The human mitochondrial translation factor TACO1 alleviates mitoribosome stalling at polyproline stretches.

The prokaryotic translation elongation factor P (EF-P) and the eukaryotic/archaeal counterparts eIF5A/aIF5A are proteins that serve a crucial role in mitigating ribosomal stalling during the translation of specific sequences, notably those containing consecutive proline residues (1,2). Although mitochondrial DNA-encoded proteins synthesized by mitochondrial ribosomes also contain polyproline stretches, an EF-P/eIF5A mitochondrial counterpart remains unidentified. Here, we show that the missing factor is TACO1, a protein causative of a juvenile form of neurodegenerative Leigh's syndrome associated with cytochrome c oxidase deficiency, until now believed to be a translational activator of COX1 mRNA. By using a combination of metabolic labeling, puromycin release and mitoribosome profiling experiments, we show that TACO1 is required for the rapid synthesis of the polyproline-rich COX1 and COX3 cytochrome c oxidase subunits, while its requirement is negligible for other mitochondrial DNA-encoded proteins. In agreement with a role in translation efficiency regulation, we show that TACO1 cooperates with the N-terminal extension of the large ribosomal subunit bL27m to provide stability to the peptidyl-transferase center during elongation. This study illuminates the translation elongation dynamics within human mitochondria, a TACO1-mediated biological mechanism in place to mitigate mitoribosome stalling at polyproline stretches during protein synthesis, and the pathological implications of its malfunction.

Emerging mechanisms in the redox regulation of mitochondrial cytochrome c oxidase assembly and function.

In eukaryotic cells, mitochondria perform cellular respiration through a series of redox reactions ultimately reducing molecular oxygen to water. The system responsible for this process is the respiratory chain or electron transport system (ETS) composed of complexes I-IV. Due to its function, the ETS is the main source of reactive oxygen species (ROS), generating them on both sides of the mitochondrial inner membrane, i.e. the intermembrane space (IMS) and the matrix. A correct balance between ROS generation and scavenging is important for keeping the cellular redox homeostasis and other important aspects of cellular physiology. However, ROS generated in the mitochondria are important signaling molecules regulating mitochondrial biogenesis and function. The IMS contains a large number of redox sensing proteins, containing specific Cys-rich domains, that are involved in ETS complex biogenesis. The large majority of these proteins function as cytochrome c oxidase (COX) assembly factors, mainly for the handling of copper ions necessary for the formation of the redox reactive catalytic centers. A particular case of ROS-regulated COX assembly factor is COA8, whose intramitochondrial levels are increased by oxidative stress, promoting COX assembly and/or protecting the enzyme from oxidative damage. In this review, we will discuss the current knowledge concerning the role played by ROS in regulating mitochondrial activity and biogenesis, focusing on the COX enzyme and with a special emphasis on the functional role exerted by the redox sensitive Cys residues contained in the COX assembly factors.

CG7630 is the Drosophila melanogaster homolog of the cytochrome c oxidase subunit COX7B.

The mitochondrial respiratory chain (MRC) is composed of four multiheteromeric enzyme complexes. According to the endosymbiotic origin of mitochondria, eukaryotic MRC derives from ancestral proteobacterial respiratory structures consisting of a minimal set of complexes formed by a few subunits associated with redox prosthetic groups. These enzymes, which are the "core" redox centers of respiration, acquired additional subunits, and increased their complexity throughout evolution. Cytochrome c oxidase (COX), the terminal component of MRC, has a highly interspecific heterogeneous composition. Mammalian COX consists of 14 different polypeptides, of which COX7B is considered the evolutionarily youngest subunit. We applied proteomic, biochemical, and genetic approaches to investigate the COX composition in the invertebrate model Drosophila melanogaster. We identified and characterized a novel subunit which is widely different in amino acid sequence, but similar in secondary and tertiary structures to COX7B, and provided evidence that this object is in fact replacing the latter subunit in virtually all protostome invertebrates. These results demonstrate that although individual structures may differ the composition of COX is functionally conserved between vertebrate and invertebrate species.

Mutation in the MICOS subunit gene APOO (MIC26) associated with an X-linked recessive mitochondrial myopathy, lactic acidosis, cognitive impairment and autistic features.

BACKGROUND: Mitochondria provide ATP through the process of oxidative phosphorylation, physically located in the inner mitochondrial membrane (IMM). The mitochondrial contact site and organising system (MICOS) complex is known as the 'mitoskeleton' due to its role in maintaining IMM architecture. APOO encodes MIC26, a component of MICOS, whose exact function in its maintenance or assembly has still not been completely elucidated. METHODS: We have studied a family in which the most affected subject presented progressive developmental delay, lactic acidosis, muscle weakness, hypotonia, weight loss, gastrointestinal and body temperature dysautonomia, repetitive infections, cognitive impairment and autistic behaviour. Other family members showed variable phenotype presentation. Whole exome sequencing was used to screen for pathological variants. Patient-derived skin fibroblasts were used to confirm the pathogenicity of the variant found in APOO. Knockout models in Drosophila melanogaster and Saccharomyces cerevisiae were employed to validate MIC26 involvement in MICOS assembly and mitochondrial function. RESULTS: A likely pathogenic c.350T>C transition was found in APOO predicting an I117T substitution in MIC26. The mutation caused impaired processing of the protein during import and faulty insertion into the IMM. This was associated with altered MICOS assembly and cristae junction disruption. The corresponding mutation in MIC26 or complete loss was associated with mitochondrial structural and functional deficiencies in yeast and D. melanogaster models. CONCLUSION: This is the first case of pathogenic mutation in APOO, causing altered MICOS assembly and neuromuscular impairment. MIC26 is involved in the assembly or stability of MICOS in humans, yeast and flies.

Mitochondrial ribosome biogenesis and redox sensing.

Mitoribosome biogenesis is a complex process involving RNA elements encoded in the mitochondrial genome and mitoribosomal proteins typically encoded in the nuclear genome. This process is orchestrated by extra-ribosomal proteins, nucleus-encoded assembly factors, which play roles across all assembly stages to coordinate ribosomal RNA processing and maturation with the sequential association of ribosomal proteins. Both biochemical studies and recent cryo-EM structures of mammalian mitoribosomes have provided insights into their assembly process. In this article, we will briefly outline the current understanding of mammalian mitoribosome biogenesis pathways and the factors involved. Special attention is devoted to the recent identification of iron-sulfur clusters as structural components of the mitoribosome and a small subunit assembly factor, the existence of redox-sensitive cysteines in mitoribosome proteins and assembly factors, and the role they may play as redox sensor units to regulate mitochondrial translation under stress.

The human mitochondrial mRNA structurome reveals mechanisms of gene expression.

The human mitochondrial genome encodes crucial oxidative phosphorylation system proteins, pivotal for aerobic energy transduction. They are translated from nine monocistronic and two bicistronic transcripts whose native structures remain unexplored, posing a gap in understanding mitochondrial gene expression. In this work, we devised the mitochondrial dimethyl sulfate mutational profiling with sequencing (mitoDMS-MaPseq) method and applied detection of RNA folding ensembles using expectation-maximization (DREEM) clustering to unravel the native mitochondrial messenger RNA (mt-mRNA) structurome in wild-type (WT) and leucine-rich pentatricopeptide repeat-containing protein (LRPPRC)-deficient cells. Our findings elucidate LRPPRC's role as a holdase contributing to maintaining mt-mRNA folding and efficient translation. mt-mRNA structural insights in WT mitochondria, coupled with metabolic labeling, unveil potential mRNA-programmed translational pausing and a distinct programmed ribosomal frameshifting mechanism. Our data define a critical layer of mitochondrial gene expression regulation. These mt-mRNA folding maps provide a reference for studying mt-mRNA structures in diverse physiological and pathological contexts.

Mitochondrial Neurodegeneration: Lessons from Drosophila melanogaster Models.

The fruit fly-i.e., Drosophila melanogaster-has proven to be a very useful model for the understanding of basic physiological processes, such as development or ageing. The availability of straightforward genetic tools that can be used to produce engineered individuals makes this model extremely interesting for the understanding of the mechanisms underlying genetic diseases in physiological models. Mitochondrial diseases are a group of yet-incurable genetic disorders characterized by the malfunction of the oxidative phosphorylation system (OXPHOS), which is the highly conserved energy transformation system present in mitochondria. The generation of D. melanogaster models of mitochondrial disease started relatively recently but has already provided relevant information about the molecular mechanisms and pathological consequences of mitochondrial dysfunction. Here, we provide an overview of such models and highlight the relevance of D. melanogaster as a model to study mitochondrial disorders.

The human mitochondrial mRNA structurome reveals mechanisms of gene expression.

The mammalian mitochondrial genome encodes thirteen oxidative phosphorylation system proteins, crucial in aerobic energy transduction. These proteins are translated from 9 monocistronic and 2 bicistronic transcripts, whose native structures remain unexplored, leaving fundamental molecular determinants of mitochondrial gene expression unknown. To address this gap, we developed a mitoDMS-MaPseq approach and used DREEM clustering to resolve the native human mitochondrial mt-mRNA structurome. We gained insights into mt-mRNA biology and translation regulatory mechanisms, including a unique programmed ribosomal frameshifting for the ATP8/ATP6 transcript. Furthermore, absence of the mt-mRNA maintenance factor LRPPRC led to a mitochondrial transcriptome structured differently, with specific mRNA regions exhibiting increased or decreased structuredness. This highlights the role of LRPPRC in maintaining mRNA folding to promote mt-mRNA stabilization and efficient translation. In conclusion, our mt-mRNA folding maps reveal novel mitochondrial gene expression mechanisms, serving as a detailed reference and tool for studying them in different physiological and pathological contexts.

Structural rather than catalytic role for mitochondrial respiratory chain supercomplexes.

Mammalian mitochondrial respiratory chain (MRC) complexes are able to associate into quaternary structures named supercomplexes (SCs), which normally coexist with non-bound individual complexes. The functional significance of SCs has not been fully clarified and the debate has been centered on whether or not they confer catalytic advantages compared with the non-bound individual complexes. Mitochondrial respiratory chain organization does not seem to be conserved in all organisms. In fact, and differently from mammalian species, mitochondria from Drosophila melanogaster tissues are characterized by low amounts of SCs, despite the high metabolic demands and MRC activity shown by these mitochondria. Here, we show that attenuating the biogenesis of individual respiratory chain complexes was accompanied by increased formation of stable SCs, which are missing in Drosophila melanogaster in physiological conditions. This phenomenon was not accompanied by an increase in mitochondrial respiratory activity. Therefore, we conclude that SC formation is necessary to stabilize the complexes in suboptimal biogenesis conditions, but not for the enhancement of respiratory chain catalysis.

Drosophila Mpv17 forms an ion channel and regulates energy metabolism.

Mutations in MPV17 are a major contributor to mitochondrial DNA (mtDNA) depletion syndromes, a group of inherited genetic conditions due to mtDNA instability. To investigate the role of MPV17 in mtDNA maintenance, we generated and characterized a Drosophila melanogaster Mpv17 (dMpv17) KO model showing that the absence of dMpv17 caused profound mtDNA depletion in the fat body but not in other tissues, increased glycolytic flux and reduced lifespan in starvation. Accordingly, the expression of key genes of glycogenolysis and glycolysis was upregulated in dMpv17 KO flies. In addition, we demonstrated that dMpv17 formed a channel in planar lipid bilayers at physiological ionic conditions, and its electrophysiological hallmarks were affected by pathological mutations. Importantly, the reconstituted channel translocated uridine but not orotate across the membrane. Our results indicate that dMpv17 forms a channel involved in translocation of key metabolites and highlight the importance of dMpv17 in energy homeostasis and mitochondrial function.