Distinct Roles of Mitochondrial HIGD1A and HIGD2A in Respiratory Complex and Supercomplex Biogenesis

Timón‐Gómez, Alba; Garlich, Joshua; Stuart, Rosemary A.; Ugalde, Cristina; Barrientos, Antoni

doi:10.1016/j.celrep.2020.107607

Cited by 56 publications

(89 citation statements)

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“…Note: For human HEK293T cell lines, a protein/detergent ratio between 1:1–2 of DDM ( Figure 2B ) or 1:2–4 of high-purity digitonin ( Figures 2A – 2C and ( Lobo-Jarne et al, 2018 ; Timón-Gómez et al, 2020 )), and 1:1–3 of LMNG is recommended ( Figure 2A ). In Saccharomyces cerevisiae mitochondria, a protein/detergent ratio of 1:1–3 for DDM or digitonin is suggested ( Figure 4B ).…”

Section: Step-by-step Methods Detailsmentioning

confidence: 99%

“…Mitochondria purified from wild-type, and HIGD1A -KO HEK293T cells were extracted with digitonin (protein/detergent ratio of 1:2) and analyzed by BN-PAGE and immunoblotting with the indicated antibodies. Replicates were generated for the quantification and analysis of Figures 1G and S3E in Timón-Gómez et al (2020) .…”

Section: Figurementioning

confidence: 99%

“…Here, we present a detailed protocol of BN-PAGE to obtain robust and reproducible results. For complete details on the use and execution of this protocol, please refer to Lobo-Jarne et al (2018) and Timón-Gómez et al (2020) .…”

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Protocol for the Analysis of Yeast and Human Mitochondrial Respiratory Chain Complexes and Supercomplexes by Blue Native Electrophoresis

et al. 2020

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SUMMARY By using negatively charged Coomassie brilliant blue G-250 dye to induce a charge shift on proteins, blue native polyacrylamide gel electrophoresis (BN-PAGE) allows resolution of enzymatically active multiprotein complexes extracted from cellular or subcellular lysates while retaining their native conformation. BN-PAGE was first developed to analyze the size, composition, and relative abundance of the complexes and supercomplexes that form the mitochondrial respiratory chain and OXPHOS system. Here, we present a detailed protocol of BN-PAGE to obtain robust and reproducible results. For complete details on the use and execution of this protocol, please refer to Lobo-Jarne et al. (2018) and Timón-Gómez et al. (2020) .

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Section: Step-by-step Methods Detailsmentioning

confidence: 99%

Section: Figurementioning

confidence: 99%

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Protocol for the Analysis of Yeast and Human Mitochondrial Respiratory Chain Complexes and Supercomplexes by Blue Native Electrophoresis

et al. 2020

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“…A myriad of assembly factors is necessary for the synthesis, translocation, stabilization and incorporation of the metal groups in both MT-CO1 and MT-CO2 [187]. Other assembly factors such as PET100, PET117 and MR-1S work on the middle stages of assembly [186] and only one, HIGD2A, is known to promote the incorporation of the MT-CO3 module in the final steps of cIV assembly [188,189].…”

Section: Accepted Articlementioning

confidence: 99%

Mitochondrial disorders of the OXPHOS system

2020

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Mitochondrial disorders are amongst the most frequent inborn errors of metabolism, their primary cause being the dysfunction of the oxidative phosphorylation system (OXPHOS). OXPHOS is composed of the electron transport chain (ETC), formed by four multimeric enzymes and two mobile electron carriers, plus an ATP synthase (also called complex V). The ETC performs the redox reactions involved in cellular respiration while generating the proton motive force used by complex V to synthesize ATP. OXPHOS biogenesis involves multiple steps, starting from the expression of genes encoded in physically separated genomes, namely the mitochondrial and nuclear DNA, to the coordinated assembly of components and cofactors building each individual complex and eventually the supercomplexes. The genetic cause underlying around half of the diagnosed mitochondrial disease cases is currently known. Many of these cases result from pathogenic variants in genes encoding structural subunits or additional factors directly involved in the assembly of the ETC complexes. Here we review the historical and most recent findings concerning the clinical phenotypes and the molecular pathological mechanisms underlying this particular group of disorders.

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“…However, our understanding of how the SCs are assembled, how they are regulated and what their physiological function in health and disease is remains limited (reviewed in [ 10 , 11 , 12 , 13 ]). Various factors described to aid in SCs formation include: Rcf1 and Rcf2 [ 14 , 15 , 16 ]; COX7A2L [ 17 , 18 , 19 , 20 ]; Cardiolipin [ 21 ]; HIGD2A [ 16 , 22 , 23 ]; MCJ/DnaJC15 [ 24 ]; and prohibitins [ 25 ]. In addition, mitochondrial dynamics and cristae morphology also have been reported to play a role in SC assembly [ 26 , 27 , 28 ].…”

Section: Introductionmentioning

confidence: 99%

Hypoxia Promotes Mitochondrial Complex I Abundance via HIF-1α in Complex III and Complex IV Deficient Cells

Saldana-Caboverde

Nissanka

Garcia

et al. 2020

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Murine fibroblasts deficient in mitochondria respiratory complexes III (CIII) and IV (CIV) produced by either the ablation of Uqcrfs1 (encoding for Rieske iron sulfur protein, RISP) or Cox10 (encoding for protoheme IX farnesyltransferase, COX10) genes, respectively, showed a pleiotropic effect in complex I (CI). Exposure to 1–5% oxygen increased the levels of CI in both RISP and COX10 KO fibroblasts. De novo assembly of the respiratory complexes occurred at a faster rate and to higher levels in 1% oxygen compared to normoxia in both RISP and COX10 KO fibroblasts. Hypoxia did not affect the levels of assembly of CIII in the COX10 KO fibroblasts nor abrogated the genetic defect impairing CIV assembly. Mitochondrial signaling involving reactive oxygen species (ROS) has been implicated as necessary for HIF-1α stabilization in hypoxia. We did not observe increased ROS production in hypoxia. Exposure to low oxygen levels stabilized HIF-1α and increased CI levels in RISP and COX10 KO fibroblasts. Knockdown of HIF-1α during hypoxic conditions abrogated the beneficial effect of hypoxia on the stability/assembly of CI. These findings demonstrate that oxygen and HIF-1α regulate the assembly of respiratory complexes.

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Distinct Roles of Mitochondrial HIGD1A and HIGD2A in Respiratory Complex and Supercomplex Biogenesis

Abstract: Highlights d HIGD1A promotes the incorporation of UQCRFS1 to mature CIII 2 d HIGD2A facilitates the assembly of the COX3 module to form holo-CIV d HIGD2A regulates modular assembly of isolated and supercomplexed CIV d HIGD1A and HIGD2A play overlapping functions in CIV assembly

Cited by 56 publications

References 57 publications

Protocol for the Analysis of Yeast and Human Mitochondrial Respiratory Chain Complexes and Supercomplexes by Blue Native Electrophoresis

Protocol for the Analysis of Yeast and Human Mitochondrial Respiratory Chain Complexes and Supercomplexes by Blue Native Electrophoresis

Mitochondrial disorders of the OXPHOS system

Hypoxia Promotes Mitochondrial Complex I Abundance via HIF-1α in Complex III and Complex IV Deficient Cells

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