Respiratory Active Mitochondrial Supercomplexes Rebeca Acín-Pérez, Patricio Fernández-Silva, Maria Luisa Peleato, Acisclo Pérez-Martos, Jose Antonio Enriquez  Molecular Cell  Volume 32, Issue 4, Pages 529-539 (November 2008) DOI: 10.1016/j.molcel.2008.10.021 Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 1 Identification of the Supercomplex Components (A) Model of the mitochondrial electron transport chain and the H+-ATPase (OXPHOS system). (B) Blue native gel electrophoresis (BNGE) analysis of the mitochondrial OXPHOS complexes from mouse cultured cells or from mouse liver mitochondria (MLM) solubilized with either dodecilmaltoside (DDM), digitonin (DIG), or growing concentrations of Triton X-100 and NP-40. (C) Detection of the respiratory complexes and potential supercomplexes, obtained with DDM, DIG, Triton X-100, and NP-40, after BNGE by western blot using sequentially specific antibodies against complex I, III, and IV. (D) Location of the respiratory complexes after 2D electrophoresis (BNGE/SDSPAGE) detected by western blot using antibodies against the indicated mitochondrial protein. (E) In gel activity analysis performed after BNGE of DIG-treated samples obtained from cultured cells (lane 1) or MLM (lanes 2 to 4), to reveal the presence of complex I (lanes 1 and 2), complex V (lane 3), or complex IV (lane 4). “n,” “a,” and “c” indicate positive reaction for NADH DH activity, ATPase activity, or complex IV activity, respectively, and at the indicated band. (F) Fluorogram showing the mitochondrial translation products labeled with [35S] methionine and resolved by BNGE (lane 2) or by 2D-electrophoresis and compared with the in gel activity analysis of complex I (lane 1) in samples obtained from cultured cells and treated with DIG. Bands 1–11 indicate the localization of the SC and individual OXPHOS complexes. On the right side of the panel the complex migration or comigration at each band is indicated. CI, CII, CIII, CIV, and CV indicate complexes I, II, III, IV, and V. COI, COII, and COIII indicate subunits I, II, and III of cytochrome c oxidase. ND1, ND2, ND3, ND4, ND4L, ND5, and ND6 indicate subunits 1, 2, 3, 4, 4L, 5, and 6 of the respiratory chain NADH dehydrogenase. A6 and A8 indicate subunits 6 and 8 of H+-ATPase; cyt b, apocytochrome b. SC-CI+, potential supercomplexes containing complex I. SC-CI−, potential supercomplexes without complex I. Molecular Cell 2008 32, 529-539DOI: (10.1016/j.molcel.2008.10.021) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 2 Immunocapture of Supercomplexes (A) BNGE analysis of the sucrose gradient fractions (1 to 10) obtained by centrifugation of digitonin-treated mouse liver mitochondria. DIG and DDM, digitonin- or dodecylmaltoside-treated whole-mouse liver mitochondria, respectively. MW, molecular weight. (B) Immunocapture (Imcp) of complex I in mouse liver mitochondria (MLM) treated with DDM or DIG and identification of the interacting OXPHOS complexes. (C) Immunocapture (Imcp) of complex II in DDM-treated MLM. (D) Immunocapture (Imcp) of complex II in the sucrose gradient fractions 2 (f2) and 3 (f3) obtained using a mitochondrial preparation treated with DIG and identification of respiratory complexes immunocaptured together with complex II. T, total; Sp, supernatant. Molecular Cell 2008 32, 529-539DOI: (10.1016/j.molcel.2008.10.021) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 3 Genetic Analysis of the Interaction between Complexes (A) BNGE followed by complex I in gel activity of the mitochondrial preparations solubilized with DIG from control (WT) and mutant mouse cultured cells. (B) BNGE followed by western blot using antibodies against the indicated protein of the mitochondrial preparations (DIG) from control and mutant mouse cultured cells. WT, wild-type; CIko, cells lacking complex I; CIIIko, cells lacking complex III; CIVko, cells lacking complex IV; ρ°, mtDNA-less cells lacking complexes I, III, IV, and V. Other abbreviations as in Figure 1. Molecular Cell 2008 32, 529-539DOI: (10.1016/j.molcel.2008.10.021) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 4 Kinetics of the Supercomplex Assembly (A) Fluorogram showing the mitochondrial translation products labeled with [35S] methionine for 1 hr in the presence of cycloheximide followed by the indicated time of chase and analyzed by BNGE of DIG-treated mitochondria obtained from cultured cells. (B) Series of 2D-electrophoresis fluorograms (BNGE followed by SDS-PAGE) of samples run as in (A) corresponding to 0.5, 2, 12, and 24 hr of chase. Abbreviations as in Figure 1. Molecular Cell 2008 32, 529-539DOI: (10.1016/j.molcel.2008.10.021) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 5 Presence of Cyt c and CoQ in Supercomplexes (A) Western blot analysis revealing the presence of the targeted protein after BNGE followed by excision of the indicated band and run in a SDS-PAGE gel. Bands 1, 3, 4, and 5 correspond to supercomplexes and band 6 to free complex I of MLM treated with DIG. (B) HPLC analysis of the presence of CoQ9 and CoQ10 in supercomplex band 3. Abs, absorbance; tr, retention time. The position of peaks corresponding to CoQ9 and CoQ10 used as standards (markers) is shown. Molecular Cell 2008 32, 529-539DOI: (10.1016/j.molcel.2008.10.021) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 6 Respiration Activity of Supercomplexes Oxygen consumption traces of the indicated band excised from the BNGE and analyzed in a Clark-type oxygen electrode. Supercomplexes obtained with DIG (A) and complexes from DDM-treated mitochondrial samples (B). ROT, rotenone; Succ, succinate; Ant A, antimycin A; TMPD, N,N,N',N'-tetramethyl-p-phenylenediamine; KCN, potassium cyanide. Molecular Cell 2008 32, 529-539DOI: (10.1016/j.molcel.2008.10.021) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 7 Plasticity Model of the Mitochondrial OXPHOS System Organization Schematic representation of the “classical” fluid (A) and solid (B) models for the organization of the OXPHOS system and a “plasticity model” (C) derived from this work. The shape and color code for representing the individual complexes can be seen in (A) (and Figure 1A); coenzyme Q is represented as small red-filled stars and cytochrome c as red-filled triangles. Supercomplexes in (C) are identified with the numbering system used in Figure 1. Only one complex unit of each type is represented in the different supercomplex associations, although the actual stoichiometry may vary. Supercomplexes 3 and 4 can be considered true respirasomes, since they can transfer directly electrons from NADH to oxygen. Molecular Cell 2008 32, 529-539DOI: (10.1016/j.molcel.2008.10.021) Copyright © 2008 Elsevier Inc. Terms and Conditions