Cells lacking Rieske iron-sulfur protein have a reactive oxygen species-associated decrease in respiratory complexes I and IV

Mitochondrial respiratory complexes of the electron transport chain (CI, CIII, and CIV) can be assembled into larger structures forming supercomplexes. We analyzed the assembly/stability of respiratory complexes in mouse lung fibroblasts lacking the Rieske iron-sulfur protein (RISP knockout [KO]cells), one of the catalytic subunits of CIII. In the absence of RISP, most of the remaining CIII subunits were able to assemble into a large precomplex that lacked enzymatic activity. CI, CIV, and supercomplexes were decreased in the RISP-deficient cells. Reintroduction of RISP into KO cells restored CIII activity and increased the levels of active CI, CIV, and supercomplexes. We found that hypoxia (1% O(2)) resulted in increased levels of CI, CIV, and supercomplex assembly in RISP KO cells. In addition, treatment of control cells with different oxidative phosphorylation (OXPHOS) inhibitors showed that compounds known to generate reactive oxygen species (ROS) (e.g., antimycin A and oligomycin) had a negative impact on CI and supercomplex levels. Accordingly, a superoxide dismutase (SOD) mimetic compound and SOD2 overexpression provided a partial increase in supercomplex levels in the RISP KO cells. Our data suggest that the stability of CI, CIV, and supercomplexes is regulated by ROS in the context of defective oxidative phosphorylation.     

Respiratory Chain Complexes in Dynamic Mitochondria Display a Patchy Distribution in Life Cells

Background

Mitochondria, the main suppliers of cellular energy, are dynamic organelles that fuse and divide frequently. Constraining these processes impairs mitochondrial is closely linked to certain neurodegenerative diseases. It is proposed that functional mitochondrial dynamics allows the exchange of compounds thereby providing a rescue mechanism.

Methodology/Principal Findings

The question discussed in this paper is whether fusion and fission of mitochondria in different cell lines result in re-localization of respiratory chain (RC) complexes and of the ATP synthase. This was addressed by fusing cells containing mitochondria with respiratory complexes labelled with different fluorescent proteins and resolving their time dependent re-localization in living cells. We found a complete reshuffling of RC complexes throughout the entire chondriome in single HeLa cells within 2–3 h by organelle fusion and fission. Polykaryons of fused cells completely re-mixed their RC complexes in 10–24 h in a progressive way. In contrast to the recently described homogeneous mixing of matrix-targeted proteins or outer membrane proteins, the distribution of RC complexes and ATP synthase in fused hybrid mitochondria, however, was not homogeneous but patterned. Thus, complete equilibration of respiratory chain complexes as integral inner mitochondrial membrane complexes is a slow process compared with matrix proteins probably limited by complete fusion. In co-expressing cells, complex II is more homogenously distributed than complex I and V, resp. Indeed, this result argues for higher mobility and less integration in supercomplexes.

Conclusion/Significance

Our results clearly demonstrate that mitochondrial fusion and fission dynamics favours the re-mixing of all RC complexes within the chondriome. This permanent mixing avoids a static situation with a fixed composition of RC complexes per mitochondrion.     

A CMC1‐knockout reveals translation‐independent control of human mitochondrial complex IV biogenesis

Defects in mitochondrial respiratory chain complex IV (CIV) frequently cause encephalocardiomyopathies. Human CIV assembly involves 14 subunits of dual genetic origin and multiple nucleus‐encoded ancillary factors. Biogenesis of the mitochondrion‐encoded copper/heme‐containing COX1 subunit initiates the CIV assembly process. Here, we show that the intermembrane space twin CX₉C protein CMC1 forms an early CIV assembly intermediate with COX1 and two assembly factors, the cardiomyopathy proteins COA3 and COX14. A TALEN‐mediated CMC1 knockout HEK293T cell line displayed normal COX1 synthesis but decreased CIV activity owing to the instability of newly synthetized COX1. We demonstrate that CMC1 stabilizes a COX1‐COA3‐COX14 complex before the incorporation of COX4 and COX5a subunits. Additionally, we show that CMC1 acts independently of CIV assembly factors relevant to COX1 metallation (COX10, COX11, and SURF1) or late stability (MITRAC7). Furthermore, whereas human COX14 and COA3 have been proposed to affect COX1 mRNA translation, our data indicate that CMC1 regulates turnover of newly synthesized COX1 prior to and during COX1 maturation, without affecting the rate of COX1 synthesis.     

Mitochondrial complex I plays an essential role in human respirasome assembly

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.     

Whole-exome sequencing identifies homozygous AFG3L2 mutations in a spastic ataxia-neuropathy syndrome linked to mitochondrial m-AAA proteases

We report an early onset spastic ataxia-neuropathy syndrome in two brothers of a consanguineous family characterized clinically by lower extremity spasticity, peripheral neuropathy, ptosis, oculomotor apraxia, dystonia, cerebellar atrophy, and progressive myoclonic epilepsy. Whole-exome sequencing identified a homozygous missense mutation (c.1847G>A; p.Y616C) in AFG3L2, encoding a subunit of an m-AAA protease. m-AAA proteases reside in the mitochondrial inner membrane and are responsible for removal of damaged or misfolded proteins and proteolytic activation of essential mitochondrial proteins. AFG3L2 forms either a homo-oligomeric isoenzyme or a hetero-oligomeric complex with paraplegin, a homologous protein mutated in hereditary spastic paraplegia type 7 (SPG7). Heterozygous loss-of-function mutations in AFG3L2 cause autosomal-dominant spinocerebellar ataxia type 28 (SCA28), a disorder whose phenotype is strikingly different from that of our patients. As defined in yeast complementation assays, the AFG3L2(Y616C) gene product is a hypomorphic variant that exhibited oligomerization defects in yeast as well as in patient fibroblasts. Specifically, the formation of AFG3L2(Y616C) complexes was impaired, both with itself and to a greater extent with paraplegin. This produced an early-onset clinical syndrome that combines the severe phenotypes of SPG7 and SCA28, in additional to other "mitochondrial" features such as oculomotor apraxia, extrapyramidal dysfunction, and myoclonic epilepsy. These findings expand the phenotype associated with AFG3L2 mutations and suggest that AFG3L2-related disease should be considered in the differential diagnosis of spastic ataxias.     

Knockdown of Human COX17 Affects Assembly and Supramolecular Organization of Cytochrome c Oxidase

Assembly of cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain, requires a concerted activity of a number of chaperones and factors for the insertion of subunits, accessory proteins, cofactors and prosthetic groups. It is now well accepted that the multienzyme complexes of the respiratory chain are organized in vivo as supramolecular functional structures, so-called supercomplexes. Here, we investigate the role of COX17 in the biogenesis of the respiratory chain in HeLa cells. In accordance with its predicted function as a copper chaperone and its role in formation of the binuclear copper centre of cytochrome c oxidase, COX17 siRNA knockdown affects activity and assembly of cytochrome c oxidase. While the abundance of cytochrome c oxidase dimers seems to be unaffected, blue native gel electrophoresis reveals the disappearance of COX-containing supercomplexes as an early response. We observe the accumulation of a novel ∼ 150 kDa complex that contains Cox1, but not Cox2. This observation may indicate that the absence of Cox17 interferes with copper delivery to Cox2, but not to Cox1. We suggest that supercomplex formation is not simply due to assembly of completely assembled complexes. An interdependent assembly scenario for the formation of supercomplexes that rather requires the coordinated synthesis and association of individual complexes, is proposed.     

Taz1, an outer mitochondrial membrane protein, affects stability and assembly of inner membrane protein complexes: implications for Barth Syndrome

The Saccharomyces cerevisiae Taz1 protein is the orthologue of human Tafazzin, a protein that when inactive causes Barth Syndrome (BTHS), a severe inherited X-linked disease. Taz1 is a mitochondrial acyltransferase involved in the remodeling of cardiolipin. We show that Taz1 is an outer mitochondrial membrane protein exposed to the intermembrane space (IMS). Transport of Taz1 into mitochondria depends on the receptor Tom5 of the translocase of the outer membrane (TOM complex) and the small Tim proteins of the IMS, but is independent of the sorting and assembly complex (SAM). TAZ1 deletion in yeast leads to growth defects on nonfermentable carbon sources, indicative of a defect in respiration. Because cardiolipin has been proposed to stabilize supercomplexes of the respiratory chain complexes III and IV, we assess supercomplexes in taz1delta mitochondria and show that these are destabilized in taz1Delta mitochondria. This leads to a selective release of a complex IV monomer from the III2IV2 supercomplex. In addition, assembly analyses of newly imported subunits into complex IV show that incorporation of the complex IV monomer into supercomplexes is affected in taz1Delta mitochondria. We conclude that inactivation of Taz1 affects both assembly and stability of respiratory chain complexes in the inner membrane of mitochondria.     

Fine-tuning of the respiratory complexes stability and supercomplexes assembly in cells defective of complex III

The respiratory complexes are organized in supramolecular assemblies called supercomplexes thought to optimize cellular metabolism under physiological and pathological conditions. In this study, we used genetically and biochemically well characterized cells bearing the pathogenic microdeletion m.15,649–15,666 (ΔI300-P305) in MT-CYB gene, to investigate the effects of an assembly-hampered CIII on the re-organization of supercomplexes. First, we found that this mutation also affects the stability of both CI and CIV, and evidences the occurrence of a preferential structural interaction between CI and CIII₂, yielding a small amount of active CI+CIII₂ supercomplex. Indeed, a residual CI+CIII combined redox activity, and a low but detectable ATP synthesis driven by CI substrates are detectable, suggesting that the assembly of CIII into the CI+CIII₂ supercomplex mitigates the detrimental effects of MT-CYB deletion. Second, measurements of oxygen consumption and ATP synthesis driven by NADH-linked and FADH₂-linked substrates alone, or in combination, indicate a common ubiquinone pool for the two respiratory pathways. Finally, we report that prolonged incubation with rotenone enhances the amount of CI and CIII₂, but reduces CIV assembly. Conversely, the antioxidant N-acetylcysteine increases CIII₂ and CIV₂ and partially restores respirasome formation. Accordingly, after NAC treatment, the rate of ATP synthesis increases by two-fold compared with untreated cell, while the succinate level, which is enhanced by the homoplasmic mutation, markedly decreases. Overall, our findings show that fine-tuning the supercomplexes stability improves the energetic efficiency of cells with the MT-CYB microdeletion.     

Lack of Cytochrome c in Mouse Fibroblasts Disrupts Assembly/Stability of Respiratory Complexes I and IV

Cytochrome c (cyt c) is a heme-containing protein that participates in electron transport in the respiratory chain and as a signaling molecule in the apoptotic cascade. Here we addressed the effect of removing mammalian cyt c on the integrity of the respiratory complexes in mammalian cells. Mitochondria from cyt c knockout mouse cells lacked fully assembled complexes I and IV and had reduced levels of complex III. A redox-deficient mutant of cyt c was unable to rescue the levels of complexes I and IV. We found that cyt c is associated with both complex IV and respiratory supercomplexes, providing a potential mechanism for the requirement for cyt c in the assembly/stability of complex IV.The mitochondrial electron transport chain consists of four multisubunit complexes, namely, NADH-ubiquinone oxidoreductase (complex I),² succinate-ubiquinone oxidoreductase (complex II), ubiquinone-cytochrome c oxidoreductase (complex III), and cytochrome c oxidase (complex IV, COX). Cytochrome c (cyt c) shuttles electrons from oxidative phosphorylation complex III to complex IV. Electrons are transferred from reduced cyt c sequentially to the Cu_A site, heme a, heme a₃, and Cu_B binuclear center in the complex IV before being finally transferred to molecular oxygen to generate water (1). Respiratory complexes are assembled into supercomplexes (also called respirasomes). These contain complex I bound to dimeric complex III and a variable copy number of complex IV (2).In Saccharomyces cerevisiae, cyt c is encoded by two genes: CYC1 and CYC7. Mutagenesis studies in yeast have shown that cyt c is required for the assembly of COX (3, 4). In yeast lacking both the cyt c genes (CYC1 and CYC7), COX assembly was absent. It was also shown that cyt c is only structurally required for COX assembly, because a catalytic mutant of cyt c (W65S) was sufficient to bring about near normal levels of COX. However, because yeast lacks complex I, they could not analyze the role of cyt c in the assembly/stability of complex I. Mammals possess two different isoforms of cyt c encoded on different chromosomes: the somatic (cyt cS)- and testis (cyt cT)-specific isoforms. In mouse, the cDNAs bear 74% homology, whereas the proteins possess 86% identity with most dissimilarity in the C terminus.Cardiolipin (CL) is an anionic phospholipid present almost exclusively in the mitochondrial membranes and constitutes 25% of its total phospholipids (5). Work from several laboratories showed that CL is essential for the membrane anchorage of the respiratory supercomplexes. CL has two main roles in the mitochondrial structure and function, namely, stabilization of mitochondrial membranes and specific interactions with proteins. CL deficiency results in inefficient energy transformation by oxidative phosphorylation, swelling of mitochondria, decreased ATP/oxygen ratio, and reduced membrane potential (6, 7). In accordance, in S. cerevisiae lacking CL synthase, the supercomplex comprising complexes III and IV is unstable (8). Assembly mutants of COX had significantly reduced CL synthase activity, whereas assembly mutants of respiratory complex III and complex V showed less inhibition (9). Subsequently, the proton gradient across the inner mitochondrial membrane was found to be important for CL formation and that CL synthase was stimulated by alkaline pH at the matrix side (10). In this study, we investigated the role of cyt c depletion on CL levels by examining its content and composition in cyt c null cells.Here we aimed to answer the following questions: What is the role of cyt c in the assembly and maintenance of the different respiratory complexes in mammals? Are there changes in the content/composition of lipids in the cyt c-ablated cells? Analysis of mouse fibroblasts revealed that cyt c is essential for the assembly/stability of COX, and a catalytically mutant form of cyt c cannot rescue the COX defect in the cyt c null cells. CL and triacylglycerols showed significant differences in the cyt c null cells, both in content and composition.     

Protein assembly of photosystem II and accumulation of subcomplexes in the absence of low molecular mass subunits PsbL and PsbJ.

The protein assembly and stability of photosystem II (PSII) (sub)complexes were studied in mature leaves of four plastid mutants of tobacco (Nicotiana tabacum L), each having one of the psbEFLJ operon genes inactivated. In the absence of psbL, no PSII core dimers or PSII-light harvesting complex (LHCII) supercomplexes were formed, and the assembly of CP43 into PSII core monomers was extremely labile. The assembly of CP43 into PSII core monomers was found to be necessary for the assembly of PsbO on the lumenal side of PSII. The two other oxygen-evolving complex (OEC) proteins, PsbP and PsbQ, were completely lacking in Delta psbL. In the absence of psbJ, both intact PSII core monomers and PSII core dimers harboring the PsbO protein were formed, whereas the LHCII antenna remained detached from the PSII dimers, as demonstrated by 77 K fluorescence measurements and by the lack of PSII-LHCII supercomplexes. The Delta psbJ mutant was characterized by a deficiency of PsbQ and a complete lack of PsbP. Thus, both the PsbL and PsbJ subunits of PSII are essential for proper assembly of the OEC. The absence of psbE and psbF resulted in a complete absence of all central PSII core and OEC proteins. In contrast, very young, vigorously expanding leaves of all psbEFLJ operon mutants accumulated at least traces of D2, CP43 and the OEC proteins PsbO and PsbQ, implying developmental control of the expression of the PSII core and OEC proteins. Despite severe problems in PSII assembly, the thylakoid membrane complexes other than PSII were present and correctly assembled in all psbEFLJ operon mutants.     

The Arabidopsis thylakoid protein PAM68 is required for efficient D1 biogenesis and photosystem II assembly

Photosystem II (PSII) is a multiprotein complex that functions as a light-driven water:plastoquinone oxidoreductase in photosynthesis. Assembly of PSII proceeds through a number of distinct intermediate states and requires auxiliary proteins. The photosynthesis affected mutant 68 (pam68) of Arabidopsis thaliana displays drastically altered chlorophyll fluorescence and abnormally low levels of the PSII core subunits D1, D2, CP43, and CP47. We show that these phenotypes result from a specific decrease in the stability and maturation of D1. This is associated with a marked increase in the synthesis of RC (the PSII reaction center-like assembly complex) at the expense of PSII dimers and supercomplexes. PAM68 is a conserved integral membrane protein found in cyanobacterial and eukaryotic thylakoids and interacts in split-ubiquitin assays with several PSII core proteins and known PSII assembly factors. Biochemical analyses of thylakoids from Arabidopsis and Synechocystis sp PCC 6803 suggest that, during PSII assembly, PAM68 proteins associate with an early intermediate complex that might contain D1 and the assembly factor LPA1. Inactivation of cyanobacterial PAM68 destabilizes RC but does not affect larger PSII assembly complexes. Our data imply that PAM68 proteins promote early steps in PSII biogenesis in cyanobacteria and plants, but their inactivation is differently compensated for in the two classes of organisms.     

The F1F0-ATP synthase complex influences the assembly state of the cytochrome bc1-cytochrome oxidase supercomplex and its association with the TIM23 machinery

The enzyme complexes involved in mitochondrial oxidative phosphorylation are organized into higher ordered assemblies termed supercomplexes. Subunits e and g (Su e and Su g, respectively) are catalytically nonessential subunits of the F1F0-ATP synthase whose presence is required to directly support the stable dimerization of the ATP synthase complex. We report here that Su g and Su e are also important for securing the correct organizational state of the cytochrome bc1-cytochrome oxidase (COX) supercomplex. Mitochondria isolated from the Delta su e and Delta su g null mutant strains exhibit decreased levels of COX enzyme activity but appear to have normal COX subunit protein levels. An altered stoichiometry of the cytochrome bc1-COX supercomplex was observed in mitochondria deficient in Su e and/or Su g, and a perturbation in the association of Cox4, a catalytically important subunit of the COX complex, was also detected. In addition, an increase in the level of the TIM23 translocase associated with the cytochrome bc1-COX supercomplex is observed in the absence of Su e and Su g. Together, our data highlight that a further level of complexity exists between the oxidative phosphorylation supercomplexes, whereby the organizational state of one complex, i.e. the ATP synthase, may influence that of another supercomplex, namely the cytochrome bc1-COX complex.     

Sealing the mitochondrial respirasome

The mitochondrial respiratory chain is organized within an array of supercomplexes that function to minimize the generation of reactive oxygen species (ROS) during electron transfer reactions. Structural models of supercomplexes are now known. Another recent advance is the discovery of non-OXPHOS complex proteins that appear to adhere to and seal the individual respiratory complexes to form stable assemblages that prevent electron leakage. This review highlights recent advances in our understanding of the structures of supercomplexes and the factors that mediate their stability.     

Proteomic approach to characterize the supramolecular organization of photosystems in higher plants

A project to investigate the supramolecular structure of photosystems was initiated, which is based on protein solubilizations by digitonin, protein separations by Blue native (BN)-polyacrylamide gel electrophoresis (PAGE) and protein identifications by mass spectrometry (MS). Under the conditions applied, nine photosystem supercomplexes could be described for chloroplasts of Arabidopsis, which have apparent molecular masses between 600 and 3200 kDa on BN gels. Identities of the supercomplexes were determined on the basis of their subunit compositions as documented by 2D BN/SDS-PAGE and BN/BN-PAGE. Two supercomplexes of 1060 and approximately 1600 kDa represent dimeric and trimeric forms of photosystem I (PSI), which include tightly bound LHCI proteins. Compared to monomeric PSI, these protein complexes are of low abundance. In contrast, photosystem II mainly forms part of dominant supercomplexes of 850, 1000, 1050 and 1300 kDa. According to our interpretation, these supercomplexes contain dimeric PSII, 1-4 LHCII trimers and additionally monomeric LHCII proteins. The 1300-kDa PSII supercomplex (containing four LHCII trimers) is partially converted into the 1000-kDa PSII supercomplex (containing two LHCII trimers) in the presence of dodecylmaltoside on 2D BN/BN gels. Analyses of peptides of the trypsinated 1300-kDa PSII supercomplex by mass spectrometry allowed to identify known subunits of the PSII core complex and additionally LHCII proteins encoded by eight different genes in Arabidopsis. Further application of this experimental approach will allow new insights into the supermolecular organization of photosystems in plants.     

Cox25 teams up with Mss51, Ssc1, and Cox14 to regulate mitochondrial cytochrome c oxidase subunit 1 expression and assembly in Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, mitochondrial cytochrome c oxidase (COX) biogenesis is translationally regulated. Mss51, a specific COX1 mRNA translational activator and Cox1 chaperone, drives the regulatory mechanism. During translation and post-translationally, newly synthesized Cox1 physically interacts with a complex of proteins involving Ssc1, Mss51, and Cox14, which eventually hand over Cox1 to the assembly pathway. This step is probably catalyzed by assembly chaperones such as Shy1 in a process coupled to the release of Ssc1-Mss51 from the complex. Impaired COX assembly results in the trapping of Mss51 in the complex, thus limiting its availability for COX1 mRNA translation. An exception is a null mutation in COX14 that does not affect Cox1 synthesis because the Mss51 trapping complexes become unstable, and Mss51 is readily available for translation. Here we present evidence showing that Cox25 is a new essential COX assembly factor that plays some roles similar to Cox14. A null mutation in COX25 by itself or in combination with other COX mutations does not affect Cox1 synthesis. Cox25 is an inner mitochondrial membrane intrinsic protein with a hydrophilic C terminus protruding into the matrix. Cox25 is an essential component of the complexes containing newly synthesized Cox1, Ssc1, Mss51, and Cox14. In addition, Cox25 is also found to interact with Shy1 and Cox5 in a complex that does not contain Mss51. These results suggest that once Ssc1-Mss51 are released from the Cox1 stabilization complex, Cox25 continues to interact with Cox14 and Cox1 to facilitate the formation of multisubunit COX assembly intermediates.     

The photosystem of Rhodobacter sphaeroides assembles with zinc bacteriochlorophyll in a bchD (magnesium chelatase) mutant

A Rhodobacter sphaeroides bchD (magnesium chelatase) mutant was studied to determine the properties of its photosystem in the absence of bacteriochlorophyll (BChl). Western blots of reaction center H, M, and L (RC H/M/L) proteins from mutant membranes showed levels of 12% RC H, 32% RC L, and 46% RC M relative to those of the wild type. Tricine-SDS-PAGE revealed 52% light-harvesting complex alpha chain and 14% beta chain proteins compared to those of the wild type. Pigment analysis of bchD cells showed the absence of BChl and bacteriopheophytin (BPhe), but zinc bacteriochlorophyll (Zn-BChl) was discovered. Zn-BChl binds to light-harvesting 1 (LH1) and 2 (LH2) complexes in place of BChl in bchD membranes, with a LH2:LH1 ratio resembling that of wild-type cells under BChl-limiting conditions. Furthermore, the RC from the bchD mutant contained Zn-BChl in the special pair and accessory BChl binding sites, as well as carotenoid and quinone, but BPhe was absent. Comparison of the bchD mutant RC absorption spectrum to that of Acidiphilium rubrum, which contains Zn-BChl in the RC, suggests the RC protein environment at L168 contributes to A. rubrum special pair absorption characteristics rather than solely Zn-BChl. We speculate that Zn-BChl is synthesized via the normal BChl biosynthetic pathway, but with ferrochelatase supplying zinc protoporphyrin IX for enzymatic steps following the nonfunctional magnesium chelatase. The absence of BPhe in bchD cells is likely related to Zn2+ stability in the chlorin macrocycle and consequently high resistance of Zn-BChl to pheophytinization (dechelation). Possible agents prevented from dechelating Zn-BChl include the RC itself, a hypothetical dechelatase enzyme, and spontaneous processes.     

SILAC-based complexome profiling dissects the structural organization of the human respiratory supercomplexes in SCAFIKO cells

The study of the mitochondrial respiratory chain (MRC) function in relation with its structural organization is of great interest due to the central role of this system in eukaryotic cell metabolism. The complexome profiling technique has provided invaluable information for our understanding of the composition and assembly of the individual MRC complexes, and also of their association into larger supercomplexes (SCs) and respirasomes. The formation of the SCs has been highly debated, and their assembly and regulation mechanisms are still unclear. Previous studies demonstrated a prominent role for COX7A2L (SCAFI) as a structural protein bridging the association of individual MRC complexes III and IV in the minor SC III₂ + IV, although its relevance for respirasome formation and function remains controversial. In this work, we have used SILAC-based complexome profiling to dissect the structural organization of the human MRC in HEK293T cells depleted of SCAFI (SCAFI^KO) by CRISPR-Cas9 genome editing. SCAFI ablation led to a preferential loss of SC III₂ + IV and of a minor subset of respirasomes without affecting OXPHOS function. Our data suggest that the loss of SCAFI-dependent respirasomes in SCAFI^KO cells is mainly due to alterations on early stages of CI assembly, without impacting the biogenesis of complexes III and IV. Contrary to the idea of SCAFI being the main player in respirasome formation, SILAC-complexome profiling showed that, in wild-type cells, the majority of respirasomes (ca. 70%) contained COX7A2 and that these species were present at roughly the same levels when SCAFI was knocked-out. We thus demonstrate the co-existence of structurally distinct respirasomes defined by the preferential binding of complex IV via COX7A2, rather than SCAFI, in human cultured cells.     

Two‐dimensional native electrophoretic analysis of respiratory supercomplexes from Yarrowia lipolytica

Mitochondria of the strictly aerobic yeast Yarrowia lipolytica contain respiratory complex I with close functional and structural similarity to the mammalian enzyme. Unlike mammalian mitochondria, however, Yarrowia mitochondria have been thought not to contain supercomplexes. Here, we identify respiratory supercomplexes composed of complexes I, III and IV also in Y. lipolytica. Evidence for dimeric complex I suggests further association of respiratory supercomplexes into respiratory strings or patches. Similar supercomplex organization in Yarrowia and mammalian mitochondria further makes this aerobic yeast a useful model for the human oxidative phosphorylation system. The analysis of supercomplexes and their constituent complexes was made possible by 2‐D native electrophoresis, i.e. by using native electrophoresis for both dimensions. Digitonin and blue‐native electrophoresis were generally applied for the initial separation of supercomplexes followed by less mild native electrophoresis variants in the second dimension to release the individual complexes from the supercomplexes. Such 2‐D native systems are useful means to identify the constituent proteins and their copy numbers in detergent‐labile physiological assemblies, since they can reduce the complexity of supramolecular systems to the level of individual complexes.