Mitochondrion

Serrano et al. (Serrano et al., 2024) use a high-fidelity somatic mtDNA mutation analysis in conplastic mice in which mtDNA was replaced with exogenous mtDNA of different mouse strains. Serrano reported apparent abundant somatic reversion mutations in the exogenous mtDNA that seemed to restore the original mito-nuclear match. If real, such a phenomenon would have important implications for health and genetics. In todays highly mixed human population, the pairing of potentially mismatched nuclear and mitochondrial genomes is widespread, so the proposed reversion mutagenesis should be commonplace. We demonstrate, however, that these reversion mutations are not real but originate from cross-contamination between samples and from NUMTs, the mtDNA pseudogenes located in the nuclear genome.

Mitochondria are key energy transforming organelles in mammalian cells. However, how defects in oxidative phosphorylation (OxPhos) and other mitochondrial functions influence whole-body energy expenditure (EE) has not been rigorously studied. Cellular and organismal responses to OxPhos defects likely involve a combination of functional downregulation to conserve energy and compensatory upregulation of stress responses. If the energy cost of compensatory responses exceeds the potential energy savings of functional downregulation, as recent work suggests, the result would be an increase in total EE. To address the hypothesis that OxPhos defects increase the energetic cost of living, we performed a meta-analysis of available studies reporting EE in animal models with mitochondrial gene defects. Of all reported experimental conditions (n = 91, from 29 studies), 51% reported a >10% elevation in EE relative to control animals, compared to 11% reporting <10% reduction in EE (p<0.0001, Chi-square). Of the experimental conditions where locomotor activity was also quantified, 39% showed that OxPhos-deficient animals had elevated EE despite reduced locomotor activity, which would be expected to decrease EE. To extend this finding in humans, we re-analyzed a high-quality clinical and multi-omics dataset (Sharma et al. 2021) of mitochondrial disease patients with the m.3243A>G mutation. This analysis similarly indicates an upregulation of energetically costly physiological, immune, and metabolic parameters in people with OxPhos deficiency. These results suggest that animals and humans with mitochondrial defects must expend more energy to sustain life, a state clinically called hypermetabolism. High-quality human energetics studies are needed to understand the magnitude, mechanisms, and modifiability of hypermetabolism in mitochondrial disorders.

The broad clinical and genetic heterogeneity of mitochondrial diseases makes diagnosis challenging. Accurate characterization of novel variants is crucial to reduce diagnostic uncertainty, guide treatment, and enable reliable genetic counseling. In this study, we validated a single-cell NGS-based analysis approach by comparison with conventional PCR-RFLP and applied it to five mtDNA VUS identified in patients evaluated at our national reference center for mitochondrial disorders (CALISSON). Variant interpretation was assessed using multiple frameworks, including Yarhams scoring, Wongs specifications, and the ClinGen guidelines, highlighting differences between these scoring systems and the limitations of current recommendations in fully integrating functional evidence and tissue segregation data. We implemented a classification approach that incorporates these aspects to achieve a more clinically meaningful interpretation. This analysis enabled the reclassification of four novel variants (m.9998T>C in MT-TG, m.7530A>G in MT-TD, and m.4271G>C and m.4305A>G in MT-TI), providing a definitive molecular diagnosis. Further validation on a larger set of variants will be required, as well as the establishment of standardized criteria for single-fiber analyses, including minimum fiber numbers, thresholds for COX-negative fibers, and statistical significance. Overall, this study underscores the critical importance of integrating robust functional evidence into mtDNA variant interpretation and provides insights for refining existing guidelines to improve diagnostic accuracy and support clinical decision-making.

Mitochondrial DNA copy number (mtDNA-CN) is a metric of mitochondrial function that has been associated with a variety of diseases including cardiovascular disease and all-cause mortality. To investigate genes and pathways affected by mtDNA-CN variation, we perturbed HEK 293T cells with ethidium bromide to deplete mtDNA. Using RNASeq and methylation microarrays, we evaluated transcriptomic and methylomic changes in treated cell lines. We observed an 8-fold decrease in mtDNA-CN and compensatory shifts in mitochondrial transcription to support mtDNA replication. Nuclear transcriptomic and methylomic analysis highlighted changes in metabolic pathways, including oxidative phosphorylation and canonical glycolysis. Longitudinal analyses revealed that the identified genes and pathways have different response timing, with nuclear response lagging behind mitochondrial response. These findings further elucidate the mechanisms behind mtDNA maintenance and responses to cellular energetics as well as mitochondrial-nuclear crosstalk dynamics.

Mitochondrial DNA depletion syndromes are severe genetic disorders associated with mutations in a variety of genes including MPV17, encoding a protein of the inner mitochondrial membrane with an unclear function. In this study, using BioID technology, we identified MPV17 interacting partners among which proteins from the MICOS complex. However, MPV17 knockout did not impact mitochondrial ultrastructure, but led to increased mitochondria-derived vesicles formation and altered mitochondrial permeability transition pore. Furthermore, MPV17 KO cells exhibited higher mitochondrial calcium levels and reactive oxygen species, leading to mtDNA degradation, a phenomenon prevented by blocking mitochondrial calcium entry or treating cells with antioxidant. We thus propose a function for MPV17 as a potential additional member of the mitochondrial permeability transition pore, whereas in the absence of the protein, the build-up of calcium inside the mitochondria would lead to mtDNA degradation caused by increased oxidative damages.

Strictly maternal inheritance and lack of intermolecular recombination of the human mitochondrial genome (mtDNA) are the assumed preconditions for molecular evolution studies, phylogenetic reconstruction and population genetic analyses. This hypothesis, however, has been challenged by investigations providing evidence for genetic recombination of mtDNA, thus sparking controversy. Using single-molecule real-time (SMRT) sequencing technology, we sequenced the entire mtDNA from blood and fibroblast cells from five individuals with biparental mtDNA transmission in three separate, multiple-generation families. After phasing the single nucleotide polymorphism (SNP) genotypes of mtDNA, no intermolecular recombination between paternal and maternal mtDNA was found when the mtDNA was transmitted in either biparental or maternal mode. Our study provides support for the argument that intermolecular mtDNA recombination is absent or extremely rare in humans. As a consequence, these results support the feasibility of mtDNA-based molecular evolution studies and phylogenetic and population genetic analyses for humans, while also avoiding inaccurate phylogenetic inferences and incorrect rejection of the molecular clock.

Background/ObjectivesWhole genome sequencing (WGS) from large cohorts enables the study of mitochondrial DNA (mtDNA) variation on human health. We aimed to investigate the influence of common, rare, and pathogenic mtDNA variants on 15 mitochondrial disease-related phenotypes. MethodsUsing WGS from 179,862 individuals from in the UK Biobank, we identified mtDNA variants using MitoHPC. We performed extensive association analyses with 15 mitochondrial disease-relevant phenotypes. We compared the results for the m.3243A>G variant with those from a clinically referred patient cohort. ResultsOf 15,881 mtDNA variants, 12 homoplasmic and one heteroplasmic variant had genome-wide significant associations. All homoplasmic variants increased aspartate aminotransferase level and three were novel, low frequency, variants (MAF[~]0.002 and beta[~]0.3 SD). Only m.3243A>G (MAF=0.0002) associated with diabetes (OR=5.6, 95%CI [3.2-9.9]), deafness (OR=12.3, 95%CI [6.2-24.4]) and heart failure (OR=39.5, 95%CI [9.76-160.1]). Multi-system disease risk and penetrance of all three traits increased with m.3243A>G level. Diabetes risk was further influenced by common nuclear genome variation. The penetrance of diabetes with m.3243A>G in the UK Biobank was lower than clinically referred patients, partly attributed to lower heteroplasmy. Of 73 pathogenic mitochondrial disease variants, most were rare in the population with low penetrance. ConclusionOur study highlights the utility of WGS for investigating mitochondrial genetics within a large, unselected population. We identified novel associations and demonstrated that pathogenic mitochondrial variants have lower penetrance in clinically unselected than clinically referred settings. m.3243A>G associated with mitochondrial-related phenotypes at higher heteroplasmy. Our findings suggest potential benefits of reporting incidentally identified m.3243A>G at high heteroplasmy levels.

Oxidative phosphorylation (OXPHOS) system localized in the inner mitochondrial membrane secures production of the majority of ATP in mammalian organisms. Individual OXPHOS complexes were shown to form supramolecular assemblies termed supercomplexes. It has been repeatedly shown that complexes are not linked only by their function but also by interdependence of individual complex biogenesis or maintenance. For instance, cytochrome c oxidase (cIV, COX) or cytochrome bc1 complex (cIII) deficiencies affect the level of fully assembled NADH dehydrogenase (cI) in monomer as well as within supercomplexes. It was hypothesized that cI is affected at the level of enzyme assembly as well as at the level of cI stability and maintenance. However, the true nature of interdependency between cI and cIV is not fully understood yet. We used HEK293 cellular model with complete knockout of COX4 subunit, which serves as an ideal system to study interdependency of cI and cIV, as early phases of cIV assembly process are disrupted. Total absence of cIV was accompanied by profound deficiency of cI, documented by selective decrease in cI subunits amount and significantly reduced amount of assembled cI. Supercomplexes assembled from cI, cIII and cIV were missing in COX4dKO due to loss of cIV and decrease in cI amount. Pulse-chase metabolic labelling of mtDNA-encoded proteins uncovered decrease of cIV and cI subunits translation. Moreover, partial impairment of mitochondrial proteosynthesis correlated with decreased level of mitochondrial ribosomal proteins. In addition, complexome profiling approach uncovered accumulation of cI assembly intermediates indicating that cI biogenesis was affected rather than stability. We propose that impairment of mitochondrial proteosynthesis caused by cIV deficiency represents one of the mechanisms which may couple biogenesis of cI and cIV.

Mitochondrial DNA (mtDNA) mutations accumulate in both mitotic and post-mitotic somatic tissues of normal individuals with age. They clonally expand within individual cells and cause mitochondrial dysfunction. In contrast, in patients with inherited disease-causing mtDNA mutations the mutation load decreases in mitotic tissues over time, whereas the mutations load in post-mitotic tissues remains relatively stable. The mechanisms underlying this decrease in mitotic tissues, and whether mitochondrial function is restored at the tissue level are unknown. Here, using a combination of homogenate tissue and single crypt/muscle fibre pyrosequencing we have shown a decrease in the mutation load of the germline heteroplasmic m.5024C>T mutation in multiple mitotic tissues of a mouse model of inherited mitochondrial disease (C5024T mice). We have then used in silico predictions to model the cellular dynamics of mtDNA mutation load in mitotic and post mitotic tissues. We demonstrate that: (1) the rate of m.5024C>T decrease correlates with the rate of tissue turnover; (2) the mutation load decrease is not associated with changes in overall cellular proliferation and apoptosis within the mitotic colonic epithelium; instead, it could be due to an upper limit of m.5024C>T load in stem cell populations; (3) the m.5024C>T mutation load is maintained in post-mitotic tissues over time with a consistent load amongst individual muscle fibres; (4) in silico modelling supports a scenario where genetic drift is accelerated in mitotic tissues by high levels of mtDNA replication coupled with mtDNA segregation at cell division. This study has advanced our understanding of the dynamics of mtDNA mutations and phenotype development in patients with mtDNA disease. Author SummaryHealthy individuals randomly accumulate pathogenic mtDNA mutations with age in dividing cells, causing mitochondrial dysfunction. Interestingly, patients with mitochondrial disease show a relative decrease in the loads of inherited mtDNA mutations in some dividing cells over time. The mechanisms underlying this decrease are unknown. Here we show a decrease in the load of the germline heteroplasmic m.5024C>T mutation in dividing cells and tissues of a mouse model of mitochondrial disease. In contrast, the mutation load in non-dividing cells and tissue remains stable. Our data are consistent with the hypothesis that a higher frequency of mtDNA replication in dividing cells, coupled with stem cells having an upper tolerance limit for m.5024C>T, causes an overall decrease in m.5024C>T load at the tissue level.

The mutational spectrum of the mitochondrial DNA (mtDNA) does not resemble any of the known mutational signatures of the nuclear genome and variation in mtDNA mutational spectra between different organisms is still incomprehensible. Since mitochondria is tightly involved in aerobic energy production, it is expected that mtDNA mutational spectra is affected by the oxidative damage. Assuming that oxidative damage increases with age, we analyze mtDNA mutagenesis of different species. Analysing (i) dozens thousands of somatic mtDNA mutations in samples of different age (ii) 70053 polymorphic synonymous mtDNA substitutions, reconstructed in 424 mammalian species with different generation length and (iii) synonymous nucleotide content of 650 complete mitochondrial genomes of mammalian species we observed that the frequency of AH>GH substitutions (H - heavy chain notation) is twice higher in species with high versus low generation length making their mtDNA more AH poor and GH rich. Considering that AH>GH substitutions are also sensitive to the time spent single stranded (TSSS) during asynchroniuos mtDNA replication we demonstrated that AH>GH substitution rate is a function of both species-specific generation length and position specific TSSS. We propose that AH>GH is a mitochondria-specific signature of oxidative damage associated with both aging and TSSS.

Mitochondrial biogenesis requires the import of [~]1,000-1,500 nuclear-encoded proteins across the Translocase of Outer Membrane (TOM) and the Translocase of Inner Membrane (TIM) 22 or 23 complexes. Protein import defects cannot only impair mitochondrial respiration but also cause mitochondrial Precursor Overaccumulation Stress (mPOS) in the cytosol. Recent studies showed that specific mutations in the nuclear-encoded Adenine Nucleotide Translocase 1 (ANT1) cause musculoskeletal and neurological diseases by clogging TOM and TIM22 and inducing mPOS. Here, we found that overexpression of MFB1, encoding the mitochondrial F-box protein 1, suppresses cell growth defect caused by a clogger allele of AAC2, the yeast homolog of Ant1. Disruption of MFB1 synergizes with a clogger allele of aac2 to inhibit cell growth. This is accompanied by increased retention of mitochondrial proteins in the cytosol, suggesting exacerbated defect in mitochondrial protein import. Proximity-dependent biotin identification (BioID) suggested that Mfb1 interacts with several mitochondrial surface proteins including Tom22, a component of the TOM complex. Loss of MFB1 under clogging conditions activates genes encoding cytosolic chaperones including HSP31. Interestingly, disruption of HSP31 creates a synthetic lethality with protein import clogging under respiring conditions. We propose that Mfb1 functions to maintain mitochondrial protein import competency under clogging conditions, whereas Hsp31 plays an important role in protecting the cytosol against mPOS. Mutations in DJ-1, the human homolog of Hsp31, and mitochondria-associated F-box proteins (eg., Fbxo7) are known to cause early-onset Parkinsons disease. Our work may help to better understand how these mutations affect cellular proteostasis and cause neurodegeneration.

An often-overlooked aspect of mitochondrial biology is the mitochondrial DNA (mtDNA). The multi-copy mtDNA is highly dynamic, with changes in supercoiling, synthesis rates, and turnover rates that are tightly associated with mitochondrial and cellular functions. To better understand the state of the mtDNA, here we describe a protocol that selectively incorporates bromodeoxyuridine into the mtDNA for subsequent measurement via an adapted Southern blot followed by immunoblotting (a.k.a. Southwestern blot). This basic protocol can be applied with slight modifications for the measurement of mtDNA synthesis, turnover or supercoiling to understand mtDNA changes.

Mutations in the TANGO2 gene are associated with a severe neurometabolic disorder in humans, often presenting with life-threatening metabolic crisis. However, the function of TANGO2 protein remains unknown. It has recently been proposed that TANGO2 transports heme within and between cells, from areas with high heme concentrations to those with lower concentrations. Here, we demonstrate that prior heme-related observations in Caenorhabditis elegans lacking TANGO2 homologs HRG-9 and HRG-10 may be better explained by a previously unreported metabolic phenotype, characterized by reduced feeding, decreased lifespan and brood sizes, and poor motility. We also show that several genes not implicated in heme transport are upregulated in the low heme state and conversely demonstrate that hrg-9 in particular is highly responsive to oxidative stress, independent of heme status. Collectively, these data implicate bioenergetic failure and oxidative stress as potential factors in the pathophysiology of TANGO2 deficiency, in alignment with observations from human patients. Our group performed several experiments in yeast and zebrafish deficient in TANGO2 homologs and was unable to replicate prior findings from these models. Overall, we believe there is insufficient evidence to support heme transport as the primary function for TANGO2. Impact statementCross-species examination of TANGO2 homologs demonstrates that phenotypes previously attributed to altered heme trafficking may instead reflect broader disturbances in mitochondrial function and cellular homeostasis.

Human biofluids contain cell-free mitochondrial DNA (cf-mtDNA) and extracellular mitochondria (ex-Mito), creating the challenge of defining their origins, destinations, mechanisms of regulation, and purposes. To expand our understanding of cf-mtDNA biology, we present a descriptive electron microscopy analysis of circulating particles from cf-mtDNA-enriched plasma (citrate, heparin, and EDTA), serum (red and gold top), and saliva collected from ten healthy people (5 females, 5 males, mean age 44.9 years). Ex-mito and extracellular vesicles (EVs) were isolated by centrifugation followed by size-exclusion chromatography, imaged by transmission electron microscopy, and morphometrically analyzed. In parallel, cf-mtDNA was quantified in each biofluid. The resulting catalog of the most common circulating particles in plasma, serum, and saliva show that circulating double-membrane extracellular particles-- consistent with mitochondrial ultrastructure--are present across human biofluids, along with EVs and other particle types. Combining imaging with cf-mtDNA quantification, we show that individuals with higher plasma cf-mtDNA concentrations tend to contain more double-membrane, ex-Mito-like particles. These preliminary results challenge the notion that, under normal conditions, the majority of cf-mtDNA exists as naked and potentially pro-inflammatory forms. Instead, these results are consistent with the concept of mitochondria transfer or signaling between cells and tissues. The image inventory provided here expands our knowledge of cell-free mitochondrial biology and provides a resource to inform biofluid selection and technical considerations in future studies quantifying ex-Mito and cf-mtDNA.

Mitochondrial single-nucleotide variants (mtSNVs) can dysregulate cellular bioenergetics and have been increasingly implicated in susceptibility to Parkinsons disease (PD). These variants may impair oxidative metabolism and respiratory chain efficiency, thus contributing to neuronal dysfunction and degeneration. In peripheral blood, mtSNVs may also reflect systemic immunometabolic alterations associated with PD; however, this aspect remains poorly explored, particularly in admixed populations with significant indigenous ancestry. In this study, we analyzed the complete mtDNA of peripheral blood samples from 179 admixed individuals (104 with PD and 75 controls) from the Brazilian Amazon. Associations between mtSNVs and PD were assessed using adjusted logistic regression models, and functional annotation was performed using the Variant Effect Predictor. Furthermore, we proposed and calculated a heteroplasmy-weighted mitochondrial polygenic risk score (mtPRS). We observed a higher mtSNV burden in PD patients, predominantly in RNR2 gene and genes of Complexes I and IV. In total, 536 unique mitochondrial SNVs were identified (214 exclusive to PD, 321 shared between PD and controls, and one exclusive to controls). Four mitochondrial SNVs were associated with PD, including three novel variants (COX1: m.6630G>A, COX2: m.7613C>T, and RNR2: m.1996C>T) and one previously reported variant (ND4: m.12112C>T). Notably, the mitochondrial polygenic risk score (mtPRS) showed a strong association with increased PD risk (OR = 3.64; FDR = 1.12 x 10-_). Taken together, these results suggest that mtSNVs may contribute to PD susceptibility in admixed Amazonian population, highlighting the relevance of mitochondrial genetic architecture in PD and emphasizing the importance of including underrepresented populations in genomic research.

During muscle contraction, increased influx of mitochondrial calcium (mtCa{superscript 2}) from the myocyte cytosol through the mitochondrial calcium uniporter (MCU) couples calcium homeostasis with high ATP provision. The mitochondrial calcium uniporter regulator 1 (MCUR1) is an integral membrane protein that promotes MCU activity. Although its function has been studied in cell models, mutations in MCUR1 have not yet been associated with human disease. Here, we present a case study of a patient exhibiting proximal muscle weakness and atrophy, who carries a novel homozygous loss-of-function mutation in MCUR1. To investigate the underlying mechanisms of muscle pathology, we examined patient fibroblasts and quadriceps muscle specimens. MCUR1 deficiency compromised mitochondrial Ca{superscript 2} uptake upon histamine exposure, but did not alter resting mitochondrial membrane potential or MCU protein complex assembly or subcellular location. Consequently, ATP production and oxygen consumption were reduced, and mitochondrial biogenesis was disturbed in muscle, with histological features of autophagic vacuoles with sarcolemmal features. Our study associates MCUR1 deficiency with mitochondrial dysfunction and autophagic vacuolar myopathy, thereby highlighting the crucial role of mitochondrial Ca{superscript 2} uptake in regulating mitochondrial function and expanding the spectrum of mitochondrial disorders in humans. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=144 HEIGHT=200 SRC="FIGDIR/small/25338070v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@9fa55org.highwire.dtl.DTLVardef@111ebb3org.highwire.dtl.DTLVardef@1895c4corg.highwire.dtl.DTLVardef@10aac6c_HPS_FORMAT_FIGEXP M_FIG C_FIG

Myalgic Encephalomyelitis (ME) / Chronic Fatigue Syndrome is a disease of uncertain aetiology that affects up to 400,000 individuals in the UK. Exposure of cultured cells to the sera of people with ME has been proposed to cause phenotypic changes in these cells in vitro when compared to sera from healthy controls. ME serum factors causing these changes could inform the development of diagnostic tests. In this study, we performed a large-scale, pre-registered replication of an experiment from Fluge et al (2016) that reported an increase in maximal respiratory capacity in healthy myoblasts after treatment with serum from people with ME compared to serum from healthy controls. We replicated the original experiment with a larger sample size, using sera from 67 people with ME and 53 controls to treat healthy cultured myoblasts, and generated results from over 1,700 mitochondrial stress tests performed with a Seahorse Bioanalyser. We observed no significant differences between treatment with ME or healthy control sera for our primary outcome of interest, oxygen consumption rate at maximal respiratory capacity. Results from our study provide strong evidence against the hypothesis that ME blood factors differentially affect healthy myoblast mitochondrial phenotypes in vitro.

Mitochondrial function is critical for cellular health, with dysfunction contributing to human diseases. Structural changes in mitochondria, such as size and shape, reflect alterations in bioenergetics, fission-fusion dynamics, and metabolic homeostasis. Existing morphological quantification is outdated, can be biased, technologically limited, or overly complex. This study presents a high content system for quantifying morphology using open-access resources and widely available equipment. Fibroblasts were stained with PKmitoTM Dye Deep Red, imaged via automated confocal microscopy, and analyzed with CellProfiler and KNIME(R). We tested different imaging conditions and found live-cell confocal imaging at 60x magnification provided the most precise measurements. Using this system, we found that human Huntington Disease fibroblast mitochondria were significantly smaller and more circular, suggesting increased fission. To confirm our results, we employed other mitochondrial assays and found elevated expression of the fission protein Drp1, reduced respiration, impaired iron uptake, and increased membrane potential. This system offers a robust, unbiased high content approach to studying mitochondrial morphology in disease.

Mitochondrial DNA encodes the genetic information necessary for mitochondrial function. In humans, mitochondrial DNA spans 16,569 base pairs while representing a small fraction of the genetic material in humans. Due to their higher mutation rates compared to nuclear DNA, Mitochondrial DNA mutations are emerging as promising biomarkers for assessing disease predisposition and progression. This in-silico analyzes the correlation between Mitochondrial DNA mutations and two conditions: Leigh syndrome, a severe neurodegenerative disorder, and carotid atherosclerosis, a major cardiovascular disease. We focus on five single nucleotide variants (SNVs) - m.14459G>A, m.13513G>A, m.12315G>A, m.1555A>G, and m.15059G>A - to explore their roles in both diseases. Our findings suggest that mutations m.14459G>A, m.1555A>G, and m.15059G>A contribute to the pathogenesis of both conditions. In contrast, m.12315G>A is linked to MELAS syndrome and carotid atherosclerosis. Interestingly, the m.13513G>A mutation is associated with Leigh syndrome but negatively correlated with atherosclerosis, suggesting a potential protective effect. These SNVs could serve as targets for diagnostic and therapeutic approaches, enhancing our understanding of the genetic basis of these diseases.

Recent evidence suggests that the coding potential of the mitogenome is underestimated. We found a downstream alternative ATG initiation codon in the +3 reading frame of the human mitochondrial nd4 gene. This newly characterized alternative open reading frame (altORF) encodes a 99-amino acids long polypeptide, MTALTND4, which is conserved in primates. This small protein is localized in mitochondria and cytoplasm and is also found in the plasma, and it impacts mitochondrial physiology. Alternative mitochondrial peptides such as MTALTND4 may offer a new framework for the investigation of mitochondrial functions and diseases.