Elucidating the contribution of ETC complexes I and II to the respirasome formation in cardiac mitochondria.
ABSTRACT: Mitochondrial electron transport chain (ETC) plays a central role in ATP synthesis, and its dysfunction is associated with human diseases. Recent studies revealed that individual ETC complexes are assembled into supercomplexes. The main supercomplex, respirasome composed of complexes I, III, and IV has been suggested to improve electron channeling and control ROS production, maintain the structural integrity of ETC complexes and prevent protein aggregation in the inner mitochondrial membrane. However, many questions related to the structural organization of the respirasome, particularly, a possible role of complexes I and II in respirasome formation remain unclear. Here, we investigated whether genetic and pharmacological inhibition of complexes I and II affect respirasome assembly in cardioblast cells and isolated cardiac mitochondria. Pharmacological inhibition of the enzymatic activity of complexes I and II stimulated disruption of the respirasome. Likewise, knockdown of the complex I subunit NDUFA11 stimulated dissociation of respirasome and reduced the activity of complexes I, III, and IV. However, silencing of the membrane-anchored SDHC subunit of complex II had no effect on the respirasome assembly but reduced the activity of complexes II and IV. Downregulation of NDUFA11 or SDHC reduced ATP production and increased mitochondrial ROS production. Overall, these studies, for the first time, provide biochemical evidence that the complex I activity, and the NDUFA11 subunit are important for assembly and stability of the respirasome. The SDHC subunit of complex II is not involved in the respirasome however the complex may play a regulatory role in respirasome formation.
Project description:The protein complexes of the mitochondrial electron transport chain exist in isolation and in higher order assemblies termed supercomplexes (SCs) or respirasomes (SC I+III<sub>2</sub>+IV). The association of complexes I, III and IV into the respirasome is regulated by unknown mechanisms. Here, we designed a nanoluciferase complementation reporter for complex III and IV proximity to determine in vivo respirasome levels. In a chemical screen, we found that inhibitors of the de novo pyrimidine synthesis enzyme dihydroorotate dehydrogenase (DHODH) potently increased respirasome assembly and activity. By-passing DHODH inhibition via uridine supplementation decreases SC assembly by altering mitochondrial phospholipid composition, specifically elevated peroxisomal-derived ether phospholipids. Cell growth rates upon DHODH inhibition depend on ether lipid synthesis and SC assembly. These data reveal that nucleotide pools signal to peroxisomes to modulate synthesis and transport of ether phospholipids to mitochondria for SC assembly, which are necessary for optimal cell growth in conditions of nucleotide limitation.
Project description:The COX7A2L (Supercomplex Assembly Factor I, SCAFI) protein has been proposed to be a mitochondrial supercomplex assembly factor required for respirasome (supercomplex containing complexes I, III, and IV) formation. In the C57BL/6 mouse strain a homozygous in-frame 6-base-pair deletion in the COX7a2l/SCAF1 gene resulting in unstable protein and suggesting loss of function was previously identified. The loss of SCAFI was shown to impede respirasome formation, a major concern for the use of C57BL mouse strains in mitochondrial research. In contradiction, another recent study suggested that supercomplex formation is independent of SCAFI isoforms. We investigated whether SCAFI isoform status affected the disease severity and supercomplex formation in the liver of Bcs1lc.232A>G knock-in mice with incomplete complex III assembly. In homozygotes (Bcs1lG/G) of mixed (C57BL/6:129/Sv) genetic background, the lifespan was similar in mice with wild-type SCAFI allele and in those homozygous (SCAFIshort/short) for the deleted SCAF1 variant (34±3 days; n = 6 vs. 32±2 days; n = 7, respectively). SCAFI heterozygosity (SCAFIlong/short) resulted in decreased SCAFI protein but respirasome assembly was unaffected. Congenic (C57BL/6) mice were of the genotype SCAFIshort/short and had no detectable SCAFI protein. In their liver mitochondria, respirasome composition was altered as compared to mixed background mice. Complex IV was mainly present as monomers and dimers, and only low amounts were found in combination with complex I and complex III or with precomplex III. The main supercomplex in the liver mitochondria of C57BL/6 mice comprised only complexes I and III. In conclusion, in liver mitochondria of C57BL/6 mice, supercomplexes had markedly reduced amount of, but were not completely depleted of, complex IV, supporting a role for COX7A2L/SCAFI in supercomplex assembly. However, the disease progression of the Bcs1l mutant mice was unrelated to SCAFI isoforms and supercomplex composition, suggesting that other genetic factors contribute to the different survival in the different genetic backgrounds.
Project description:The biogenesis and function of the mitochondrial respiratory chain (RC) involve the organization of RC enzyme complexes in supercomplexes or respirasomes through an unknown biosynthetic process. This leads to structural interdependences between RC complexes, which are highly relevant from biological and biomedical perspectives, because RC defects often lead to severe neuromuscular disorders. We show that in human cells, respirasome biogenesis involves a complex I assembly intermediate acting as a scaffold for the combined incorporation of complexes III and IV subunits, rather than originating from the association of preassembled individual holoenzymes. The process ends with the incorporation of complex I NADH dehydrogenase catalytic module, which leads to the respirasome activation. While complexes III and IV assemble either as free holoenzymes or by incorporation of free subunits into supercomplexes, the respirasomes constitute the structural units where complex I is assembled and activated, thus explaining the significance of the respirasomes for RC function.
Project description:Mitochondrial protein interactions and complexes facilitate mitochondrial function. These complexes range from simple dimers to the respirasome supercomplex consisting of oxidative phosphorylation complexes I, III, and IV. To improve understanding of mitochondrial function, we used chemical cross-linking mass spectrometry to identify 2,427 cross-linked peptide pairs from 327 mitochondrial proteins in whole, respiring murine mitochondria. In situ interactions were observed in proteins throughout the electron transport chain membrane complexes, ATP synthase, and the mitochondrial contact site and cristae organizing system (MICOS) complex. Cross-linked sites showed excellent agreement with empirical protein structures and delivered complementary constraints for in silico protein docking. These data established direct physical evidence of the assembly of the complex I-III respirasome and enabled prediction of in situ interfacial regions of the complexes. Finally, we established a database and tools to harness the cross-linked interactions we observed as molecular probes, allowing quantification of conformation-dependent protein interfaces and dynamic protein complex assembly.
Project description:The existence of specific respiratory supercomplexes in mitochondria of most organisms has gained much momentum. However, its functional significance is still poorly understood. The availability of many deletion mutants in complex I (NADH:ubiquinone oxidoreductase) of Neurospora crassa, distinctly affected in the assembly process, offers unique opportunities to analyze the biogenesis of respiratory supercomplexes. Herein, we describe the role of complex I in assembly of respiratory complexes and supercomplexes as suggested by blue and colorless native polyacrylamide gel electrophoresis and mass spectrometry analyses of mildly solubilized mitochondria from the wild type and eight deletion mutants. As an important refinement of the fungal respirasome model, we found that the standard respiratory chain of N. crassa comprises putative complex I dimers in addition to I-III-IV and III-IV supercomplexes. Three Neurospora mutants able to assemble a complete complex I, lacking only the disrupted subunit, have respiratory supercomplexes, in particular I-III-IV supercomplexes and complex I dimers, like the wild-type strain. Furthermore, we were able to detect the I-III-IV supercomplexes in the nuo51 mutant with no overall enzymatic activity, representing the first example of inactive respirasomes. In addition, III-IV supercomplexes were also present in strains lacking an assembled complex I, namely, in four membrane arm subunit mutants as well as in the peripheral arm nuo30.4 mutant. In membrane arm mutants, high-molecular-mass species of the 30.4-kDa peripheral arm subunit comigrating with III-IV supercomplexes and/or the prohibitin complex were detected. The data presented herein suggest that the biogenesis of complex I is linked with its assembly into supercomplexes.
Project description:The respirasome is a multisubunit supercomplex of the respiratory chain in mitochondria. Here we report the 3D reconstruction of the bovine heart respirasome, composed of dimeric complex III and single copies of complex I and IV, at about 2.2-nm resolution, determined by cryoelectron tomography and subvolume averaging. Fitting of X-ray structures of single complexes I, III(2), and IV with high fidelity allows interpretation of the model at the level of secondary structures and shows how the individual complexes interact within the respirasome. Surprisingly, the distance between cytochrome c binding sites of complexes III(2) and IV is about 10 nm. Modeling indicates a loose interaction between the three complexes and provides evidence that lipids are gluing them at the interfaces.
Project description:In vivo associations of respiratory complexes forming higher supramolecular structures are generally accepted nowadays. Supercomplexes (SC) built by complexes I, III and IV and the so-called respirasome (I/III<sub>2</sub>/IV) have been described in mitochondria from several model organisms (yeasts, mammals and green plants), but information is scarce in other lineages. Here we studied the supramolecular associations between the complexes I, III, IV and V from the secondary photosynthetic flagellate Euglena gracilis with an approach that involves the extraction with several mild detergents followed by native electrophoresis. Despite the presence of atypical subunit composition and additional structural domains described in Euglena complexes I, IV and V, canonical associations into III<sub>2</sub>/IV, III<sub>2</sub>/IV<sub>2</sub> SCs and I/III<sub>2</sub>/IV respirasome were observed together with two oligomeric forms of the ATP synthase (V<sub>2</sub> and V<sub>4</sub>). Among them, III<sub>2</sub>/IV SC could be observed by electron microscopy. The respirasome was further purified by two-step liquid chromatography and showed in-vitro oxygen consumption independent of the addition of external cytochrome c.
Project description:<h4>Aims</h4>Mitochondrial dysfunction is a major factor in heart failure (HF). A pronounced variability of mitochondrial electron transport chain (ETC) defects is reported to occur in severe acquired cardiomyopathies without a consistent trend for depressed activity or expression. The aim of this study was to define the defect in the integrative function of cardiac mitochondria in coronary microembolization-induced HF.<h4>Methods and results</h4>Studies were performed in the canine coronary microembolization-induced HF model of moderate severity. Oxidative phosphorylation was assessed as the integrative function of mitochondria, using a comprehensive variety of substrates in order to investigate mitochondrial membrane transport, dehydrogenase activity and electron-transport coupled to ATP synthesis. The supramolecular organization of the mitochondrial ETC also was investigated by native gel electrophoresis. We found a dramatic decrease in ADP-stimulated respiration that was not relieved by an uncoupler. Moreover, the ADP/O ratio was normal, indicating no defect in the phosphorylation apparatus. The data point to a defect in oxidative phosphorylation within the ETC. However, the individual activities of ETC complexes were normal. The amount of the supercomplex consisting of complex I/complex III dimer/complex IV, the major form of respirasome considered essential for oxidative phosphorylation, was decreased.<h4>Conclusions</h4>We propose that the mitochondrial defect lies in the supermolecular assembly rather than in the individual components of the ETC.
Project description:Mitochondrial respiratory complexes assemble into supercomplexes (SC). Q-respirasome (III2 + IV) requires the supercomplex assembly factor (SCAF1) protein. The role of this factor in the N-respirasome (I + III2 + IV) and the physiological role of SCs are controversial. Here, we study C57BL/6J mice harboring nonfunctional SCAF1, the full knockout for SCAF1, or the wild-type version of the protein and found that exercise performance is SCAF1 dependent. By combining quantitative data-independent proteomics, 2D Blue native gel electrophoresis, and functional analysis of enriched respirasome fractions, we show that SCAF1 confers structural attachment between III2 and IV within the N-respirasome, increases NADH-dependent respiration, and reduces reactive oxygen species (ROS). Furthermore, the expression of AOX in cells and mice confirms that CI-CIII superassembly segments the CoQ in two pools and modulates CI-NADH oxidative capacity.
Project description:Although mitochondrial DNA is known to encode a limited number (<20) of the polypeptide components of respiratory complexes I, III, IV, and V, genes for components of complex II [succinate dehydrogenase (ubiquinone); succinate:ubiquinone oxidoreductase, EC 184.108.40.206] are conspicuously lacking in mitochondrial genomes so far characterized. Here we show that the same three subunits of complex II are encoded in the mitochondrial DNA of two phylogenetically distant eukaryotes, Porphyra purpurea (a photosynthetic red alga) and Reclinomonas americana (a heterotrophic zooflagellate). These complex II genes, sdh2, sdh3, and sdh4, are homologs, respectively, of Escherichia coli sdhB, sdhC, and sdhD. In E. coli, sdhB encodes the iron-sulfur subunit of succinate dehydrogenase (SDH), whereas sdhC and sdhD specify, respectively, apocytochrome b558 and a hydrophobic 13-kDa polypeptide, which together anchor SDH to the inner mitochondrial membrane. Amino acid sequence similarities indicate that sdh2, sdh3, and sdh4 were originally encoded in the protomitochondrial genome and have subsequently been transferred to the nuclear genome in most eukaryotes. The data presented here are consistent with the view that mitochondria constitute a monophyletic lineage.