We are searching data for your request:
Upon completion, a link will appear to access the found materials.
I have read wikipedia but don't understand it well. Do we mean Complex I, II,III and IV when we say respirasomes?
What the article is saying, is that there are several respirasomes, each of which consists of multiple other mitochondrial complexes or cytochromes. The article makes an example of three commonly observed respirasomes when it says:
The most common supercomplexes observed are Complex I/III, Complex I/III/IV, and Complex III/IV.
So, the short answer to your question is that a respirasome refers to a set of multiple cytochromes, whose combined action leads to respiration.
This paper goes into more detail, and reiterates that the term "respirasome" is used to describe multiple cytochrome supercomplexes.
Identification and Characterization of Respirasomes in Potato Mitochondria
In: Plant Physiology , Vol. 134, No. 4, 2004, p. 1450-1459.
Research output : Contribution to journal › Article
T1 - Identification and Characterization of Respirasomes in Potato Mitochondria
N2 - Plant mitochondria were previously shown to comprise respiratory supercomplexes containing cytochrome c reductase (complex III) and NADH dehydrogenase (complex I) of I1III2 and I2III4, composition. Here we report the discovery of additional supercomplexes in potato (Solanum tuberosum) mitochondria, which are of lower abundance and include cytochrome c oxidase (complex IV). Highly active mitochondria were isolated from potato tubers and stems, solubilized by digitonin, and subsequently analyzed by Blue-native (BN) polyacrylamide gel electrophoresis (PAGE). Visualization of supercomplexes by in-gel activity stains for complex IV revealed five novel supercomplexes of 850, 1,200, 1,850, 2,200, and 3,000 kD in potato tuber mitochondria. These supercomplexes have III2IV1, III2IV2, I1III2V1, I1III2IV2, and I1III2IV4 compositions as shown by two-dimensional BN/sodium dodecyl sulfate (SDS)-PAGE and BN/BN-PAGE in combination with activity stains for cytochrome c oxidase. Potato stem mitochondria include similar supercomplexes, but complex IV is partially present in a smaller version that lacks the Cox6b protein and possibly other subunits. However, in mitochondria from potato tubers and stems, about 90% of complex IV was present in monomeric form. It was suggested that the I1III2IV4 supercomplex represents a basic unit for respiration in mammalian mitochondria termed respirasome. Respirasomes also occur in potato mitochondria but were of low concentrations under all conditions applied. We speculate that respirasomes are more abundant under in vivo conditions.
AB - Plant mitochondria were previously shown to comprise respiratory supercomplexes containing cytochrome c reductase (complex III) and NADH dehydrogenase (complex I) of I1III2 and I2III4, composition. Here we report the discovery of additional supercomplexes in potato (Solanum tuberosum) mitochondria, which are of lower abundance and include cytochrome c oxidase (complex IV). Highly active mitochondria were isolated from potato tubers and stems, solubilized by digitonin, and subsequently analyzed by Blue-native (BN) polyacrylamide gel electrophoresis (PAGE). Visualization of supercomplexes by in-gel activity stains for complex IV revealed five novel supercomplexes of 850, 1,200, 1,850, 2,200, and 3,000 kD in potato tuber mitochondria. These supercomplexes have III2IV1, III2IV2, I1III2V1, I1III2IV2, and I1III2IV4 compositions as shown by two-dimensional BN/sodium dodecyl sulfate (SDS)-PAGE and BN/BN-PAGE in combination with activity stains for cytochrome c oxidase. Potato stem mitochondria include similar supercomplexes, but complex IV is partially present in a smaller version that lacks the Cox6b protein and possibly other subunits. However, in mitochondria from potato tubers and stems, about 90% of complex IV was present in monomeric form. It was suggested that the I1III2IV4 supercomplex represents a basic unit for respiration in mammalian mitochondria termed respirasome. Respirasomes also occur in potato mitochondria but were of low concentrations under all conditions applied. We speculate that respirasomes are more abundant under in vivo conditions.
Comparative Profiling of N-Respirasomes Predicts Aberrant Mitochondrial Bioenergetics at Single-Cell Resolution
Mitochondria sustain the energy demand of the cell. The composition and functional state of the mitochondrial oxidative phosphorylation system are informative indicators of organelle homeostasis and bioenergetic capacity. Here we describe a highly sensitive and reproducible method for single-cell visualization and quantification of mitochondrial respiratory supercomplexes as a novel means of measuring mitochondrial respiratory chain integrity. We apply a proximity ligation assay (PLA) and perform comparative studies of mitochondrial CI, CIII, CIV-containing supercomplexes (or N-respirasomes) in fixed human and mouse brain tissues, tumorigenic cells, iPSCs and iPSC-derived NPCs and neurons. Our optimized approach enables quantitative in-situ assessments of even subtle mitochondrial lesions associated with aberrant respiration. By combining quantitative proteomics with single cell imaging analysis, we also report the mechanistic contribution of the MICOS complex subunit CHCHD3 in regulating N-respirasomes. Overall, our PLA-based profiling of N-respirasomes establishes a sensitive and complementary technique for detecting cell-type specific mitochondrial perturbations in fixed materials.
Keywords: brain, Mitochondria, mitochondrial diseases, mitochondrial respiratory supercomplexes, proximity ligation assay
Related Biology Terms
- Electron transport train – Sometimes called respiratory chain, this refers to a series of proteins located on the inner mitochondrial membrane that receive high-energy electrons produced by the citric acid cycle. As the electrons move through multiple members of this chain, they gradually lose energy, which in turn, is used to generate a proton gradient across the inner mitochondrial membrane.
- F1Fo-ATP synthase – The enzyme present on mitochondrial cristae responsible for the synthesis of adenosine triphosphate (ATP).
- Mitochondria – Eukaryotic organelles wrapped in a double membrane and found in the cell cytoplasm. Their main function is to participate in aerobic respiration and generate ATP.
1. On which of these mitochondrial structures are cristae found?
A. The outer membrane
B. The inner membrane
C. The matrix
D. All of the above
2. Which statement is true about cristae?
A. The wrinkled form of cristae increases the surface area of the inner mitochondrial membrane.
B. Cristae are located inside the nucleus.
C. Cristae do not play a role in allowing mitochondria to communicate with other mitochondria in the cells.
D. Cristae are part of the plasma membrane.
3. Which of the following statements is true?
A. Cristae membrane surface area size is proportional to a cell’s capacity for ATP generation.
B. Cristae membranes are deformed in diseases such as Parkinson’s and Alzheimer’s.
C. The cylindrical connections between cristae membranes and the inner membrane boundary of mitochondria are called cristae junctions.
D. All of the above.
Ustilago maydis is an aerobic basidiomycete that depends on oxidative phosphorylation for its ATP supply, pointing to the mitochondrion as a key player in its energy metabolism. Mitochondrial respiratory complexes I, III2, and IV occur in supramolecular structures named respirasome. In this work, we characterized the subunit composition and the kinetics of NADH:Q oxidoreductase activity of the digitonine-solubilized respirasome (1600 kDa) and the free-complex I (990 kDa). In the presence of 2,6-dimethoxy-1,4-benzoquinone (DBQ) and cytochrome c, both the respirasome NADH:O2 and the NADH:DBQ oxidoreductase activities were inhibited by rotenone, antimycin A or cyanide. A value of 2.4 for the NADH oxidized/oxygen reduced ratio was determined for the respirasome activity, while ROS production was less than 0.001% of the oxygen consumption rate. Analysis of the NADH:DBQ oxidoreductase activity showed that respirasome was 3-times more active and showed higher affinity than free-complex I. The results suggest that the contacts between complexes I, III2 and IV in the respirasome increase the catalytic efficiency of complex I and regulate its activity to prevent ROS production.
The CI-deficient cell line (CI-KD) harbors a homoplasmic m.4681TϬ mutation in the MT-ND2 subunit gene that leads to a severe CI assembly defect due to a p.L71P substitution (Ugalde et al., 2007). The CIII mutant (CIII-KO) cell line contains a homoplasmic 4se pair deletion in the MT-CYB gene affecting the de novo synthesis of cytochrome b (Rana et al., 2000). The CIV mutant cell line (CIV-KO) lacks holo-COX due to the homoplasmic m.6930GϪ transition in the MT-COI gene, which creates a stop codon that results in a predicted loss of the last 170 amino acids of the COX1 polypeptide (Bruno et al., 1999). HeLa cells, either transduced with the empty pWPXLd-ires-PuroR vector or overexpressing LYRM7-001-HA (MZM1L-HA), were generated as previously described (Sanchez et al., 2013).
Cells were cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM, Life Technologies) supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 1 mM sodium pyruvate, and antibiotics. To block mitochondrial translation, 15 μg/ml doxycycline was added for six days to the culture medium. Cells were grown in exponential conditions and harvested at the indicated time points.
In vitro Import
Radiolabeled COX7A2L, COX6A and RISP proteins were obtained by coupled transcription and translation in the presence of 35 S-methionine (PerkinElmer) using TNT SP6 Quick Coupled System (Promega). Import experiments were performed on freshly isolated mitochondria from heart tissue as described before (Mourier et al., 2014a).
One milligram of mitochondrial protein from HEK293 transduced cells was solubilized in 600 μl of 4 g /g digitonin-to-protein buffer as for BNE analyses. After centrifugation for 30 min at 13,000 rpm at 4 ଌ, 50 μg of the supernatant was separated as the input fraction. The remainder supernatant was co-immunoprecipitated in resin spin columns (Pierce Co-IP Kit, Thermo Scientific) in which 15 μg of antibodies against DDK-tag (Oncogene), CORE2 or COX1 had been previously immobilized. The mixture was gently incubated overnight at 4ଌ in a rotating shaker and centrifuged at 1000 g for 1 min to separate the flow-through fraction. The column was washed three times with Lysis Buffer containing 1% NP-40 and proteins were eluted. The immunoprecipitate was divided into three aliquots, treated with 5× Loading Sample Buffer and heated at 95 ଌ for 5 minutes prior to loading.
Blue Native Electrophoresis and In-Gel Activity Assays
Mitochondrial pellets and blue native analyses were performed as described before (Moreno-Lastres et al., 2012 Mourier et al., 2014a). Native PAGE™ Novex® 3-12% Bis-Tris Protein Gels (Life Technologies) or self-made 4-10% polyacrylamide gradient gels were loaded with 60-80 μg of mitochondrial protein. After electrophoresis, proteins were transferred to nitrocellulose or PVDF membranes at 40 V overnight and probed with specific antibodies.
Western blot was performed using primary antibodies raised against COX7A2L (ProteinTech), Myc (Origene), turbo-GFP (Origene), HA (Roche), β-actin (Sigma), and against the following human OXPHOS subunits: NDUFS1 (GeneTex) NDUFA9, NDUFB8, CORE2, RISP, CYC1, UQCRB, UQCRQ, COX1, COX4, COX5A, COX6C, SDHA, SDHB (Mitosciences) and COX5B (Santa Cruz). Peroxidase-conjugated anti-mouse and anti-rabbit IgGs were used as secondary antibodies (Molecular Probes). Immunoreactive bands were detected with an ECL prime Western Blotting Detection Reagent (Amersham) in a ChemiDoc™ MP Imager (Biorad). Optical densities of the immunoreactive bands were measured using the ImageLab™ (Biorad) and ImageJ analysis softwares.
Cells were fixed with 4% paraformaldehyde for 15 min, permeabilized for 15 min with 0.1% Triton X-100, and incubated in blocking buffer containing 10% goat serum for 1h. Cover slips were incubated with an antibody against monoclonal complex V α subunit and a Texas Red-conjugated anti-mouse secondary antibody (Abcam). Cover slips were rinsed, mounted in ProLong Gold antifade reagent (Molecular Probes) on glass slides, and cells were viewed with a Zeiss LSM 510 Meta confocal microscope and a 63× planapochromat oil inmersion objective (NA: 1.42). Sequential scanning of green and red channels was performed to avoid bleed-through effect. Cells were imaged randomly with 0,5-1,0 μm slices and 1024 pixels resolution. For colocalization analysis, the “Merge channels” plugin from the ImageJ 1.48v software was used.
Statistical Data Analysis
All experiments were performed at least in triplicate and results were presented as mean ± standard deviation (SD) values. Statistical p values were obtained by application of the Friedman and Mann-Whitney U tests using the SPSS v21.0 program.
This study was performed in accordance with the guidelines of the Federation of European Laboratory Animal Science Associations. The protocol was approved by the Landesamt für Natur, Umwelt und Verbraucherschutz in Nordrhein-Westfalen in Germany.
COX7A2L preferentially interacts with respiratory chain complex III
COX7A2L is essential to stabilize the III2+IV supercomplex
COX7A2L is not necessary for biogenesis or maintenance of the respirasome
Biogenesis of the III2+IV supercomplex is not necessary for respirasome formation
Analysis of Mitochondrial Respiratory Chain Supercomplexes Using Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE)
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.
The respiratory chain supercomplex organization is independent of COX7a2l isoforms
The organization of individual respiratory chain complexes into supercomplexes or respirasomes has attracted great interest because of the implications for cellular energy conversion. Recently, it was reported that commonly used mouse strains harbor a short COX7a2l (SCAFI) gene isoform that supposedly precludes the formation of complex IV-containing supercomplexes. This claim potentially has serious implications for numerous mouse studies addressing important topics in metabolism, including adaptation to space flights. Using several complementary experimental approaches, we show that mice with the short COX7a2l isoform have normal biogenesis and steady-state levels of complex IV-containing supercomplexes and consequently have normal respiratory chain function. Furthermore, we use a mouse knockout of Lrpprc and show that loss of complex IV compromises respirasome formation. We conclude that the presence of the short COX7a2l isoform in the commonly used C57BL/6 mouse strains does not prevent their use in metabolism research.
Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.
Different Cox7a2l Isoforms Have No…
Different Cox7a2l Isoforms Have No Differential Effects on Respiratory Chain Activity (A) PCR…
The Short Cox7a2l Isoform Does…
The Short Cox7a2l Isoform Does Not Affect the Supramolecular Organization of the Respiratory…
Complex IV-Containing Respirasomes Are Present…
Complex IV-Containing Respirasomes Are Present in Mouse Strains Harboring the Short Cox7a2l Isoform…
The Assembly of Complex IV…
The Assembly of Complex IV Containing Respirasomes Is Unaffected in Mouse Strains Harboring…
The bovine heart I + III2 + IV supercomplex was purified to homogeneity by BN-PAGE, followed by electroelution, a prerequisite to perform a cryoelectron microscopy study. Preparation of frozen hydrated cryo-EM grids by adsorption of the purified sample onto holey carbon film grids and plunge-freezing, however, resulted in inhomogeneously distributed particles in the grid holes. We therefore used holey carbon film grids that were covered with a thinner second carbon film, to which the particles could adsorb. This approach resulted in evenly distributed molecules (Fig. 1). Single particle analysis, including classification and averaging of two-dimensional projections, showed that the respirasome molecules were preferentially oriented in a few positions on carbon support film. The most abundant projection type has a triangular shape (Fig. 1, Inset), as also previously noticed in negatively stained samples (5). We performed electron tomography on these cryo-EM specimens and from 21 tilt series the 3D volumes of the samples were reconstructed. From these tomograms, 2,466 subtomograms containing single respirasomes were extracted for 3D averaging. Combination of XMIPP (X-Window-based Microscopy Image Processing Package) maximum likelihood (ML) global alignment (11) and local refinement by cross-correlation with AV3 toolbox (15) was used to create an average structure. The resolution of the respirasome is anisotropic (Fig. S1) and correlates to the angular sampling (Fig. S2). The highest achievable resolution of our average structure in the direction perpendicular to the incident beam is 1.7 nm and the lowest one is 2.55 nm parallel to the beam. We have filtered the entire reconstruction to 2.2 nm, which is at the first zero of the contrast transfer function.
Cryoelectron micrograph of isolated bovine respirasomes. (Scale bar: 100 nm.) (Inset) Two-dimensional projection map of the respirasome in a top view position from single particle image analysis. (Scale bar: 10 nm.)
The reconstruction shows a respirasome with dimensions of 27 × 21 nm in the membrane plane (Fig. 2 B and C) and a maximal height of 23 nm (Fig. 2B), made by the giant hydrophilic domain of complex I. By comparison with known 3D structures of the individual complexes, many relevant features are recognizable, such as protruding densities at the center, related by a local twofold symmetry axis (Fig. 2, arrows), that point to the position of the core I and II subunits of dimeric complex III. Complex III2 sits in the arc of the membrane arm of complex I and complex IV is attached to the tip of NADH dehydrogenase and complex III2 (Fig. 2, arrowheads). To closely interpret the cryo-EM map, we superimposed the high-resolution X-ray structures of bovine complexes III2 (12) and IV (13). For modeling of the complex I moiety, the medium-resolution X-ray data from the yeast Yarrowia lipolytica were used (14). Fitting was accomplished with correlation coefficients between 0.83–0.92 for the bovine structures, indicating a unique, close fit with high confidence of components in the final hybrid structure (Fig. 3 A, B, and D, and Fig. S3). No substantial parts of the single complexes extend the 3D cryo-EM reconstruction, indicating that the respirasome is basically the sum of its components. Based on the fitting, our 3D structure presents a complete respirasome, including the peripheral arm domain of complex I (Fig. 3 A and B, arrowheads), which sometimes is partly lacking in negatively stained samples (16). Some minor differences exist between the EM reconstruction and the X-ray maps (Fig. 3 A, B, and D, orange arrowheads), which are most likely detergent molecules surrounding the respirasome. Iterative multireference alignment and classification (17) showed the presence of the peripheral arm in all five classes with a slight amount of variation (Fig. S4). Subtraction of two reconstructions representing two different classes revealed a density (Fig. S4, red asterisk), which can be accounted for a possible conformational change in the hydrophilic domain of complex I. To prove it, additional experiments are required. The full complex I moiety of bovine and Yarrowia are equal the peripheral matrix arms make the same angle (Fig. 3A, yellow) and the bend of the membrane arm is closely the same (Fig. 3D, double arrowhead). The superposition of the smaller prokaryotic complex I from Thermus thermophilus indicates a similar overall shape (Fig. S5) and, in addition, clearly shows the locations of the accessory subunits of the eukaryotic enzymes are located. These accessory proteins appear to be arranged mostly in clusters.
Cryo-EM map of I + III2 + IV supercomplex: (A and B) seen from aside, (C) seen from the matrix side arrow points to complex III2, arrowhead to complex IV, and double arrowhead to the curved membrane arm of complex I, respectively. (D) Seen from aside from the tip. Contour level is 0.1. Horizontal lines on A and D indicate the position of the membrane. (Scale bar: 10 nm.)
Fitting of the high- and medium-resolution structures of complexes I, III2, and IV to the 3D cryo-EM map of I + III2 + IV supercomplex: (A) side view, arrowhead points to flavoproteins (B) side view from the membrane, arrows point to core I and II subunits of complex III2, arrowhead to flavoproteins (C) section through the space-filling model of respirasome on the level of membrane, demonstrating gaps between complexes within the supercomplex (D) top view from the intermembrane space, double arrowhead points to the bend of complex I in membrane (E) space-filling model of respirasome seen from the membrane, red and light-blue arrowheads show the level of sections in C and F (F) section through the space-filling model of respirasome on the level of matrix. In green, X-ray structure of the bovine dimeric complex III in purple, X-ray structure of bovine monomeric complex IV in yellow, the density map of complex I from Yarrowia lipolytica. Horizontal lines on E indicate the position of the membrane. Orange arrowheads on A, B, and D point to the position of detergent micelles. (Scale bar: 10 nm.)
Conceptualization: MO, SB, and FF Methodology: JB, AA, AB, FF, and MO Validation: FF and MO Formal Analysis: JB, AA, and SR Investigation: JB, AA, SR, LM-B, HD, JD, VK, and FF Resources: AB, SB, FF, and MO Data Curation: JB, AA, SR, FF, and MO Writing—Original Draft: JB, AA, and MO Writing—Review and Editing: JB, AA, HD, VK, AB, SB, FF, and MO Visualization: JB, AA, and MO Supervision: SB, FF, and MO Project Administration: MO Funding Acquisition: AA, VK, AB, SB, FF, and MO.