bims-cytox1 2023-01-15 papers

Nucleic Acids Res. 2023 Jan 09. pii: gkac1229. [Epub ahead of print]

DPC29 promotes post-initiation mitochondrial translation in Saccharomyces cerevisiae.

Mitochondrial ribosomes synthesize essential components of the oxidative phosphorylation (OXPHOS) system in a tightly regulated process. In the yeast Saccharomyces cerevisiae, mitochondrial mRNAs require specific translational activators, which orchestrate protein synthesis by recognition of their target gene's 5'-untranslated region (UTR). Most of these yeast genes lack orthologues in mammals, and only one such gene-specific translational activator has been proposed in humans-TACO1. The mechanism by which TACO1 acts is unclear because mammalian mitochondrial mRNAs do not have significant 5'-UTRs, and therefore must promote translation by alternative mechanisms. In this study, we examined the role of the TACO1 orthologue in yeast. We found this 29 kDa protein to be a general mitochondrial translation factor, Dpc29, rather than a COX1-specific translational activator. Its activity was necessary for the optimal expression of OXPHOS mtDNA reporters, and mutations within the mitoribosomal large subunit protein gene MRP7 produced a global reduction of mitochondrial translation in dpc29Δ cells, indicative of a general mitochondrial translation factor. Northern-based mitoribosome profiling of dpc29Δ cells showed higher footprint frequencies at the 3' ends of mRNAs, suggesting a role in translation post-initiation. Additionally, human TACO1 expressed at native levels rescued defects in dpc29Δ yeast strains, suggesting that the two proteins perform highly conserved functions.

DOI: https://doi.org/10.1093/nar/gkac1229

Bio Protoc. 2022 Dec 20. pii: e4578. [Epub ahead of print]12(24):

Monitoring Mitochondrial Protein Import Using Mitochondrial Targeting Sequence (MTS)-eGFP.

Jonas B Michaelis, Süleyman Bozkurt, Jasmin A Schäfer, Christian Münch.

Mitochondria are cellular organelles essential for the function and survival of eukaryotic cells. Nearly all mitochondrial proteins are nuclear-encoded and require mitochondrial import upon their synthesis in the cytosol. Various approaches have been described to study mitochondrial protein import, such as monitoring the entry of radiolabeled proteins into purified mitochondria or quantifying newly synthesized proteins within mitochondria by proteomics. Here, we provide a detailed protocol for a commonly used and straightforward assay that quantitatively examines mitochondrial protein import by monitoring the co-localization of mitochondrially targeted enhanced green fluorescent protein (eGFP) with the mitochondrial fluorescence dye MitoTracker TM Deep Red FM by live cell imaging. We describe the preparation and use of a stable mammalian cell line inducibly expressing a mitochondrial targeting sequence (MTS)-eGFP, followed by quantitative image analysis using an open-source ImageJ-based plugin. This inducible expression system avoids the need for transient transfection while enabling titration of MTS-eGFP expression and thereby avoiding protein folding stress. Overall, the assay provides a simple and robust approach to assess mitochondrial import capacity of cells in various disease-related settings. This protocol was validated in: Mol Cell (2021), DOI: 10.1016/j.molcel.2021.11.004 Graphical abstract.

Keywords: Live cell imaging ; Microscopy ; Mitochondria ; Mitochondrial protein import ; Protein translocation

DOI: https://doi.org/10.21769/BioProtoc.4578

Nucleic Acids Res. 2023 Jan 11. pii: gkac1233. [Epub ahead of print]

Translation initiation of leaderless and polycistronic transcripts in mammalian mitochondria.

Cristina Remes, Anas Khawaja, Sarah F Pearce, Adam M Dinan, Shreekara Gopalakrishna, Miriam Cipullo, Vasileios Kyriakidis, Jingdian Zhang, Xaquin Castro Dopico, Olessya Yukhnovets, Ilian Atanassov, Andrew E Firth, Barry Cooperman, Joanna Rorbach.

The synthesis of mitochondrial OXPHOS complexes is central to cellular metabolism, yet many molecular details of mitochondrial translation remain elusive. It has been commonly held view that translation initiation in human mitochondria proceeded in a manner similar to bacterial systems, with the mitoribosomal small subunit bound to the initiation factors, mtIF2 and mtIF3, along with initiator tRNA and an mRNA. However, unlike in bacteria, most human mitochondrial mRNAs lack 5' leader sequences that can mediate small subunit binding, raising the question of how leaderless mRNAs are recognized by mitoribosomes. By using novel in vitro mitochondrial translation initiation assays, alongside biochemical and genetic characterization of cellular knockouts of mitochondrial translation factors, we describe unique features of translation initiation in human mitochondria. We show that in vitro, leaderless mRNA transcripts can be loaded directly onto assembled 55S mitoribosomes, but not onto the mitoribosomal small subunit (28S), in a manner that requires initiator fMet-tRNAMet binding. In addition, we demonstrate that in human cells and in vitro, mtIF3 activity is not required for translation of leaderless mitochondrial transcripts but is essential for translation of ATP6 in the case of the bicistronic ATP8/ATP6 transcript. Furthermore, we show that mtIF2 is indispensable for mitochondrial protein synthesis. Our results demonstrate an important evolutionary divergence of the mitochondrial translation system and further our fundamental understanding of a process central to eukaryotic metabolism.

DOI: https://doi.org/10.1093/nar/gkac1233

Mol Genet Metab Rep. 2023 Mar;34 100951

Total and reduced/oxidized forms of coenzyme Q10 in fibroblasts of patients with mitochondrial disease.

Chika Watanabe, Hitoshi Osaka, Miyuki Watanabe, Akihiko Miyauchi, Eriko F Jimbo, Takeshi Tokuyama, Hideki Uosaki, Yoshihito Kishita, Yasushi Okazaki, Takanori Onuki, Tomohiro Ebihara, Kenichi Aizawa, Kei Murayama, Akira Ohtake, Takanori Yamagata.

Coenzyme Q10 (CoQ10) is involved in ATP production through electron transfer in the mitochondrial respiratory chain complex. CoQ10 receives electrons from respiratory chain complex I and II to become the reduced form, and then transfers electrons at complex III to become the oxidized form. The redox state of CoQ10 has been reported to be a marker of the mitochondrial metabolic state, but to our knowledge, no reports have focused on the individual quantification of reduced and oxidized CoQ10 or the ratio of reduced to total CoQ10 (reduced/total CoQ10) in patients with mitochondrial diseases. We measured reduced and oxidized CoQ10 in skin fibroblasts from 24 mitochondrial disease patients, including 5 primary CoQ10 deficiency patients and 10 respiratory chain complex deficiency patients, and determined the reduced/total CoQ10 ratio. In primary CoQ10 deficiency patients, total CoQ10 levels were significantly decreased, however, the reduced/total CoQ10 ratio was not changed. On the other hand, in mitochondrial disease patients other than primary CoQ10 deficiency patients, total CoQ10 levels did not decrease. However, the reduced/total CoQ10 ratio in patients with respiratory chain complex IV and V deficiency was higher in comparison to those with respiratory chain complex I deficiency. Measurement of CoQ10 in fibroblasts proved useful for the diagnosis of primary CoQ10 deficiency. In addition, the reduced/total CoQ10 ratio may reflect the metabolic status of mitochondrial disease.

Keywords: Coenzyme Q10; Forward electron transport; Mitochondrial disease; Primary coenzyme Q10 deficiency; Reduced/total CoQ10; Reverse electron transport

DOI: https://doi.org/10.1016/j.ymgmr.2022.100951

Commun Biol. 2023 Jan 12. 6(1): 22

OxPhos defects cause hypermetabolism and reduce lifespan in cells and in patients with mitochondrial diseases.

Patients with primary mitochondrial oxidative phosphorylation (OxPhos) defects present with fatigue and multi-system disorders, are often lean, and die prematurely, but the mechanistic basis for this clinical picture remains unclear. By integrating data from 17 cohorts of patients with mitochondrial diseases (n = 690) we find evidence that these disorders increase resting energy expenditure, a state termed hypermetabolism. We examine this phenomenon longitudinally in patient-derived fibroblasts from multiple donors. Genetically or pharmacologically disrupting OxPhos approximately doubles cellular energy expenditure. This cell-autonomous state of hypermetabolism occurs despite near-normal OxPhos coupling efficiency, excluding uncoupling as a general mechanism. Instead, hypermetabolism is associated with mitochondrial DNA instability, activation of the integrated stress response (ISR), and increased extracellular secretion of age-related cytokines and metabokines including GDF15. In parallel, OxPhos defects accelerate telomere erosion and epigenetic aging per cell division, consistent with evidence that excess energy expenditure accelerates biological aging. To explore potential mechanisms for these effects, we generate a longitudinal RNASeq and DNA methylation resource dataset, which reveals conserved, energetically demanding, genome-wide recalibrations. Taken together, these findings highlight the need to understand how OxPhos defects influence the energetic cost of living, and the link between hypermetabolism and aging in cells and patients with mitochondrial diseases.

DOI: https://doi.org/10.1038/s42003-022-04303-x