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.

ESYT1 tethers the ER to mitochondria and is required for mitochondrial lipid and calcium homeostasis.

Mitochondria interact with the ER at structurally and functionally specialized membrane contact sites known as mitochondria-ER contact sites (MERCs). Combining proximity labelling (BioID), co-immunoprecipitation, confocal microscopy and subcellular fractionation, we found that the ER resident SMP-domain protein ESYT1 was enriched at MERCs, where it forms a complex with the outer mitochondrial membrane protein SYNJ2BP. BioID analyses using ER-targeted, outer mitochondrial membrane-targeted, and MERC-targeted baits, confirmed the presence of this complex at MERCs and the specificity of the interaction. Deletion of ESYT1 or SYNJ2BP reduced the number and length of MERCs. Loss of the ESYT1-SYNJ2BP complex impaired ER to mitochondria calcium flux and provoked a significant alteration of the mitochondrial lipidome, most prominently a reduction of cardiolipins and phosphatidylethanolamines. Both phenotypes were rescued by reexpression of WT ESYT1 and an artificial mitochondria-ER tether. Together, these results reveal a novel function for ESYT1 in mitochondrial and cellular homeostasis through its role in the regulation of MERCs.

BOLA3 and NFU1 link mitoribosome iron-sulfur cluster assembly to multiple mitochondrial dysfunctions syndrome.

The human mitochondrial ribosome contains three [2Fe-2S] clusters whose assembly pathway, role, and implications for mitochondrial and metabolic diseases are unknown. Here, structure-function correlation studies show that the clusters play a structural role during mitoribosome assembly. To uncover the assembly pathway, we have examined the effect of silencing the expression of Fe-S cluster biosynthetic and delivery factors on mitoribosome stability. We find that the mitoribosome receives its [2Fe-2S] clusters from the GLRX5-BOLA3 node. Additionally, the assembly of the small subunit depends on the mitoribosome biogenesis factor METTL17, recently reported containing a [4Fe-4S] cluster, which we propose is inserted via the ISCA1-NFU1 node. Consistently, fibroblasts from subjects suffering from 'multiple mitochondrial dysfunction' syndrome due to mutations in BOLA3 or NFU1 display previously unrecognized attenuation of mitochondrial protein synthesis that contributes to their cellular and pathophysiological phenotypes. Finally, we report that, in addition to their structural role, one of the mitoribosomal [2Fe-2S] clusters and the [4Fe-4S] cluster in mitoribosome assembly factor METTL17 sense changes in the redox environment, thus providing a way to regulate organellar protein synthesis accordingly.

The role of the mitochondrial outer membrane protein SLC25A46 in mitochondrial fission and fusion.

Mutations in SLC25A46 underlie a wide spectrum of neurodegenerative diseases associated with alterations in mitochondrial morphology. We established an SLC25A46 knock-out cell line in human fibroblasts and studied the pathogenicity of three variants (p.T142I, p.R257Q, and p.E335D). Mitochondria were fragmented in the knock-out cell line and hyperfused in all pathogenic variants. The loss of SLC25A46 led to abnormalities in the mitochondrial cristae ultrastructure that were not rescued by the expression of the variants. SLC25A46 was present in discrete puncta at mitochondrial branch points and tips of mitochondrial tubules, co-localizing with DRP1 and OPA1. Virtually, all fission/fusion events were demarcated by a SLC25A46 focus. SLC25A46 co-immunoprecipitated with the fusion machinery, and loss of function altered the oligomerization state of OPA1 and MFN2. Proximity interaction mapping identified components of the ER membrane, lipid transfer proteins, and mitochondrial outer membrane proteins, indicating that it is present at interorganellar contact sites. SLC25A46 loss of function led to altered mitochondrial lipid composition, suggesting that it may facilitate interorganellar lipid flux or play a role in membrane remodeling associated with mitochondrial fusion and fission.

Identification of Proximity Interactors of Mammalian Nucleoid Proteins by BioID.

Mitochondrial nucleoids are compact nucleoprotein complexes, in which mtDNA is located, replicated, and transcribed. Several proteomic approaches have been previously employed to identify nucleoid proteins; however, a consensus list of nucleoid-associated proteins has not been generated. Here we describe a proximity-biotinylation assay, BioID, which allows identification of proximity interactors of mitochondrial nucleoid proteins. It uses a promiscuous biotin ligase fused to a protein of interest which covalently attaches biotin to lysine residues of its proximal neighbors. Biotinylated proteins can be further enriched by a biotin-affinity purification and identified by mass-spectrometry. BioID can identify transient and weak interactions and can be used to identify changes in the interactions upon different cellular treatments, for different protein isoforms or for pathogenic variants.

ATAD3A has a scaffolding role regulating mitochondria inner membrane structure and protein assembly.

The ATPase Family AAA Domain Containing 3A (ATAD3A), is a mitochondrial inner membrane protein conserved in metazoans. ATAD3A has been associated with several mitochondrial functions, including nucleoid organization, cholesterol metabolism, and mitochondrial translation. To address its primary role, we generated a neuronal-specific conditional knockout (Atad3 nKO) mouse model, which developed a severe encephalopathy by 5 months of age. Pre-symptomatic mice showed aberrant mitochondrial cristae morphogenesis in the cortex as early as 2 months. Using a multi-omics approach in the CNS of 2-to-3-month-old mice, we found early alterations in the organelle membrane structure. We also show that human ATAD3A associates with different components of the inner membrane, including OXPHOS complex I, Letm1, and prohibitin complexes. Stochastic Optical Reconstruction Microscopy (STORM) shows that ATAD3A is regularly distributed along the inner mitochondrial membrane, suggesting a critical structural role in inner mitochondrial membrane and its organization, most likely in an ATPase-dependent manner.

A proximity-dependent biotinylation map of a human cell.

Compartmentalization is a defining characteristic of eukaryotic cells, and partitions distinct biochemical processes into discrete subcellular locations. Microscopy1 and biochemical fractionation coupled with mass spectrometry2-4 have defined the proteomes of a variety of different organelles, but many intracellular compartments have remained refractory to such approaches. Proximity-dependent biotinylation techniques such as BioID provide an alternative approach to define the composition of cellular compartments in living cells5-7. Here we present a BioID-based map of a human cell on the basis of 192 subcellular markers, and define the intracellular locations of 4,145 unique proteins in HEK293 cells. Our localization predictions exceed the specificity of previous approaches, and enabled the discovery of proteins at the interface between the mitochondrial outer membrane and the endoplasmic reticulum that are crucial for mitochondrial homeostasis. On the basis of this dataset, we created humancellmap.org as a community resource that provides online tools for localization analysis of user BioID data, and demonstrate how this resource can be used to understand BioID results better.

A High-Density Human Mitochondrial Proximity Interaction Network.

We used BioID, a proximity-dependent biotinylation assay with 100 mitochondrial baits from all mitochondrial sub-compartments, to create a high-resolution human mitochondrial proximity interaction network. We identified 1,465 proteins, producing 15,626 unique high-confidence proximity interactions. Of these, 528 proteins were previously annotated as mitochondrial, nearly half of the mitochondrial proteome defined by Mitocarta 2.0. Bait-bait analysis showed a clear separation of mitochondrial compartments, and correlation analysis among preys across all baits allowed us to identify functional clusters involved in diverse mitochondrial functions and to assign uncharacterized proteins to specific modules. We demonstrate that this analysis can assign isoforms of the same mitochondrial protein to different mitochondrial sub-compartments and show that some proteins may have multiple cellular locations. Outer membrane baits showed specific proximity interactions with cytosolic proteins and proteins in other organellar membranes, suggesting specialization of proteins responsible for contact site formation between mitochondria and individual organelles.

Human GTPBP5 (MTG2) fuels mitoribosome large subunit maturation by facilitating 16S rRNA methylation.

Biogenesis of mammalian mitochondrial ribosomes (mitoribosomes) involves several conserved small GTPases. Here, we report that the Obg family protein GTPBP5 or MTG2 is a mitochondrial protein whose absence in a TALEN-induced HEK293T knockout (KO) cell line leads to severely decreased levels of the 55S monosome and attenuated mitochondrial protein synthesis. We show that a fraction of GTPBP5 co-sediments with the large mitoribosome subunit (mtLSU), and crosslinks specifically with the 16S rRNA, and several mtLSU proteins and assembly factors. Notably, the latter group includes MTERF4, involved in monosome assembly, and MRM2, the methyltransferase that catalyzes the modification of the 16S mt-rRNA A-loop U1369 residue. The GTPBP5 interaction with MRM2 was also detected using the proximity-dependent biotinylation (BioID) assay. In GTPBP5-KO mitochondria, the mtLSU lacks bL36m, accumulates an excess of the assembly factors MTG1, GTPBP10, MALSU1 and MTERF4, and contains hypomethylated 16S rRNA. We propose that GTPBP5 primarily fuels proper mtLSU maturation by securing efficient methylation of two 16S rRNA residues, and ultimately serves to coordinate subunit joining through the release of late-stage mtLSU assembly factors. In this way, GTPBP5 provides an ultimate quality control checkpoint function during mtLSU assembly that minimizes premature subunit joining to ensure the assembly of the mature 55S monosome.

Stomatin-like protein 2 deficiency results in impaired mitochondrial translation.

Mitochondria translate the RNAs for 13 core polypeptides of respiratory chain and ATP synthase complexes that are essential for the assembly and function of these complexes. This process occurs in close proximity to the mitochondrial inner membrane. However, the mechanisms and molecular machinery involved in mitochondrial translation are not fully understood, and defects in this process can result in severe diseases. Stomatin-like protein (SLP)-2 is a mainly mitochondrial protein that forms cardiolipin- and prohibitin-enriched microdomains in the mitochondrial inner membrane that are important for the formation of respiratory supercomplexes and their function. Given this regulatory role of SLP-2 in processes closely associated with the mitochondrial inner membrane, we hypothesized that the function of SLP-2 would have an impact on mitochondrial translation. 35S-Methionine/cysteine pulse labeling of resting or activated T cells from T cell-specific Slp-2 knockout mice showed a significant impairment in the production of several mitochondrial DNA-encoded polypeptides following T cell activation, including Cytb, COXI, COXII, COXIII, and ATP6. Measurement of mitochondrial DNA stability and mitochondrial transcription revealed that this impairment was at the post-transcriptional level. Examination of mitochondrial ribosome assembly showed that SLP-2 migrated in sucrose-density gradients similarly to the large ribosomal subunit but that its deletion at the genetic level did not affect mitochondrial ribosome assembly. Functionally, the impairment in mitochondrial translation correlated with decreased interleukin-2 production in activated T cells. Altogether, these data show that SLP-2 acts as a general regulator of mitochondrial translation.

A pseudouridine synthase module is essential for mitochondrial protein synthesis and cell viability.

Pseudouridylation is a common post-transcriptional modification in RNA, but its functional consequences at the cellular level remain largely unknown. Using a proximity-biotinylation assay, we identified a protein module in mitochondrial RNA granules, platforms for post-transcriptional RNA modification and ribosome assembly, containing several proteins of unknown function including three uncharacterized pseudouridine synthases, TRUB2, RPUSD3, and RPUSD4. TRUB2 and RPUSD4 were previously identified as core essential genes in CRISPR/Cas9 screens. Depletion of the individual enzymes produced specific mitochondrial protein synthesis and oxidative phosphorylation assembly defects without affecting mitochondrial mRNA levels. Investigation of the molecular targets in mitochondrial RNA by pseudouridine-Seq showed that RPUSD4 plays a role in the pseudouridylation of a single residue in the 16S rRNA, a modification that is essential for its stability and assembly into the mitochondrial ribosome, while TRUB2/RPUSD3 were similarly involved in pseudouridylating specific residues in mitochondrial mRNAs. These results establish essential roles for epitranscriptomic modification of mitochondrial RNA in mitochondrial protein synthesis, oxidative phosphorylation, and cell survival.

A Mutation in the Flavin Adenine Dinucleotide-Dependent Oxidoreductase FOXRED1 Results in Cell-Type-Specific Assembly Defects in Oxidative Phosphorylation Complexes I and II.

Complex I (NADH ubiquinone oxidoreductase) is a large multisubunit enzyme that catalyzes the first step in oxidative phosphorylation (OXPHOS). In mammals, complex I biogenesis occurs in a stepwise manner, a process that requires the participation of several nucleus-encoded accessory proteins. The FAD-dependent oxidoreductase-containing domain 1 (FOXRED1) protein is a complex I assembly factor; however, its specific role in the assembly pathway remains poorly understood. We identified a homozygous missense mutation, c.1308 G→A (p.V421M) in FOXRED1 in a patient who presented with epilepsy and severe psychomotor retardation. A patient myoblast line showed a severe reduction in complex I, associated with the accumulation of subassemblies centered around ∼340 kDa, and a milder decrease in complex II, all of which were rescued by retroviral expression of wild-type FOXRED1. Two additional assembly factors, AIFM1 and ACAD9, coimmunoprecipitated with FOXRED1, and all were associated with a 370-kDa complex I subassembly that, together with a 315-kDa subassembly, forms the 550-kDa subcomplex. Loss of FOXRED1 function prevents efficient formation of this midassembly subcomplex. Although we could not identify subassemblies of complex II, our results establish that FOXRED1 function is both broader than expected, involving the assembly of two flavoprotein-containing OXPHOS complexes, and cell type specific.

Whole-exome sequencing identifies novel ECHS1 mutations in Leigh syndrome.

Leigh syndrome (LS) is a rare heterogeneous progressive neurodegenerative disorder usually presenting in infancy or early childhood. Clinical presentation is variable and includes psychomotor delay or regression, acute neurological or acidotic episodes, hypotonia, ataxia, spasticity, movement disorders, and corresponding anomalies of the basal ganglia and brain stem on magnetic resonance imaging. To date, 35 genes have been associated with LS, mostly involved in mitochondrial respiratory chain function and encoded in either nuclear or mitochondrial DNA. We used whole-exome sequencing to identify disease-causing variants in four patients with basal ganglia abnormalities and clinical presentations consistent with LS. Compound heterozygote variants in ECHS1, encoding the enzyme enoyl-CoA hydratase were identified. One missense variant (p.Thr180Ala) was common to all four patients and the haplotype surrounding this variant was also shared, suggesting a common ancestor of French-Canadian origin. Rare mutations in ECHS1 as well as in HIBCH, the enzyme downstream in the valine degradation pathway, have been associated with LS or LS-like disorders. A clear clinical overlap is observed between our patients and the reported cases with ECHS1 or HIBCH deficiency. The main clinical features observed in our cohort are T2-hyperintense signal in the globus pallidus and putamen, failure to thrive, developmental delay or regression, and nystagmus. Respiratory chain studies are not strikingly abnormal in our patients: one patient had a mild reduction of complex I and III and another of complex IV. The identification of four additional patients with mutations in ECHS1 highlights the emerging importance of this pathway in LS.

An N-terminal formyl methionine on COX 1 is required for the assembly of cytochrome c oxidase.

Protein synthesis in mitochondria is initiated by formylmethionyl-tRNA(Met) (fMet-tRNA(Met)), which requires the activity of the enzyme MTFMT to formylate the methionyl group. We investigated the molecular consequences of mutations in MTFMT in patients with Leigh syndrome or cardiomyopathy. All patients studied were compound heterozygotes. Levels of MTFMT in patient fibroblasts were almost undetectable by immunoblot analysis, and BN-PAGE analysis showed a combined oxidative phosphorylation (OXPHOS) assembly defect involving complexes I, IV and V. The synthesis of only a subset of mitochondrial polypeptides (ND5, ND4, ND1, COXII) was decreased, whereas all others were translated at normal or even increased rates. Expression of the wild-type cDNA rescued the biochemical phenotype when MTFMT was expressed near control levels, but overexpression produced a dominant-negative phenotype, completely abrogating assembly of the OXPHOS complexes, suggesting that MTFMT activity must be tightly regulated. fMet-tRNA(Met) was almost undetectable in control cells and absent in patient cells by high-resolution northern blot analysis, but accumulated in cells overexpressing MTFMT. Newly synthesized COXI was under-represented in complex IV immunoprecipitates from patient fibroblasts, and two-dimensional BN-PAGE analysis of newly synthesized mitochondrial translation products showed an accumulation of free COXI. Quantitative mass spectrophotometry of an N-terminal COXI peptide showed that the ratio of formylated to unmodified N-termini in the assembled complex IV was ∼350:1 in controls and 4:1 in patient cells. These results show that mitochondrial protein synthesis can occur with inefficient formylation of methionyl-tRNA(Met), but that assembly of complex IV is impaired if the COXI N-terminus is not formylated.

Mitochondrial RNA Granules Are Centers for Posttranscriptional RNA Processing and Ribosome Biogenesis.

Cytoplasmic RNA granules play a central role in mRNA metabolism, but the importance of mitochondrial RNA granules remains relatively unexplored. We characterized their proteome and found that they contain a large toolbox of proteins dedicated to RNA metabolism. Investigation of four uncharacterized putative RNA-binding proteins-two RNA helicases, DHX30 and DDX28, and two proteins of the Fas-activated serine-threonine kinase (FASTKD) family, FASTKD2 and FASTKD5-demonstrated that both helicases and FASTKD2 are required for mitochondrial ribosome biogenesis. RNA-sequencing (RNA-seq) analysis showed that DDX28 and FASTKD2 bound the 16S rRNA. FASTKD5 is required for maturing precursor mRNAs that are not flanked by tRNAs and that therefore cannot be processed by the canonical mRNA maturation pathway. Silencing FASTKD5 rendered mature COX I mRNA almost undetectable, which severely reduced the synthesis of COX I, resulting in a complex IV assembly defect. These data demonstrate that mitochondrial RNA granules are centers for posttranscriptional RNA processing and the biogenesis of mitochondrial ribosomes.

The 3' addition of CCA to mitochondrial tRNASer(AGY) is specifically impaired in patients with mutations in the tRNA nucleotidyl transferase TRNT1.

Addition of the trinucleotide cytosine/cytosine/adenine (CCA) to the 3' end of transfer RNAs (tRNAs) is essential for translation and is catalyzed by the enzyme TRNT1 (tRNA nucleotidyl transferase), which functions in both the cytoplasm and mitochondria. Exome sequencing revealed TRNT1 mutations in two unrelated subjects with different clinical features. The first presented with acute lactic acidosis at 3 weeks of age and developed severe developmental delay, hypotonia, microcephaly, seizures, progressive cortical atrophy, neurosensorial deafness, sideroblastic anemia and renal Fanconi syndrome, dying at 21 months. The second presented at 3.5 years with gait ataxia, dysarthria, gross motor regression, hypotonia, ptosis and ophthalmoplegia and had abnormal signals in brainstem and dentate nucleus. In subject 1, muscle biopsy showed combined oxidative phosphorylation (OXPHOS) defects, but there was no OXPHOS deficiency in fibroblasts from either subject, despite a 10-fold-reduction in TRNT1 protein levels in fibroblasts of the first subject. Furthermore, in normal controls, TRNT1 protein levels are 10-fold lower in muscle than in fibroblasts. High resolution northern blots of subject fibroblast RNA suggested incomplete CCA addition to the non-canonical mitochondrial tRNA(Ser(AGY)), but no obvious qualitative differences in other mitochondrial or cytoplasmic tRNAs. Complete knockdown of TRNT1 in patient fibroblasts rendered mitochondrial tRNA(Ser(AGY)) undetectable, and markedly reduced mitochondrial translation, except polypeptides lacking Ser(AGY) codons. These data suggest that the clinical phenotypes associated with TRNT1 mutations are largely due to impaired mitochondrial translation, resulting from defective CCA addition to mitochondrial tRNA(Ser(AGY)), and that the severity of this biochemical phenotype determines the severity and tissue distribution of clinical features.

RMND1 deficiency associated with neonatal lactic acidosis, infantile onset renal failure, deafness, and multiorgan involvement.

RMND1 is an integral inner membrane mitochondrial protein that assembles into a large 240 kDa complex to support translation of the 13 polypeptides encoded on mtDNA, all of which are essential subunits of the oxidative phosphorylation (OXPHOS) complexes. Variants in RMND1 produce global defects in mitochondrial translation and were first reported in patients with severe neurological phenotypes leading to mortality in the first months of life. Using whole-exome sequencing, we identified compound heterozygous RMND1 variants in a 4-year-old patient with congenital lactic acidosis, severe myopathy, hearing loss, renal failure, and dysautonomia. The levels of mitochondrial ribosome proteins were reduced in patient fibroblasts, causing a translation defect, which was rescued by expression of the wild-type cDNA. RMND1 was almost undetectable by immunoblot analysis in patient muscle and fibroblasts. BN-PAGE analysis showed a severe combined OXPHOS assembly defect that was more prominent in patient muscle than in fibroblasts. Immunofluorescence experiments showed that RMND1 localizes to discrete foci in the mitochondrial network, juxtaposed to RNA granules where the primary mitochondrial transcripts are processed. RMND1 foci were not detected in patient fibroblasts. We hypothesize that RMND1 acts to anchor or stabilize the mitochondrial ribosome near the sites where the mRNAs are matured, spatially coupling post-transcriptional handling mRNAs with their translation, and that loss of function variants in RMND1 are associated with a unique constellation of clinical phenotypes that vary with the severity of the mitochondrial translation defect.

Tissue-specific responses to the LRPPRC founder mutation in French Canadian Leigh Syndrome.

French Canadian Leigh Syndrome (LSFC) is an early-onset, progressive neurodegenerative disorder with a distinct pattern of tissue involvement. Most cases are caused by a founder missense mutation in LRPPRC. LRPPRC forms a ribonucleoprotein complex with SLIRP, another RNA-binding protein, and this stabilizes polyadenylated mitochondrial mRNAs. LSFC fibroblasts have reduced levels of LRPPRC and a specific complex IV assembly defect; however, further depletion of mutant LRPPRC results in a complete failure to assemble a functional oxidative phosphorylation system, suggesting that LRPPRC levels determine the nature of the biochemical phenotype. We tested this hypothesis in cultured muscle cells and tissues from LSFC patients. LRPPRC levels were reduced in LSFC muscle cells, resulting in combined complex I and IV deficiencies. A similar combined deficiency was observed in skeletal muscle. Complex IV was only moderately reduced in LSFC heart, but was almost undetectable in liver. Both of these tissues showed elevated levels of complexes I and III. Despite the marked biochemical differences, the steady-state levels of LRPPRC and mitochondrial mRNAs were extremely low, LRPPRC was largely detergent-insoluble, and SLIRP was undetectable in all LSFC tissues. The level of the LRPPRC/SLIRP complex appeared much reduced in control tissues by the first dimension blue-native polyacrylamide gel electrophoresis (BN-PAGE) analysis compared with fibroblasts, and even by second dimension analysis it was virtually undetectable in control heart. These results point to tissue-specific pathways for the post-transcriptional handling of mitochondrial mRNAs and suggest that the biochemical defects in LSFC reflect the differential ability of tissues to adapt to the mutation.

Novel mutations in SCO1 as a cause of fatal infantile encephalopathy and lactic acidosis.

Isolated cytochrome c oxidase (COX) deficiency is a common cause of mitochondrial disease, yet its genetic basis remains unresolved in many patients. Here, we identified novel compound heterozygous mutations in SCO1 (p.M294V, p.Val93*) in one such patient with fatal encephalopathy. The patient lacked the severe hepatopathy (p.P174L) or hypertrophic cardiomyopathy (p.G132S) observed in previously reported SCO1 cases, so we investigated whether allele-specific defects in SCO1 function might underlie the genotype-phenotype relationships. Fibroblasts expressing p.M294V had a relatively modest decrease in COX activity compared with those expressing p.P174L, whereas both SCO1 lines had marked copper deficiencies. Overexpression of known pathogenic variants in SCO1 fibroblasts showed that p.G132S exacerbated the COX deficiency, whereas COX activity was partially or fully restored by p.P174L and p.M294V, respectively. These data suggest that the clinical phenotypes in SCO1 patients might reflect the residual capacity of the pathogenic alleles to perform one or both functions of SCO1.