/0003-2697(91)90094-A
  Schon, E.A. and Dencher, N.A. 2009. Heavy breathing: Energy conversion by mitochondrial respiratory supercomplexes. Cell Metab. 9:1‐3. doi: 10.1016/j.cmet.2008.12.011
  Simonian, M.H. and Smith, J.A. 2006. Spectrophotometric and colorimetric determination of protein concentration. Curr. Protoc. Mol. Biol. 76:10.1A.1‐10.1A.9. doi: 10.1002/0471142727.mb1001as76
  Wittig, I., Karas, M., and Schagger, 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
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