The functional significance of mitochondrial respiratory chain supercomplexes.

The mitochondrial respiratory chain (MRC) is a key energy transducer in eukaryotic cells. Four respiratory chain complexes cooperate in the transfer of electrons derived from various metabolic pathways to molecular oxygen, thereby establishing an electrochemical gradient over the inner mitochondrial membrane that powers ATP synthesis. This electron transport relies on mobile electron carries that functionally connect the complexes. While the individual complexes can operate independently, they are in situ organized into large assemblies termed respiratory supercomplexes. Recent structural and functional studies have provided some answers to the question of whether the supercomplex organization confers an advantage for cellular energy conversion. However, the jury is still out, regarding the universality of these claims. In this review, we discuss the current knowledge on the functional significance of MRC supercomplexes, highlight experimental limitations, and suggest potential new strategies to overcome these obstacles.

Multiple pathways coordinate assembly of human mitochondrial complex IV and stabilization of respiratory supercomplexes.

Mitochondrial respiratory chain complexes I, III, and IV can associate into larger structures termed supercomplexes or respirasomes, thereby generating structural interdependences among the individual complexes yet to be understood. In patients, nonsense mutations in complex IV subunit genes cause severe encephalomyopathies randomly associated with pleiotropic complex I defects. Using complexome profiling and biochemical analyses, we have explored the structural rearrangements of the respiratory chain in human cell lines depleted of the catalytic complex IV subunit COX1 or COX2. In the absence of a functional complex IV holoenzyme, several supercomplex I+III2 species coexist, which differ in their content of COX subunits and COX7A2L/HIGD2A assembly factors. The incorporation of an atypical COX1-HIGD2A submodule attenuates supercomplex I+III2 turnover rate, indicating an unexpected molecular adaptation for supercomplexes stabilization that relies on the presence of COX1 independently of holo-complex IV formation. Our data set the basis for complex I structural dependence on complex IV, revealing the co-existence of alternative pathways for the biogenesis of "supercomplex-associated" versus individual complex IV, which could determine physiological adaptations under different stress and disease scenarios.

Mitochondrial respiratory supercomplexes of the yeast Saccharomyces cerevisiae.

The functional and structural relationship among the individual components of the mitochondrial respiratory chain constitutes a central aspect of our understanding of aerobic catabolism. This interplay has been a subject of intense debate for over 50 years. It is well established that individual respiratory enzymes associate into higher-order structures known as respiratory supercomplexes, which represent the evolutionarily conserved organizing principle of the mitochondrial respiratory chain. In the yeast Saccharomyces cerevisiae, supercomplexes are formed by a complex III homodimer flanked by one or two complex IV monomers, and their high-resolution structures have been recently elucidated. Despite the wealth of structural information, several proposed supercomplex functions remain speculative and our understanding of their physiological relevance is still limited. Recent advances in the field were made possible by the construction of yeast strains where the association of complex III and IV into supercomplexes is impeded, leading to diminished respiratory capacity and compromised cellular competitive fitness. Here, we discuss the experimental evidence and hypotheses relative to the functional roles of yeast respiratory supercomplexes. Moreover, we review the current models of yeast complex III and IV assembly in the context of supercomplex formation and highlight the data scattered throughout the literature suggesting the existence of cross talk between their biogenetic processes.

APOL1 risk variants affect podocyte lipid homeostasis and energy production in focal segmental glomerulosclerosis.

Lipotoxicity was recently reported in several forms of kidney disease, including focal segmental glomerulosclerosis (FSGS). Susceptibility to FSGS in African Americans is associated with the presence of genetic variants of the Apolipoprotein L1 gene (APOL1) named G1 and G2. If and how endogenous APOL1 may alter mitochondrial function by the modifying cellular lipid metabolism is unknown. Using transgenic mice expressing the APOL1 variants (G0, G1 or G2) under endogenous promoter, we show that APOL1 risk variant expression in transgenic mice does not impair kidney function at baseline. However, APOL1 G1 expression worsens proteinuria and kidney function in mice characterized by the podocyte inducible expression of nuclear factor of activated T-cells (NFAT), which we have found to cause FSGS. APOL1 G1 expression in this FSGS-model also results in increased triglyceride and cholesterol ester contents in kidney cortices, where lipid accumulation correlated with loss of renal function. In vitro, we show that the expression of endogenous APOL1 G1/G2 in human urinary podocytes is associated with increased cellular triglyceride content and is accompanied by mitochondrial dysfunction in the presence of compensatory oxidative phosphorylation (OXPHOS) complexes elevation. Our findings indicate that APOL1 risk variant expression increases the susceptibility to lipid-dependent podocyte injury, ultimately leading to mitochondrial dysfunction.

Respiratory supercomplexes enhance electron transport by decreasing cytochrome c diffusion distance.

Respiratory chains are crucial for cellular energy conversion and consist of multi-subunit complexes that can assemble into supercomplexes. These structures have been intensively characterized in various organisms, but their physiological roles remain unclear. Here, we elucidate their function by leveraging a high-resolution structural model of yeast respiratory supercomplexes that allowed us to inhibit supercomplex formation by mutation of key residues in the interaction interface. Analyses of a mutant defective in supercomplex formation, which still contains fully functional individual complexes, show that the lack of supercomplex assembly delays the diffusion of cytochrome c between the separated complexes, thus reducing electron transfer efficiency. Consequently, competitive cellular fitness is severely reduced in the absence of supercomplex formation and can be restored by overexpression of cytochrome c. In sum, our results establish how respiratory supercomplexes increase the efficiency of cellular energy conversion, thereby providing an evolutionary advantage for aerobic organisms.

The translational activator Sov1 coordinates mitochondrial gene expression with mitoribosome biogenesis.

Mitoribosome biogenesis is an expensive metabolic process that is essential to maintain cellular respiratory capacity and requires the stoichiometric accumulation of rRNAs and proteins encoded in two distinct genomes. In yeast, the ribosomal protein Var1, alias uS3m, is mitochondrion-encoded. uS3m is a protein universally present in all ribosomes, where it forms part of the small subunit (SSU) mRNA entry channel and plays a pivotal role in ribosome loading onto the mRNA. However, despite its critical functional role, very little is known concerning VAR1 gene expression. Here, we demonstrate that the protein Sov1 is an in bona fide VAR1 mRNA translational activator and additionally interacts with newly synthesized Var1 polypeptide. Moreover, we show that Sov1 assists the late steps of mtSSU biogenesis involving the incorporation of Var1, an event necessary for uS14 and mS46 assembly. Notably, we have uncovered a translational regulatory mechanism by which Sov1 fine-tunes Var1 synthesis with its assembly into the mitoribosome.

The mitoribosome-specific protein mS38 is preferentially required for synthesis of cytochrome c oxidase subunits.

Message-specific translational regulation mechanisms shape the biogenesis of multimeric oxidative phosphorylation (OXPHOS) enzyme in mitochondria from the yeast Saccharomyces cerevisiae. These mechanisms, driven mainly by the action of mRNA-specific translational activators, help to coordinate synthesis of OXPHOS catalytic subunits by the mitoribosomes with both the import of their nucleus-encoded partners and their assembly to form the holocomplexes. However, little is known regarding the role that the mitoribosome itself may play in mRNA-specific translational regulation. Here, we show that the mitoribosome small subunit protein Cox24/mS38, known to be necessary for mitoribosome-specific intersubunit bridge formation and 15S rRNA H44 stabilization, is required for efficient mitoribogenesis. Consequently, mS38 is necessary to sustain the overall mitochondrial protein synthesis rate, despite an adaptive ∼2-fold increase in mitoribosome abundance in mS38-deleted cells. Additionally, the absence of mS38 preferentially disturbs translation initiation of COX1, COX2, and COX3 mRNAs, without affecting the levels of mRNA-specific translational activators. We propose that mS38 confers the mitochondrial ribosome an intrinsic capacity of translational regulation, probably acquired during evolution from bacterial ribosomes to facilitate the translation of mitochondrial mRNAs, which lack typical anti-Shine-Dalgarno sequences.

The DEAD-box helicase Mss116 plays distinct roles in mitochondrial ribogenesis and mRNA-specific translation.

Members of the DEAD-box family are often multifunctional proteins involved in several RNA transactions. Among them, yeast Saccharomyces cerevisiae Mss116 participates in mitochondrial intron splicing and, under cold stress, also in mitochondrial transcription elongation. Here, we show that Mss116 interacts with the mitoribosome assembly factor Mrh4, is required for efficient mitoribosome biogenesis, and consequently, maintenance of the overall mitochondrial protein synthesis rate. Additionally, Mss116 is required for efficient COX1 mRNA translation initiation and elongation. Mss116 interacts with a COX1 mRNA-specific translational activator, the pentatricopeptide repeat protein Pet309. In the absence of Mss116, Pet309 is virtually absent, and although mitoribosome loading onto COX1 mRNA can occur, activation of COX1 mRNA translation is impaired. Mutations abolishing the helicase activity of Mss116 do not prevent the interaction of Mss116 with Pet309 but also do not allow COX1 mRNA translation. We propose that Pet309 acts as an adaptor protein for Mss116 action on the COX1 mRNA 5΄-UTR to promote efficient Cox1 synthesis. Overall, we conclude that the different functions of Mss116 in the biogenesis and functioning of the mitochondrial translation machinery depend on Mss116 interplay with its protein cofactors.

Insights into the genotype-phenotype correlation and molecular function of SLC25A46.

Recessive SLC25A46 mutations cause a spectrum of neurodegenerative disorders with optic atrophy as a core feature. We report a patient with optic atrophy, peripheral neuropathy, ataxia, but not cerebellar atrophy, who is on the mildest end of the phenotypic spectrum. By studying seven different nontruncating mutations, we found that the stability of the SLC25A46 protein inversely correlates with the severity of the disease and the patient's variant does not markedly destabilize the protein. SLC25A46 belongs to the mitochondrial transporter family, but it is not known to have transport function. Apart from this possible function, SLC25A46 forms molecular complexes with proteins involved in mitochondrial dynamics and cristae remodeling. We demonstrate that the patient's mutation directly affects the SLC25A46 interaction with MIC60. Furthermore, we mapped all of the reported substitutions in the protein onto a 3D model and found that half of them fall outside of the signature carrier motifs associated with transport function. We thus suggest that there are two distinct molecular mechanisms in SLC25A46-associated pathogenesis, one that destabilizes the protein while the other alters the molecular interactions of the protein. These results have the potential to inform clinical prognosis of such patients and indicate a pathway to drug target development.

Elongator-dependent modification of cytoplasmic tRNALysUUU is required for mitochondrial function under stress conditions.

To gain a wider view of the pathways that regulate mitochondrial function, we combined the effect of heat stress on respiratory capacity with the discovery potential of a genome-wide screen in Saccharomyces cerevisiae. We identified 105 new genes whose deletion impairs respiratory growth at 37°C by interfering with processes such as transcriptional regulation, ubiquitination and cytosolic tRNA wobble uridine modification via 5-methoxycarbonylmethyl-2-thiouridine formation. The latter process, specifically required for efficient decoding of AA-ending codons under stress conditions, was covered by multiple genes belonging to the Elongator (e.g. ELP3) and urmylation (e.g., NCS6) pathways. ELP3 or NCS6 deletants had impaired mitochondrial protein synthesis. Their respiratory deficiency was selectively rescued by overexpression of tRNA(Lys) UUU as well by overexpression of genes (BCK1 and HFM1) with a strong bias for the AAA codon read by this tRNA. These data extend the mitochondrial regulome, demonstrate that heat stress can impair respiration by disturbing cytoplasmic translation of proteins critically involved in mitochondrial function and document, for the first time, the involvement in such process of the Elongator and urmylation pathways. Given the conservation of these pathways, the present findings may pave the way to a better understanding of the human mitochondrial regulome in health and disease.

hCOA3 stabilizes cytochrome c oxidase 1 (COX1) and promotes cytochrome c oxidase assembly in human mitochondria.

Cytochrome c oxidase (COX) or complex IV of the mitochondrial respiratory chain plays a fundamental role in energy production of aerobic cells. In humans, COX deficiency is the most frequent cause of mitochondrial encephalomyopathies. Human COX is composed of 13 subunits of dual genetic origin, whose assembly requires an increasing number of nuclear-encoded accessory proteins known as assembly factors. Here, we have identified and characterized human CCDC56, an 11.7-kDa mitochondrial transmembrane protein, as a new factor essential for COX biogenesis. CCDC56 shares sequence similarity with the yeast COX assembly factor Coa3 and was termed hCOA3. hCOA3-silenced cells display a severe COX functional alteration owing to a decreased stability of newly synthesized COX1 and an impairment in the holoenzyme assembly process. We show that hCOA3 physically interacts with both the mitochondrial translation machinery and COX structural subunits. We conclude that hCOA3 stabilizes COX1 co-translationally and promotes its assembly with COX partner subunits. Finally, our results identify hCOA3 as a new candidate when screening for genes responsible for mitochondrial diseases associated with COX deficiency.

Defects in mitochondrial fatty acid synthesis result in failure of multiple aspects of mitochondrial biogenesis in Saccharomyces cerevisiae.

Mitochondrial fatty acid synthesis (mtFAS) shares acetyl-CoA with the Krebs cycle as a common substrate and is required for the production of octanoic acid (C8) precursors of lipoic acid (LA) in mitochondria. MtFAS is a conserved pathway essential for respiration. In a genetic screen in Saccharomyces cerevisiae designed to further elucidate the physiological role of mtFAS, we isolated mutants with defects in mitochondrial post-translational gene expression processes, indicating a novel link to mitochondrial gene expression and respiratory chain biogenesis. In our ensuing analysis, we show that mtFAS, but not lipoylation per se, is required for respiratory competence. We demonstrate that mtFAS is required for mRNA splicing, mitochondrial translation and respiratory complex assembly, and provide evidence that not LA per se, but fatty acids longer than C8 play a role in these processes. We also show that mtFAS- and LA-deficient strains suffer from a mild haem deficiency that may contribute to the respiratory complex assembly defect. Based on our data and previously published information, we propose a model implicating mtFAS as a sensor for mitochondrial acetyl-CoA availability and a co-ordinator of nuclear and mitochondrial gene expression by adapting the mitochondrial compartment to changes in the metabolic status of the cell.

Metabolic Analysis and Renal Protective Effects of Linagliptin and Empagliflozin in Alport Syndrome.

BACKGROUND: We previously demonstrated that Empagliflozin (Empa), a sodium-glucose cotransporter-2 (SGLT2) inhibitor, reduces intrarenal lipid accumulation and slows kidney function decline in experimental Alport syndrome (AS). In this study, we aimed to evaluate the renal protective benefits of Linagliptin (Lina), a dipeptidyl peptidase-4 (DPP4) inhibitor in AS and compare it to Empa. METHODS: Metabolite distribution in kidney cortices was assessed using mass spectrometry imaging. We examined albuminuria and histological changes in kidneys from AS mice treated with Lina and/or Empa or vehicle. RESULTS: Several metabolites, including adrenic acid (AdA) and glucose, were increased in renal cortices of AS mice when compared to wildtype (WT) mice, while eicosapentaenoic acid (EPA) levels were decreased. In addition, a redistribution of AdA from the glomerular compartment in WT mice to the tubulointerstitial compartment in AS mice was observed. Both Lina and Empa treatments were found to reduce albuminuria, to extend the survival of AS mice for about 10 days, and to decrease glomerulosclerosis and tubulointerstitial fibrosis compared to WT mice. There were no significant differences with regard to the renal phenotype observed between Empa and Lina treated AS mice, and the combination of Lina and Empa was not superior to individual treatments. In vitro experiments revealed that DPP4 is expressed in podocytes and tubular cells derived from both AS and WT mice. Differently from what we have reported for Empa, Lina treatment was found to reduce glucose-driven respiration in AS tubular cells, but not in AS podocytes. CONCLUSIONS: Renal expression patterns and spatial distribution of several metabolites differ in AS compared to WT mice. While Lina and Empa treatments similarly partially slow the progression of kidney disease in AS, the metabolic mechanisms conferring the protective effect may be different.

Biogenesis and assembly of eukaryotic cytochrome c oxidase catalytic core.

Eukaryotic cytochrome c oxidase (COX) is the terminal enzyme of the mitochondrial respiratory chain. COX is a multimeric enzyme formed by subunits of dual genetic origin which assembly is intricate and highly regulated. The COX catalytic core is formed by three mitochondrial DNA encoded subunits, Cox1, Cox2 and Cox3, conserved in the bacterial enzyme. Their biogenesis requires the action of messenger-specific and subunit-specific factors which facilitate the synthesis, membrane insertion, maturation or assembly of the core subunits. The study of yeast strains and human cell lines from patients carrying mutations in structural subunits and COX assembly factors has been invaluable to identify these ancillary factors. Here we review the current state of knowledge of the biogenesis and assembly of the eukaryotic COX catalytic core and discuss the degree of conservation of the players and mechanisms operating from yeast to human. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.

Mechanisms of mitochondrial translational regulation.

The mitochondrial oxidative phosphorylation system is formed by multimeric enzymes. In the yeast Saccharomyces cerevisiae, the bc1 complex, cytochrome c oxidase and the F1 FO ATP synthase contain subunits of dual genetic origin. It has been recently established that key subunits of these enzymes, translated on mitochondrial ribosomes, are the subjects of assembly-dependent translational regulation. This type of control of gene expression plays a pivotal role in optimizing the biogenesis of mitochondrial respiratory membranes by coordinating protein synthesis and complex assembly and by limiting the accumulation of potentially harmful assembly intermediates. Here, the author will discuss the mechanisms governing translational regulation in yeast mitochondria in the light of the most recent discoveries in the field.

Metabolic Labeling of Mitochondrial Translation Products in Whole Cells and Isolated Organelles.

Mitochondria retain their own genome and translational apparatus that is highly specialized in the synthesis of a handful of proteins, essential components of the oxidative phosphorylation system. During evolution, the players and mechanisms involved in mitochondrial translation have acquired some unique features, which we have only partially disclosed. The study of the mitochondrial translation process has been historically hampered by the lack of an in vitro translational system and has largely relied on the analysis of the incorporation rate of radiolabeled amino acids into mitochondrial proteins in cellulo or in organello. In this chapter, we describe methods to monitor mitochondrial translation by labeling newly synthesized mitochondrial polypeptides with [S35]-methionine in either yeast or mammalian whole cells or isolated mitochondria.

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.

Efficient elimination of MELAS-associated m.3243G mutant mitochondrial DNA by an engineered mitoARCUS nuclease.

Nuclease-mediated editing of heteroplasmic mitochondrial DNA (mtDNA) seeks to preferentially cleave and eliminate mutant mtDNA, leaving wild-type genomes to repopulate the cell and shift mtDNA heteroplasmy. Various technologies are available, but many suffer from limitations based on size and/or specificity. The use of ARCUS nucleases, derived from naturally occurring I-CreI, avoids these pitfalls due to their small size, single-component protein structure and high specificity resulting from a robust protein-engineering process. Here we describe the development of a mitochondrial-targeted ARCUS (mitoARCUS) nuclease designed to target one of the most common pathogenic mtDNA mutations, m.3243A>G. mitoARCUS robustly eliminated mutant mtDNA without cutting wild-type mtDNA, allowing for shifts in heteroplasmy and concomitant improvements in mitochondrial protein steady-state levels and respiration. In vivo efficacy was demonstrated using a m.3243A>G xenograft mouse model with mitoARCUS delivered systemically by adeno-associated virus. Together, these data support the development of mitoARCUS as an in vivo gene-editing therapeutic for m.3243A>G-associated diseases.

Empagliflozin reduces podocyte lipotoxicity in experimental Alport syndrome.

Sodium-glucose cotransporter-2 inhibitors (SGLT2i) are anti-hyperglycemic agents that prevent glucose reabsorption in proximal tubular cells. SGLT2i improves renal outcomes in both diabetic and non-diabetic patients, indicating it may have beneficial effects beyond glycemic control. Here, we demonstrate that SGLT2i affects energy metabolism and podocyte lipotoxicity in experimental Alport syndrome (AS). In vitro, we found that the SGLT2 protein was expressed in human and mouse podocytes to a similar extent in tubular cells. Newly established immortalized podocytes from Col4a3 knockout mice (AS podocytes) accumulate lipid droplets along with increased apoptosis when compared to wild-type podocytes. Treatment with SGLT2i empagliflozin reduces lipid droplet accumulation and apoptosis in AS podocytes. Empagliflozin inhibits the utilization of glucose/pyruvate as a metabolic substrate in AS podocytes but not in AS tubular cells. In vivo, we demonstrate that empagliflozin reduces albuminuria and prolongs the survival of AS mice. Empagliflozin-treated AS mice show decreased serum blood urea nitrogen and creatinine levels in association with reduced triglyceride and cholesterol ester content in kidney cortices when compared to AS mice. Lipid accumulation in kidney cortices correlates with a decline in renal function. In summary, empagliflozin reduces podocyte lipotoxicity and improves kidney function in experimental AS in association with the energy substrates switch from glucose to fatty acids in podocytes.