bims-cytox1 Biomed News
on Cytochrome oxidase subunit 1
Issue of 2021‒04‒25
thirteen papers selected by
Gavin McStay
Staffordshire University

  1. Biochim Biophys Acta Mol Basis Dis. 2021 Apr 15. pii: S0925-4439(21)00080-6. [Epub ahead of print] 166147
      The mitochondrial respiratory chain (MRC) complex III (CIII) associates with complexes I and IV (CI and CIV) into supercomplexes. We identified a novel homozygous missense mutation (c.665G>C; p.Gly222Ala) in UQCRC2 coding for structural subunit Core 2 in a patient with severe encephalomyopathy. The structural data suggest that the Gly222Ala exchange might result in an altered spatial arrangement in part of the UQCRC2 subunit, which could impact specific protein-protein interactions. Accordingly, we have found decreased levels of CIII and accumulation of CIII-specific subassemblies comprising MT-CYB, UQCRB, UQCRQ, UQCR10 and CYC1 subunits, but devoid of UQCRC1, UQCRC2, and UQCRFS1 in the patient's fibroblasts. The lack of UQCRC1 subunit-containing subassemblies could result from an impaired interaction with mutant UQCRC2Gly222Ala and subsequent degradation of both subunits by mitochondrial proteases. Indeed, we show an elevated amount of matrix CLPP protease, suggesting the activation of the mitochondrial protein quality control machinery in UQCRC2Gly222Ala fibroblasts. In line with growing evidence, we observed a rate-limiting character of CIII availability for the supercomplex formation, accompanied by a diminished amount of CI. Furthermore, we found impaired electron flux between CI and CIII in skeletal muscle and fibroblasts of the UQCRC2Gly222Ala patient. The ectopic expression of wild-type UQCRC2 in patient cells rescued maximal respiration rate, demonstrating the deleterious effect of the mutation on MRC. Our study expands the phenotypic spectrum of human disease caused by CIII Core protein deficiency, provides insight into the assembly pathway of human CIII, and supports the requirement of assembled CIII for a proper accumulation of CI.
    Keywords:  Core 2); Mitochondrial dysfunction; UQCRC2 (Core2; caseinolytic mitochondrial matrix peptidase proteolytic subunit (CLPP); mitochondrial complex III; mitochondrial protein quality control; respiratory supercomplexes
  2. Proc Natl Acad Sci U S A. 2021 Apr 27. pii: e2019665118. [Epub ahead of print]118(17):
      Mitochondrial disease is a debilitating condition with a diverse genetic etiology. Here, we report that TMEM126A, a protein that is mutated in patients with autosomal-recessive optic atrophy, participates directly in the assembly of mitochondrial complex I. Using a combination of genome editing, interaction studies, and quantitative proteomics, we find that loss of TMEM126A results in an isolated complex I deficiency and that TMEM126A interacts with a number of complex I subunits and assembly factors. Pulse-labeling interaction studies reveal that TMEM126A associates with the newly synthesized mitochondrial DNA (mtDNA)-encoded ND4 subunit of complex I. Our findings indicate that TMEM126A is involved in the assembly of the ND4 distal membrane module of complex I. In addition, we find that the function of TMEM126A is distinct from its paralogue TMEM126B, which acts in assembly of the ND2-module of complex I.
    Keywords:  complex I; membrane protein; mitochondria; optic atrophy; oxidative phosphorylation
  3. Plant Physiol. 2019 Nov 25. 181(4): 1632-1650
      Complex I biogenesis requires the expression of both nuclear and mitochondrial genes, the import of proteins, cofactor biosynthesis, and the assembly of at least 49 individual subunits. Assembly factors interact with subunits of Complex I but are not part of the final holocomplex. We show that in Arabidopsis (Arabidopsis thaliana), a mitochondrial matrix protein (EMB1793, At1g76060), which we term COMPLEX I ASSEMBLY FACTOR 1 (CIAF1), contains a LYR domain and is required for Complex I assembly. T-DNA insertion mutants of CIAF1 lack Complex I and the Supercomplex I+III. Biochemical characterization shows that the assembly of Complex I is stalled at 650 and 800 kD intermediates in mitochondria isolated from ciaf1 mutant lines.I. Yeast-two-hybrid interaction and complementation assays indicate that CIAF1 specifically interacts with the 23-kD TYKY-1 matrix domain subunit of Complex I and likely plays a role in Fe-S insertion into this subunit. These data show that CIAF1 plays an essential role in assembling the peripheral matrix arm Complex I subunits into the Complex I holoenzyme.
  4. Plant Physiol. 2020 Oct 05. 184(2): 1042-1055
      In yeast (Saccharomyces cerevisiae) and human (Homo sapiens) mitochondria, Oxidase assembly protein1 (Oxa1) is the general insertase for protein insertion from the matrix side into the inner membrane while Cytochrome c oxidase assembly protein18 (Cox18/Oxa2) is specifically involved in the topogenesis of the complex IV subunit, Cox2. Arabidopsis (Arabidopsis thaliana) mitochondria contain four OXA homologs: OXA1a, OXA1b, OXA2a, and OXA2b. OXA2a and OXA2b are unique members of the Oxa1 superfamily, in that they possess a tetratricopeptide repeat (TPR) domain at their C termini. Here, we determined the role of OXA2a by studying viable mutant plants generated by partial complementation of homozygous lethal OXA2a transfer-DNA insertional mutants using the developmentally regulated ABSCISIC ACID INSENSITIVE3 (ABI3) promoter. The ABI3p:OXA2a plants displayed growth retardation due to a reduction in the steady-state abundances of both c-type cytochromes, cytochrome c  1 and cytochrome c. The observed reduction in the steady-state abundance of complex III could be attributed to cytochrome c  1 being one of its subunits. Expression of a soluble heme lyase from an organism with cytochrome c maturation system III could functionally complement the lack of OXA2a. This implies that OXA2a is required for the system I cytochrome c maturation of Arabidopsis. Due to the interaction of OXA2a with Cytochrome c maturation protein CcmF C-terminal-like protein (CCMFC) in a yeast split-ubiquitin based interaction assay, we propose that OXA2a aids in the membrane insertion of CCMFC, which is presumed to form the heme lyase component of the cytochrome c maturation pathway. In contrast with the crucial role played by the TPR domain of OXA2b, the TPR domain of OXA2a is not essential for its functionality.
  5. G3 (Bethesda). 2019 Jul 01. 9(7): 2225-2234
      Drosophila melanogaster, like most animal species, displays considerable genetic variation in both nuclear and mitochondrial DNA (mtDNA). Here we tested whether any of four natural mtDNA variants was able to modify the effect of the phenotypically mild, nuclear tko25t mutation, affecting mitochondrial protein synthesis. When combined with tko25t, the mtDNA from wild strain KSA2 produced pupal lethality, accompanied by the presence of melanotic nodules in L3 larvae. KSA2 mtDNA, which carries a substitution at a conserved residue of cytochrome b that is predicted to be involved in subunit interactions within respiratory complex III, conferred drastically decreased respiratory capacity and complex III activity in the tko25t but not a wild-type nuclear background. The complex III inhibitor antimycin A was able to phenocopy effects of the tko25t mutation in the KSA2 mtDNA background. This is the first report of a lethal, nuclear-mitochondrial interaction within a metazoan species, representing a paradigm for understanding genetic interactions between nuclear and mitochondrial genotype relevant to human health and disease.
    Keywords:  cybrid; cytochrome b; melanotic nodules; mtDNA copy number; respiration
  6. Plant Physiol. 2020 Sep 08. 184(1): 148-164
      Protein homeostasis in eukaryotic organelles and their progenitor prokaryotes is regulated by a series of proteases including the caseinolytic protease (CLPP). CLPP has essential roles in chloroplast biogenesis and maintenance, but the significance of the plant mitochondrial CLPP remains unknown and factors that aid coordination of nuclear- and mitochondrial-encoded subunits for complex assembly in mitochondria await discovery. We generated knockout lines of the single gene for the mitochondrial CLP protease subunit, CLPP2, in Arabidopsis (Arabidopsis thaliana). Mutants showed a higher abundance of transcripts from mitochondrial genes encoding oxidative phosphorylation protein complexes, whereas nuclear genes encoding other subunits of the same complexes showed no change in transcript abundance. By contrast, the protein abundance of specific nuclear-encoded subunits in oxidative phosphorylation complexes I and V increased in CLPP2 knockouts, without accumulation of mitochondrial-encoded counterparts in the same complex. Complexes with subunits mainly or entirely encoded in the nucleus were unaffected. Analysis of protein import and function of complex I revealed that while function was retained, protein homeostasis was disrupted, leading to accumulation of soluble subcomplexes of nuclear-encoded subunits. Therefore, CLPP2 contributes to the mitochondrial protein degradation network through supporting coordination and homeostasis of protein complexes encoded across mitochondrial and nuclear genomes.
  7. Biol Cell. 2021 Apr 18.
      Mitochondria are organelles involved in various functions related to cellular metabolism and homeostasis. Though mitochondria contain own genome, their nuclear counterparts encode most of the different mitochondrial proteins. These are synthesized as precursors in the cytosol and have to be delivered into the mitochondria. These organelles hence have elaborate machineries for the import of precursor proteins from cytosol. The protein import machineries present in both mitochondrial membrane and aqueous compartments show great variability in pre-protein recognition, translocation and sorting across or into it. Mitochondrial protein import machineries also interact transiently with other protein complexes of the respiratory chain or those involved in the maintenance of membrane architecture. Hence mitochondrial protein translocation is an indispensable part of the regulatory network that maintains protein biogenesis, bioenergetics, membrane dynamics and quality control of the organelle. Various stress conditions and diseases that are associated with mitochondrial import defects lead to changes in cellular transcriptomic and proteomic profiles. Dysfunction in mitochondrial protein import also causes over-accumulation of precursor proteins and their aggregation in the cytosol. Multiple pathways may be activated for buffering these harmful consequences. Here we present a comprehensive picture of import machinery and its role in cellular quality control in response to defective mitochondrial import. We also discuss the pathological consequences of dysfunctional mitochondrial protein import in neurodegeneration and cancer. This article is protected by copyright. All rights reserved.
    Keywords:  Intracellular compartmentalization; Mitochondria; Protein degradation/proteases
  8. Hum Mol Genet. 2021 Apr 22. pii: ddab116. [Epub ahead of print]
      UBQLN2 mutations cause amyotrophic lateral sclerosis (ALS) with frontotemporal dementia (FTD), but the pathogenic mechanisms by which they cause disease remain unclear. Proteomic profiling identified 'mitochondrial proteins' as comprising the largest category of protein changes in the spinal cord (SC) of the P497S UBQLN2 mouse model of ALS/FTD. Immunoblots confirmed P497S animals have global changes in proteins predictive of a severe decline in mitochondrial health, including oxidative phosphorylation (OXPHOS), mitochondrial protein import, and network dynamics. Functional studies confirmed mitochondria purified from the SC of P497S animals have age-dependent decline in nearly all steps of OXPHOS. Mitochondria cristae deformities were evident in spinal motor neurons of aged P497S animals. Knockout (KO) of UBQLN2 in HeLa cells resulted in changes in mitochondrial proteins and OXPHOS activity similar to those seen in the SC. KO of UBQLN2 also compromised targeting and processing of the mitochondrial import factor, TIMM44, resulting in accumulation in abnormal foci. The functional OXPHOS deficits and TIMM44 targeting defects were rescued by re-expression of WT UBQLN2 but not by ALS/FTD mutant UBQLN2 proteins. In-vitro binding assays revealed ALS/FTD mutant UBQLN2 proteins bind weaker with TIMM44 than WT UBQLN2 protein, suggesting that the loss of UBQLN2 binding may underlie the import and/or delivery defect of TIMM44 to mitochondria. Our studies indicate a potential key pathogenic disturbance in mitochondrial health caused by UBQLN2 mutations.
  9. Mol Cell. 2021 Apr 09. pii: S1097-2765(21)00263-X. [Epub ahead of print]
      The mitochondrial translation system originates from a bacterial ancestor but has substantially diverged in the course of evolution. Here, we use single-particle cryo-electron microscopy (cryo-EM) as a screening tool to identify mitochondrial translation termination mechanisms and to describe them in molecular detail. We show how mitochondrial release factor 1a releases the nascent chain from the ribosome when it encounters the canonical stop codons UAA and UAG. Furthermore, we define how the peptidyl-tRNA hydrolase ICT1 acts as a rescue factor on mitoribosomes that have stalled on truncated messages to recover them for protein synthesis. Finally, we present structural models detailing the process of mitochondrial ribosome recycling to explain how a dedicated elongation factor, mitochondrial EFG2 (mtEFG2), has specialized for cooperation with the mitochondrial ribosome recycling factor to dissociate the mitoribosomal subunits at the end of the translation process.
    Keywords:  ICT1; cryo-EM; mitochondria; mtEFG2; mtRF1a; mtRRF; recycling; ribosome; termination; translation
  10. Cell Rep. 2021 Apr 20. pii: S2211-1247(21)00316-8. [Epub ahead of print]35(3): 109002
      Complex I (CI) is the largest enzyme of the mitochondrial respiratory chain, and its defects are the main cause of mitochondrial disease. To understand the mechanisms regulating the extremely intricate biogenesis of this fundamental bioenergetic machine, we analyze the structural and functional consequences of the ablation of NDUFS3, a non-catalytic core subunit. We show that, in diverse mammalian cell types, a small amount of functional CI can still be detected in the complete absence of NDUFS3. In addition, we determine the dynamics of CI disassembly when the amount of NDUFS3 is gradually decreased. The process of degradation of the complex occurs in a hierarchical and modular fashion in which the ND4 module remains stable and bound to TMEM126A. We, thus, uncover the function of TMEM126A, the product of a disease gene causing recessive optic atrophy as a factor necessary for the correct assembly and function of CI.
    Keywords:  CI; CI modules; NDUFS3; SILAC; TMEM126A; assembly factor; optic atrophy type 7; respiratory complex I
  11. Reproduction. 2021 Apr 01. pii: REP-20-0635.R1. [Epub ahead of print]
      The birth of Dolly the sheep in 1996 elicited a tsunami of commentaries, both in the popular media and academic journals, including responses to the prospect of human reproductive cloning. Much of the anxiety expressed over this imagined consequence of Dolly's genesis revealed fundamental concerns about our losing our commitments to certain ethical goods, such as human dignity, or even 'what it means to be human'. Over the last 25 years, the focus of much of the ethical debate over human biotechnology has slowly shifted towards other genetic technologies that aim to influence inheritance, such as mitochondrial replacement techniques (MRT) and heritable genome editing. Genome editing, in particular, is a technology with multiple fields of application, actual and potential, in research and innovation. In this review, I suggest that many of the fundamental concerns about the possibility of human reproductive cloning that were precipitated by Dolly persist today in the arguments of those who oppose MRT and any use of heritable human genome editing (HHGE). Whilst I do not accept that an understanding of human nature and dignity alone can demonstrate the ethical unacceptability of such assisted reproductive technologies, there are themes of justice, which extend into our relationships with animals, that demand continued wide-ranging examination and public deliberation. Dolly has cast a long shadow over such discussions, but I suggest that the general existential angst over human uses of biotechnology that she came to symbolise is neither compulsory, nor a reliable guide for how to think about biotechnologies today.
  12. Proc Natl Acad Sci U S A. 2021 Apr 13. pii: e2009329118. [Epub ahead of print]118(15):
      Stabilization of messenger RNA is an important step in posttranscriptional gene regulation. In the nucleus and cytoplasm of eukaryotic cells it is generally achieved by 5' capping and 3' polyadenylation, whereas additional mechanisms exist in bacteria and organelles. The mitochondrial mRNAs in the yeast Saccharomyces cerevisiae comprise a dodecamer sequence element that confers RNA stability and 3'-end processing via an unknown mechanism. Here, we isolated the protein that binds the dodecamer and identified it as Rmd9, a factor that is known to stabilize yeast mitochondrial RNA. We show that Rmd9 associates with mRNA around dodecamer elements in vivo and that recombinant Rmd9 specifically binds the element in vitro. The crystal structure of Rmd9 bound to its dodecamer target reveals that Rmd9 belongs to the family of pentatricopeptide (PPR) proteins and uses a previously unobserved mode of specific RNA recognition. Rmd9 protects RNA from degradation by the mitochondrial 3'-exoribonuclease complex mtEXO in vitro, indicating that recognition and binding of the dodecamer element by Rmd9 confers stability to yeast mitochondrial mRNAs.
    Keywords:  PAR-CLIP; PPR proteins; gene expression; mitochondria; protein-RNA complex
  13. Front Genet. 2021 ;12 627050
      Gene editing of the mitochondrial genome using the CRISPR-Cas9 system is highly challenging mainly due to sub-efficient delivery of guide RNA and Cas9 enzyme complexes into the mitochondria. In this study, we were able to perform gene editing in the mitochondrial DNA by appending an NADH-ubiquinone oxidoreductase chain 4 (ND4) targeting guide RNA to an RNA transport-derived stem loop element (RP-loop) and expressing the Cas9 enzyme with a preceding mitochondrial localization sequence. We observe mitochondrial colocalization of RP-loop gRNA and a marked reduction of ND4 expression in the cells carrying a 11205G variant in their ND4 sequence coincidently decreasing the mtDNA levels. This proof-of-concept study suggests that a stem-loop element added sgRNA can be transported to the mitochondria and functionally interact with Cas9 to mediate sequence-specific mtDNA cleavage. Using this novel approach to target the mtDNA, our results provide further evidence that CRISPR-Cas9-mediated gene editing might potentially be used to treat mitochondrial-related diseases.
    Keywords:  PNPase; RP-loop; chimeric guide RNA; heteroplasmic mutations; mitochondria