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5-Lipoxygenase contributes to PPAR [gamma] activation in macrophages in response to apoptotic cells
(2012)
Background: One hallmark contributing to immune suppression during the late phase of sepsis is macrophage polarization to an anti-inflammatory phenotype upon contact with apoptotic cells (AC). Taking the important role of the nuclear receptor PPARγ for this phenotype switch into consideration, it remains elusive how AC activate PPARγ in macrophages. Therefore, we were interested to characterize the underlying principle.
Methods: Apoptosis was induced by treatment of Jurkat T cells for 3 hours with 0.5 μg/ml staurosporine. Necrotic cells (NC) were prepared by heating cells for 20 minutes to 65°C. PPARγ activation was followed by stably transducing RAW264.7 macrophages with a vector encoding the red fluorescent protein mRuby after PPARγ binding to 4 × PPRE sites downstream of the reporter gene sequence. This readout was established by treatment with the PPARγ agonist rosiglitazone (1 μM) and AC (5:1). Twenty-four hours after stimulation, mRuby expression was analysed by fluorescence microscopy. Lipid rafts of AC, NC, as well as living cells (LC) were enriched by sucrose gradient centrifugation. Fractions were analysed for lipid raft-associated marker proteins. Lipid rafts were incubated with transduced RAW264.7 macrophages as described above. 5-Lipoxygenase (5-LO) involvement was verified by pharmacological inhibition (MK-866, 1 μM) and overexpression.
Results: Assuming that the molecule responsible for PPARγ activation in macrophages is localized in the cell membrane of AC, most probably associated to lipid rafts, we isolated lipid rafts from AC, NC and LC. Mass spectrometric analysis of lipid rafts of AC showed the expression of 5-LO, whereas lipid rafts of LC did not. Moreover, incubating macrophages with lipid rafts of AC induced mRuby expression. In contrast, lipid rafts of NC and LC did not. To verify the involvement of 5-LO in activating PPARγ in macrophages, Jurkat T cells were incubated for 30 minutes with the 5-LO inhibitor MK-866 (1 μM) before apoptosis induction. In line with our hypothesis, these AC did not induce mRuby expression. Finally, although living Jurkat T cells overexpressing 5-LO did not activate PPARγ in macrophages, mRuby expression was significantly increased when AC were generated from 5-LO overexpressing compared with wild-type Jurkat cells.
Conclusion: Our results suggest that induction of apoptosis activates 5-LO, localizing to lipid rafts, necessary for PPARγ activation in macrophages. Therefore, it will be challenging to determine whether 5-LO activity in AC, generated from other cell types, correlates with PPARγ activation, contributing to an immune-suppressed phenotype in macrophages.
The accumulation of functionally impaired mitochondria is a key event in aging. Previous works with the fungal aging model Podospora anserina demonstrated pronounced age-dependent changes of mitochondrial morphology and ultrastructure, as well as alterations of transcript and protein levels, including individual proteins of the oxidative phosphorylation (OXPHOS). The identified protein changes do not reflect the level of the whole protein complexes as they function in-vivo. In the present study, we investigated in detail the age-dependent changes of assembled mitochondrial protein complexes, using complexome profiling. We observed pronounced age-depen-dent alterations of the OXPHOS complexes, including the loss of mitochondrial respiratory supercomplexes (mtRSCs) and a reduction in the abundance of complex I and complex IV. Additionally, we identified a switch from the standard complex IV-dependent respiration to an alternative respiration during the aging of the P. anserina wild type. Interestingly, we identified proteasome components, as well as endoplasmic reticulum (ER) proteins, for which the recruitment to mitochondria appeared to be increased in the mitochondria of older cultures. Overall, our data demonstrate pronounced age-dependent alterations of the protein complexes involved in energy transduction and suggest the induction of different non-mitochondrial salvage pathways, to counteract the age-dependent mitochondrial impairments which occur during aging.
Long non-coding RNAs (lncRNAs) contribute to cardiac (patho)physiology. Aging is the major risk factor for cardiovascular disease with cardiomyocyte apoptosis as one underlying cause. Here, we report the identification of the aging-regulated lncRNA Sarrah (ENSMUST00000140003) that is anti-apoptotic in cardiomyocytes. Importantly, loss of SARRAH (OXCT1-AS1) in human engineered heart tissue results in impaired contractile force development. SARRAH directly binds to the promoters of genes downregulated after SARRAH silencing via RNA-DNA triple helix formation and cardiomyocytes lacking the triple helix forming domain of Sarrah show an increase in apoptosis. One of the direct SARRAH targets is NRF2, and restoration of NRF2 levels after SARRAH silencing partially rescues the reduction in cell viability. Overexpression of Sarrah in mice shows better recovery of cardiac contractile function after AMI compared to control mice. In summary, we identified the anti-apoptotic evolutionary conserved lncRNA Sarrah, which is downregulated by aging, as a regulator of cardiomyocyte survival.
Rationale: The AMP-activated protein kinase (AMPK) is stimulated by hypoxia, and although the AMPKα1 catalytic subunit has been implicated in angiogenesis, little is known about the role played by the AMPKα2 subunit in vascular repair.
Objective: To determine the role of the AMPKα2 subunit in vascular repair.
Methods and Results: Recovery of blood flow after femoral artery ligation was impaired (>80%) in AMPKα2-/- versus wild-type mice, a phenotype reproduced in mice lacking AMPKα2 in myeloid cells (AMPKα2ΔMC). Three days after ligation, neutrophil infiltration into ischemic limbs of AMPKα2ΔMC mice was lower than that in wild-type mice despite being higher after 24 hours. Neutrophil survival in ischemic tissue is required to attract monocytes that contribute to the angiogenic response. Indeed, apoptosis was increased in hypoxic neutrophils from AMPKα2ΔMC mice, fewer monocytes were recruited, and gene array analysis revealed attenuated expression of proangiogenic proteins in ischemic AMPKα2ΔMC hindlimbs. Many angiogenic growth factors are regulated by hypoxia-inducible factor, and hypoxia-inducible factor-1α induction was attenuated in AMPKα2-deficient cells and accompanied by its enhanced hydroxylation. Also, fewer proteins were regulated by hypoxia in neutrophils from AMPKα2ΔMC mice. Mechanistically, isocitrate dehydrogenase expression and the production of α-ketoglutarate, which negatively regulate hypoxia-inducible factor-1α stability, were attenuated in neutrophils from wild-type mice but remained elevated in cells from AMPKα2ΔMC mice.
Conclusions: AMPKα2 regulates α-ketoglutarate generation, hypoxia-inducible factor-1α stability, and neutrophil survival, which in turn determine further myeloid cell recruitment and repair potential. The activation of AMPKα2 in neutrophils is a decisive event in the initiation of vascular repair after ischemia.
Hundreds of genes have been associated with respiratory chain disease (RCD), the most common inborn error of metabolism so far. Elimination of the respiratory electron chain by depleting the entire mitochondrial DNA (mtDNA, ρ0 cells) has therefore one of the most severe impacts on the energy metabolism in eukaryotic cells. In this study, proteomic data sets including the post-translational modifications (PTMs) phosphorylation and ubiquitination were integrated with metabolomic data sets and selected enzyme activities in the osteosarcoma cell line 143B.TK−. A shotgun based SILAC LC-MS proteomics and a targeted metabolomics approach was applied to elucidate the consequences of the ρ0 state. Pathway and protein–protein interaction (PPI) network analyses revealed a nonuniform down-regulation of the respiratory electron chain, the tricarboxylic acid (TCA) cycle, and the pyruvate metabolism in ρ0 cells. Metabolites of the TCA cycle were dysregulated, such as a reduction of citric acid and cis-aconitic acid (six and 2.5-fold), and an increase of lactic acid, oxalacetic acid (both twofold), and succinic acid (fivefold) in ρ0 cells. Signaling pathways such as GPCR, EGFR, G12/13 alpha, and Rho GTPases were up-regulated in ρ0 cells, which could be indicative for the mitochondrial retrograde response, a pathway of communication from mitochondria to the nucleus. This was supported by our phosphoproteome data, which revealed two main processes, GTPase-related signal transduction and cytoskeleton organization. Furthermore, a general de-ubiquitination in ρ0 cells was observed, for example, 80S ribosomal proteins were in average threefold and SLC amino acid transporters fivefold de-ubiquitinated. The latter might cause the observed significant increase of amino acid levels in ρ0 cells. We conclude that an elimination of the respiratory electron chain, e.g. mtDNA depletion, not only leads to an uneven down-regulation of mitochondrial energy pathways, but also triggers the retrograde response.
Mitochondrial cristae morphology is highly variable and altered under numerous pathological conditions. The protein complexes involved are largely unknown or only insufficiently characterized. Using complexome profiling we identified apolipoprotein O (APOO) and apolipoprotein O-like protein (APOOL) as putative components of the Mitofilin/MINOS protein complex which was recently implicated in determining cristae morphology. We show that APOOL is a mitochondrial membrane protein facing the intermembrane space. It specifically binds to cardiolipin in vitro but not to the precursor lipid phosphatidylglycerol. Overexpression of APOOL led to fragmentation of mitochondria, a reduced basal oxygen consumption rate, and altered cristae morphology. Downregulation of APOOL impaired mitochondrial respiration and caused major alterations in cristae morphology. We further show that APOOL physically interacts with several subunits of the MINOS complex, namely Mitofilin, MINOS1, and SAMM50. We conclude that APOOL is a cardiolipin-binding component of the Mitofilin/MINOS protein complex determining cristae morphology in mammalian mitochondria. Our findings further assign an intracellular role to a member of the apolipoprotein family in mammals.
TMEM70 is involved in the biogenesis of mitochondrial ATP synthase and mutations in the TMEM70 gene impair oxidative phosphorylation. Herein, we report on pathology and treatment of ATP synthase deficiency in four siblings. A consanguineous family of Roma (Gipsy) ethnic origin gave birth to 6 children of which 4 were affected presenting with dysmorphic features, failure to thrive, cardiomyopathy, metabolic crises, and 3-methylglutaconic aciduria as clinical symptoms. Genetic testing revealed a homozygous mutation (c.317-2A>G) in the TMEM70 gene. While light microscopy was unremarkable, ultrastructural investigation of muscle tissue revealed accumulation of swollen degenerated mitochondria with lipid crystalloid inclusions, cristae aggregation, and exocytosis of mitochondrial material. Biochemical analysis of mitochondrial complexes showed an almost complete ATP synthase deficiency. Despite harbouring the same mutation, the clinical outcome in the four siblings was different. Two children died within 60 h after birth; the other two had recurrent life-threatening metabolic crises but were successfully managed with supplementation of anaplerotic amino acids, lipids, and symptomatic treatment during metabolic crisis. In summary, TMEM70 mutations can cause distinct ultrastructural mitochondrial degeneration and almost complete deficiency of ATP synthase but are still amenable to treatment.
Isolated complex I deficiency is a common biochemical phenotype observed in pediatric mitochondrial disease and often arises as a consequence of pathogenic variants affecting one of the ∼65 genes encoding the complex I structural subunits or assembly factors. Such genetic heterogeneity means that application of next-generation sequencing technologies to undiagnosed cohorts has been a catalyst for genetic diagnosis and gene-disease associations. We describe the clinical and molecular genetic investigations of four unrelated children who presented with neuroradiological findings and/or elevated lactate levels, highly suggestive of an underlying mitochondrial diagnosis. Next-generation sequencing identified bi-allelic variants in NDUFA6, encoding a 15 kDa LYR-motif-containing complex I subunit that forms part of the Q-module. Functional investigations using subjects’ fibroblast cell lines demonstrated complex I assembly defects, which were characterized in detail by mass-spectrometry-based complexome profiling. This confirmed a marked reduction in incorporated NDUFA6 and a concomitant reduction in other Q-module subunits, including NDUFAB1, NDUFA7, and NDUFA12. Lentiviral transduction of subjects’ fibroblasts showed normalization of complex I. These data also support supercomplex formation, whereby the ∼830 kDa complex I intermediate (consisting of the P- and Q-modules) is in complex with assembled complex III and IV holoenzymes despite lacking the N-module. Interestingly, RNA-sequencing data provided evidence that the consensus RefSeq accession number does not correspond to the predominant transcript in clinically relevant tissues, prompting revision of the NDUFA6 RefSeq transcript and highlighting not only the importance of thorough variant interpretation but also the assessment of appropriate transcripts for analysis.
Biallelic mutations in TMEM126B cause severe complex i deficiency with a variable clinical phenotype
(2016)
Complex I deficiency is the most common biochemical phenotype observed in individuals with mitochondrial disease. With 44 structural subunits and over 10 assembly factors, it is unsurprising that complex I deficiency is associated with clinical and genetic heterogeneity. Massively parallel sequencing (MPS) technologies including custom, targeted gene panels or unbiased whole-exome sequencing (WES) are hugely powerful in identifying the underlying genetic defect in a clinical diagnostic setting, yet many individuals remain without a genetic diagnosis. These individuals might harbor mutations in poorly understood or uncharacterized genes, and their diagnosis relies upon characterization of these orphan genes. Complexome profiling recently identified TMEM126B as a component of the mitochondrial complex I assembly complex alongside proteins ACAD9, ECSIT, NDUFAF1, and TIMMDC1. Here, we describe the clinical, biochemical, and molecular findings in six cases of mitochondrial disease from four unrelated families affected by biallelic (c.635G>T [p.Gly212Val] and/or c.401delA [p.Asn134Ilefs∗2]) TMEM126B variants. We provide functional evidence to support the pathogenicity of these TMEM126B variants, including evidence of founder effects for both variants, and establish defects within this gene as a cause of complex I deficiency in association with either pure myopathy in adulthood or, in one individual, a severe multisystem presentation (chronic renal failure and cardiomyopathy) in infancy. Functional experimentation including viral rescue and complexome profiling of subject cell lines has confirmed TMEM126B as the tenth complex I assembly factor associated with human disease and validates the importance of both genome-wide sequencing and proteomic approaches in characterizing disease-associated genes whose physiological roles have been previously undetermined.
BIAM switch assay coupled to mass spectrometry identifies novel redox targets of NADPH oxidase 4
(2019)
Aim: NADPH oxidase (Nox) -derived reactive oxygen species have been implicated in redox signaling via cysteine oxidation in target proteins. Although the importance of oxidation of target proteins is well known, the specificity of such events is often debated. Only a limited number of Nox-oxidized proteins have been identified thus far; especially little is known concerning redox-targets of the constitutively active NADPH oxidase Nox4.
In this study, HEK293 cells with tetracycline-inducible Nox4 overexpression (HEK-tet-Nox4), as well as podocytes of WT and Nox4-/- mice, were utilized to identify Nox4-dependent redox-modified proteins.
Results: TGFβ1 induced an elevation in Nox4 expression in podocytes from WT but not Nox4-/- mice. Using BIAM based redox switch assay in combination with mass spectrometry and western blot analysis, 142 proteins were identified as differentially oxidized in podocytes from wild type vs. Nox4-/- mice and 131 proteins were differentially oxidized in HEK-tet-Nox4 cells upon Nox4 overexpression. A predominant overlap was found for peroxiredoxins and thioredoxins, as expected. More interestingly, the GRB2-associated-binding protein 1 (Gab1) was identified as being differentially oxidized in both approaches. Further analysis using mass spectrometry-coupled BIAM switch assay and site directed mutagenesis, revealed Cys374 and Cys405 as the major Nox4 targeted oxidation sites in Gab1.
Innovation & conclusion: BIAM switch assay coupled to mass spectrometry is a powerful and versatile tool to identify differentially oxidized proteins in a global untargeted way. Nox4, as a source of hydrogen peroxide, changes the redox-state of numerous proteins. Of those, we identified Gab1 as a novel redox target of Nox4.
Broad AOX expression in a genetically tractable mouse model does not disturb normal physiology
(2017)
Plants and many lower organisms, but not mammals, express alternative oxidases (AOXs) that branch the mitochondrial respiratory chain, transferring electrons directly from ubiquinol to oxygen without proton pumping. Thus, they maintain electron flow under conditions when the classical respiratory chain is impaired, limiting excess production of oxygen radicals and supporting redox and metabolic homeostasis. AOX from Ciona intestinalis has been used to study and mitigate mitochondrial impairments in mammalian cell lines, Drosophila disease models and, most recently, in the mouse, where multiple lentivector-AOX transgenes conferred substantial expression in specific tissues. Here, we describe a genetically tractable mouse model in which Ciona AOX has been targeted to the Rosa26 locus for ubiquitous expression. The AOXRosa26 mouse exhibited only subtle phenotypic effects on respiratory complex formation, oxygen consumption or the global metabolome, and showed an essentially normal physiology. AOX conferred robust resistance to inhibitors of the respiratory chain in organello; moreover, animals exposed to a systemically applied LD50 dose of cyanide did not succumb. The AOXRosa26 mouse is a useful tool to investigate respiratory control mechanisms and to decipher mitochondrial disease aetiology in vivo.
Complexome profiling is an emerging ‘omics’ approach that systematically interrogates the composition of protein complexes (the complexome) of a sample, by combining biochemical separation of native protein complexes with mass-spectrometry based quantitation proteomics. The resulting fractionation profiles hold comprehensive information on the abundance and composition of the complexome, and have a high potential for reuse by experimental and computational researchers. However, the lack of a central resource that provides access to these data, reported with adequate descriptions and an analysis tool, has limited their reuse. Therefore, we established the ComplexomE profiling DAta Resource (CEDAR, www3.cmbi.umcn.nl/cedar/), an openly accessible database for depositing and exploring mass spectrometry data from complexome profiling studies. Compatibility and reusability of the data is ensured by a standardized data and reporting format containing the “minimum information required for a complexome profiling experiment” (MIACE). The data can be accessed through a user-friendly web interface, as well as programmatically using the REST API portal. Additionally, all complexome profiles available on CEDAR can be inspected directly on the website with the profile viewer tool that allows the detection of correlated profiles and inference of potential complexes. In conclusion, CEDAR is a unique, growing and invaluable resource for the study of protein complex composition and dynamics across biological systems.
Complexome profiling is an emerging ‘omics approach that systematically interrogates the composition of protein complexes (the complexome) of a sample, by combining biochemical separation of native protein complexes with mass-spectrometry based quantitation proteomics. The resulting fractionation profiles hold comprehensive information on the abundance and composition of the complexome, and have a high potential for reuse by experimental and computational researchers. However, the lack of a central resource that provides access to these data, reported with adequate descriptions and an analysis tool, has limited their reuse. Therefore, we established the ComplexomE profiling DAta Resource (CEDAR, www3.cmbi.umcn.nl/cedar/), an openly accessible database for depositing and exploring mass spectrometry data from complexome profiling studies. Compatibility and reusability of the data is ensured by a standardized data and reporting format containing the “minimum information required for a complexome profiling experiment” (MIACE). The data can be accessed through a user-friendly web interface, as well as programmatically using the REST API portal. Additionally, all complexome profiles available on CEDAR can be inspected directly on the website with the profile viewer tool that allows the detection of correlated profiles and inference of potential complexes. In conclusion, CEDAR is a unique, growing and invaluable resource for the study of protein complex composition and dynamics across biological systems.
SR proteins function in nuclear pre-mRNA processing, mRNA export, and translation. To investigate their cellular dynamics, we developed a quantitative assay, which detects differences in nucleocytoplasmic shuttling among seven canonical SR protein family members. As expected, SRSF2 and SRSF5 shuttle poorly in HeLa cells but surprisingly display considerable shuttling in pluripotent murine P19 cells. Combining individual-resolution cross-linking and immunoprecipitation (iCLIP) and mass spectrometry, we show that elevated arginine methylation of SRSF5 and lower phosphorylation levels of cobound SRSF2 enhance shuttling of SRSF5 in P19 cells by modulating protein-protein and protein-RNA interactions. Moreover, SRSF5 is bound to pluripotency-specific transcripts such as Lin28a and Pou5f1/Oct4 in the cytoplasm. SRSF5 depletion reduces and overexpression increases their cytoplasmic mRNA levels, suggesting that enhanced mRNA export by SRSF5 is required for the expression of pluripotency factors. Remarkably, neural differentiation of P19 cells leads to dramatically reduced SRSF5 shuttling. Our findings indicate that posttranslational modification of SR proteins underlies the regulation of their mRNA export activities and distinguishes pluripotent from differentiated cells.
We disassembled monomeric and dimeric yeast ATP synthase under mild conditions to identify labile proteins and transiently stable subcomplexes that had not been observed before. Specific removal of subunits α, β, oligomycin sensitivity conferring protein (OSCP), and h disrupted the ATP synthase at the γ-α3β3 rotor-stator interface. Loss of two F1-parts from dimeric ATP synthase led to the isolation of a dimeric subcomplex containing membrane and peripheral stalk proteins thus identifying the membrane/peripheral stalk sectors immediately as the dimerizing parts of ATP synthase. Almost all subunit a was found associated with a ring of 10 c-subunits in two-dimensional blue native/SDS gels. We therefore postulate that c10a1-complex is a stable structure in resting ATP synthase until the entry of protons induces a breaking of interactions and stepwise rotation of the c-ring relative to the a-subunit in the catalytic mechanism. Dimeric subunit a was identified in SDS gels in association with two c10-rings suggesting that a c10a2c10-complex may constitute an important part of the monomer-monomer interface in dimeric ATP synthase that seems to be further tightened by subunits b, i, e, g, and h. In contrast to the monomer-monomer interface, the interface between dimers in higher oligomeric structures remains largely unknown. However, we could show that the natural inhibitor protein Inh1 is not required for oligomerization.
Background: Mitochondrial acyl-CoA dehydrogenase family member 9 (ACAD9) is essential for the assembly of mitochondrial respiratory chain complex I. Disease causing biallelic variants in ACAD9 have been reported in individuals presenting with lactic acidosis and cardiomyopathy.
Results: We describe the genetic, clinical and biochemical findings in a cohort of 70 patients, of whom 29 previously unpublished. We found 34 known and 18 previously unreported variants in ACAD9. No patients harbored biallelic loss of function mutations, indicating that this combination is unlikely to be compatible with life. Causal pathogenic variants were distributed throughout the entire gene, and there was no obvious genotype-phenotype correlation.
Most of the patients presented in the first year of life. For this subgroup the survival was poor (50% not surviving the first 2 years) comparing to patients with a later presentation (more than 90% surviving 10 years). The most common clinical findings were cardiomyopathy (85%), muscular weakness (75%) and exercise intolerance (72%). Interestingly, severe intellectual deficits were only reported in one patient and severe developmental delays in four patients. More than 70% of the patients were able to perform the same activities of daily living when compared to peers.
Conclusions: Our data show that riboflavin treatment improves complex I activity in the majority of patient-derived fibroblasts tested. This effect was also reported for most of the treated patients and is mirrored in the survival data. In the patient group with disease-onset below 1 year of age, we observed a statistically-significant better survival for patients treated with riboflavin.
The yeast bc1 complex (complex III) and cytochrome oxidase (complex IV) are mosaics of core subunits encoded by the mitochondrial genome and additional nuclear-encoded proteins imported from the cytosol. Both complexes build in the mitochondrial inner membrane various supramolecular assemblies. The formation of the individual complexes and their supercomplexes depends on the activity of dedicated assembly factors. We identified a so far uncharacterized mitochondrial protein (open reading frame YDR381C-A) as an important assembly factor for complex III, complex IV, and their supercomplexes. Therefore, we named this protein Cox interacting (Coi) 1. Deletion of COI1 results in decreased respiratory growth, reduced membrane potential, and hampered respiration, as well as slow fermentative growth at low temperature. In addition, coi1Δ cells harbour reduced steady-state levels of subunits of complexes III and IV as well as of the assembled complexes and supercomplexes. Interaction of Coi1 with respiratory chain subunits seems transient, as it appears to be a stoichiometric subunit neither of complex III nor of complex IV. Collectively, this work identifies a novel protein that plays a role in the assembly of the mitochondrial respiratory chain.
Many proteins have been found to operate in a complex with various biomolecules such as proteins, nucleic acids, carbohydrates, or lipids. Protein complexes can be transient, stable or dynamic and their association is controlled under variable cellular conditions. Complexome profiling is a recently developed mass spectrometry-based method that combines mild separation techniques, native gel electrophoresis, and density gradient centrifugation with quantitative mass spectrometry to generate inventories of protein assemblies within a cell or subcellular fraction. This review summarizes applications of complexome profiling with respect to assembly ranging from single subunits to large macromolecular complexes, as well as their stability, and remodeling in health and disease.
The inner boundary and the cristae membrane are connected by pore-like structures termed crista junctions (CJs). The MICOS complex is required for CJ formation and enriched at CJs. Here, we address the roles of the MICOS subunits Mic27 and Mic10. We observe a positive genetic interaction between Mic27 and Mic60 and deletion of Mic27 results in impaired formation of CJs and altered cristae membrane curvature. Mic27 acts in an antagonistic manner to Mic60 as it promotes oligomerization of the F1FO-ATP synthase and partially restores CJ formation in cells lacking Mic60. Mic10 impairs oligomerization of the F1FO-ATP synthase similar to Mic60. Applying complexome profiling, we observed that deletion of Mic27 destabilizes the MICOS complex but does not impair formation of a high molecular weight Mic10 subcomplex. Moreover, this Mic10 subcomplex comigrates with the dimeric F1FO-ATP synthase in a Mic27-independent manner. Further, we observed a chemical crosslink of Mic10 to Mic27 and of Mic10 to the F1FO-ATP synthase subunit e. We corroborate the physical interaction of the MICOS complex and the F1FO-ATP synthase. We propose a model in which part of the F1FO-ATP synthase is linked to the MICOS complex via Mic10 and Mic27 and by that is regulating CJ formation.
Biogenesis of mitochondrial cytochrome c oxidase (COX) is a complex process involving the coordinate expression and assembly of numerous subunits (SU) of dual genetic origin. Moreover, several auxiliary factors are required to recruit and insert the redox-active metal compounds, which in most cases are buried in their protein scaffold deep inside the membrane. Here we used a combination of gel electrophoresis and pull-down assay techniques in conjunction with immunostaining as well as complexome profiling to identify and analyze the composition of assembly intermediates in solubilized membranes of the bacterium Paracoccus denitrificans. Our results show that the central SUI passes through at least three intermediate complexes with distinct subunit and cofactor composition before formation of the holoenzyme and its subsequent integration into supercomplexes. We propose a model for COX biogenesis in which maturation of newly translated COX SUI is initially assisted by CtaG, a chaperone implicated in CuB site metallation, followed by the interaction with the heme chaperone Surf1c to populate the redox-active metal-heme centers in SUI. Only then the remaining smaller subunits are recruited to form the mature enzyme which ultimately associates with respiratory complexes I and III into supercomplexes.