Sengers Syndrome-Associated Mitochondrial Acylglycerol Kinase Is a Subunit of the Human TIM22 Protein Import Complex.

Acylglycerol kinase (AGK) is a mitochondrial lipid kinase that catalyzes the phosphorylation of monoacylglycerol and diacylglycerol to lysophosphatidic acid and phosphatidic acid, respectively. Mutations in AGK cause Sengers syndrome, which is characterized by congenital cataracts, hypertrophic cardiomyopathy, skeletal myopathy, exercise intolerance, and lactic acidosis. Here we identified AGK as a subunit of the mitochondrial TIM22 protein import complex. We show that AGK functions in a kinase-independent manner to maintain the integrity of the TIM22 complex, where it facilitates the import and assembly of mitochondrial carrier proteins. Mitochondria isolated from Sengers syndrome patient cells and tissues show a destabilized TIM22 complex and defects in the biogenesis of carrier substrates. Consistent with this phenotype, we observe perturbations in the tricarboxylic acid (TCA) cycle in cells lacking AGK. Our identification of AGK as a bona fide subunit of TIM22 provides an exciting and unexpected link between mitochondrial protein import and Sengers syndrome.

Sideroflexin 4 is a complex I assembly factor that interacts with the MCIA complex and is required for the assembly of the ND2 module.

SignificanceMitochondria are double-membraned eukaryotic organelles that house the proteins required for generation of ATP, the energy currency of cells. ATP generation within mitochondria is performed by five multisubunit complexes (complexes I to V), the assembly of which is an intricate process. Mutations in subunits of these complexes, or the suite of proteins that help them assemble, lead to a severe multisystem condition called mitochondrial disease. We show that SFXN4, a protein that causes mitochondrial disease when mutated, assists with the assembly of complex I. This finding explains why mutations in SFXN4 cause mitochondrial disease and is surprising because SFXN4 belongs to a family of amino acid transporter proteins, suggesting that it has undergone a dramatic shift in function through evolution.

Accessory subunits are integral for assembly and function of human mitochondrial complex I.

Complex I (NADH:ubiquinone oxidoreductase) is the first enzyme of the mitochondrial respiratory chain and is composed of 45 subunits in humans, making it one of the largest known multi-subunit membrane protein complexes. Complex I exists in supercomplex forms with respiratory chain complexes III and IV, which are together required for the generation of a transmembrane proton gradient used for the synthesis of ATP. Complex I is also a major source of damaging reactive oxygen species and its dysfunction is associated with mitochondrial disease, Parkinson's disease and ageing. Bacterial and human complex I share 14 core subunits that are essential for enzymatic function; however, the role and necessity of the remaining 31 human accessory subunits is unclear. The incorporation of accessory subunits into the complex increases the cellular energetic cost and has necessitated the involvement of numerous assembly factors for complex I biogenesis. Here we use gene editing to generate human knockout cell lines for each accessory subunit. We show that 25 subunits are strictly required for assembly of a functional complex and 1 subunit is essential for cell viability. Quantitative proteomic analysis of cell lines revealed that loss of each subunit affects the stability of other subunits residing in the same structural module. Analysis of proteomic changes after the loss of specific modules revealed that ATP5SL and DMAC1 are required for assembly of the distal portion of the complex I membrane arm. Our results demonstrate the broad importance of accessory subunits in the structure and function of human complex I. Coupling gene-editing technology with proteomics represents a powerful tool for dissecting large multi-subunit complexes and enables the study of complex dysfunction at a cellular level.

Modelling Mitochondrial Disease in Human Pluripotent Stem Cells: What Have We Learned?

Mitochondrial diseases disrupt cellular energy production and are among the most complex group of inherited genetic disorders. Affecting approximately 1 in 5000 live births, they are both clinically and genetically heterogeneous, and can be highly tissue specific, but most often affect cell types with high energy demands in the brain, heart, and kidneys. There are currently no clinically validated treatment options available, despite several agents showing therapeutic promise. However, modelling these disorders is challenging as many non-human models of mitochondrial disease do not completely recapitulate human phenotypes for known disease genes. Additionally, access to disease-relevant cell or tissue types from patients is often limited. To overcome these difficulties, many groups have turned to human pluripotent stem cells (hPSCs) to model mitochondrial disease for both nuclear-DNA (nDNA) and mitochondrial-DNA (mtDNA) contexts. Leveraging the capacity of hPSCs to differentiate into clinically relevant cell types, these models permit both detailed investigation of cellular pathomechanisms and validation of promising treatment options. Here we catalogue hPSC models of mitochondrial disease that have been generated to date, summarise approaches and key outcomes of phenotypic profiling using these models, and discuss key criteria to guide future investigations using hPSC models of mitochondrial disease.

Mitochondrial energy generation disorders: genes, mechanisms, and clues to pathology.

Inherited disorders of oxidative phosphorylation cause the clinically and genetically heterogeneous diseases known as mitochondrial energy generation disorders, or mitochondrial diseases. Over the last three decades, mutations causing these disorders have been identified in almost 290 genes, but many patients still remain without a molecular diagnosis. Moreover, while our knowledge of the genetic causes is continually expanding, our understanding into how these defects lead to cellular dysfunction and organ pathology is still incomplete. Here, we review recent developments in disease gene discovery, functional characterization, and shared pathogenic parameters influencing disease pathology that offer promising avenues toward the development of effective therapies.

ATAD3 gene cluster deletions cause cerebellar dysfunction associated with altered mitochondrial DNA and cholesterol metabolism.

Although mitochondrial disorders are clinically heterogeneous, they frequently involve the central nervous system and are among the most common neurogenetic disorders. Identifying the causal genes has benefited enormously from advances in high-throughput sequencing technologies; however, once the defect is known, researchers face the challenge of deciphering the underlying disease mechanism. Here we characterize large biallelic deletions in the region encoding the ATAD3C, ATAD3B and ATAD3A genes. Although high homology complicates genomic analysis of the ATAD3 defects, they can be identified by targeted analysis of standard single nucleotide polymorphism array and whole exome sequencing data. We report deletions that generate chimeric ATAD3B/ATAD3A fusion genes in individuals from four unrelated families with fatal congenital pontocerebellar hypoplasia, whereas a case with genomic rearrangements affecting the ATAD3C/ATAD3B genes on one allele and ATAD3B/ATAD3A genes on the other displays later-onset encephalopathy with cerebellar atrophy, ataxia and dystonia. Fibroblasts from affected individuals display mitochondrial DNA abnormalities, associated with multiple indicators of altered cholesterol metabolism. Moreover, drug-induced perturbations of cholesterol homeostasis cause mitochondrial DNA disorganization in control cells, while mitochondrial DNA aggregation in the genetic cholesterol trafficking disorder Niemann-Pick type C disease further corroborates the interdependence of mitochondrial DNA organization and cholesterol. These data demonstrate the integration of mitochondria in cellular cholesterol homeostasis, in which ATAD3 plays a critical role. The dual problem of perturbed cholesterol metabolism and mitochondrial dysfunction could be widespread in neurological and neurodegenerative diseases.

Characterization of mitochondrial FOXRED1 in the assembly of respiratory chain complex I.

Human mitochondrial complex I is the largest enzyme of the respiratory chain and is composed of 44 different subunits. Complex I subunits are encoded by both nuclear and mitochondrial (mt) DNA and their assembly requires a number of additional proteins. FAD-dependent oxidoreductase domain-containing protein 1 (FOXRED1) was recently identified as a putative assembly factor and FOXRED1 mutations in patients cause complex I deficiency; however, its role in assembly is unknown. Here, we demonstrate that FOXRED1 is involved in mid-late stages of complex I assembly. In a patient with FOXRED1 mutations, the levels of mature complex I were markedly decreased, and a smaller ∼475 kDa subcomplex was detected. In the absence of FOXRED1, mtDNA-encoded complex I subunits are still translated and transiently assembled into a late stage ∼815 kDa intermediate; but instead of transitioning further to the mature complex I, the intermediate breaks down to an ∼475 kDa complex. As the patient cells contained residual assembled complex I, we disrupted the FOXRED1 gene in HEK293T cells through TALEN-mediated gene editing. Cells lacking FOXRED1 had ∼10% complex I levels, reduced complex I activity, and were unable to grow on galactose media. Interestingly, overexpression of FOXRED1 containing the patient mutations was able to rescue complex I assembly. In addition, FOXRED1 was found to co-immunoprecipitate with a number of complex I subunits. Our studies reveal that FOXRED1 is a crucial component in the productive assembly of complex I and that mutations in FOXRED1 leading to partial loss of function cause defects in complex I biogenesis.

COA6 is a mitochondrial complex IV assembly factor critical for biogenesis of mtDNA-encoded COX2.

Biogenesis of complex IV of the mitochondrial respiratory chain requires assembly factors for subunit maturation, co-factor attachment and stabilization of intermediate assemblies. A pathogenic mutation in COA6, leading to substitution of a conserved tryptophan for a cysteine residue, results in a loss of complex IV activity and cardiomyopathy. Here, we demonstrate that the complex IV defect correlates with a severe loss in complex IV assembly in patient heart but not fibroblasts. Complete loss of COA6 activity using gene editing in HEK293T cells resulted in a profound growth defect due to complex IV deficiency, caused by impaired biogenesis of the copper-bound mitochondrial DNA-encoded subunit COX2 and subsequent accumulation of complex IV assembly intermediates. We show that the pathogenic mutation in COA6 does not affect its import into mitochondria but impairs its maturation and stability. Furthermore, we show that COA6 has the capacity to bind copper and can associate with newly translated COX2 and the mitochondrial copper chaperone SCO1. Our data reveal that COA6 is intricately involved in the copper-dependent biogenesis of COX2.

Mutations in the UQCC1-interacting protein, UQCC2, cause human complex III deficiency associated with perturbed cytochrome b protein expression.

Mitochondrial oxidative phosphorylation (OXPHOS) is responsible for generating the majority of cellular ATP. Complex III (ubiquinol-cytochrome c oxidoreductase) is the third of five OXPHOS complexes. Complex III assembly relies on the coordinated expression of the mitochondrial and nuclear genomes, with 10 subunits encoded by nuclear DNA and one by mitochondrial DNA (mtDNA). Complex III deficiency is a debilitating and often fatal disorder that can arise from mutations in complex III subunit genes or one of three known complex III assembly factors. The molecular cause for complex III deficiency in about half of cases, however, is unknown and there are likely many complex III assembly factors yet to be identified. Here, we used Massively Parallel Sequencing to identify a homozygous splicing mutation in the gene encoding Ubiquinol-Cytochrome c Reductase Complex Assembly Factor 2 (UQCC2) in a consanguineous Lebanese patient displaying complex III deficiency, severe intrauterine growth retardation, neonatal lactic acidosis and renal tubular dysfunction. We prove causality of the mutation via lentiviral correction studies in patient fibroblasts. Sequence-profile based orthology prediction shows UQCC2 is an ortholog of the Saccharomyces cerevisiae complex III assembly factor, Cbp6p, although its sequence has diverged substantially. Co-purification studies show that UQCC2 interacts with UQCC1, the predicted ortholog of the Cbp6p binding partner, Cbp3p. Fibroblasts from the patient with UQCC2 mutations have deficiency of UQCC1, while UQCC1-depleted cells have reduced levels of UQCC2 and complex III. We show that UQCC1 binds the newly synthesized mtDNA-encoded cytochrome b subunit of complex III and that UQCC2 patient fibroblasts have specific defects in the synthesis or stability of cytochrome b. This work reveals a new cause for complex III deficiency that can assist future patient diagnosis, and provides insight into human complex III assembly by establishing that UQCC1 and UQCC2 are complex III assembly factors participating in cytochrome b biogenesis.

Modelling biochemical features of mitochondrial neuropathology.

BACKGROUND: The neuropathology of mitochondrial disease is well characterised. However, pathophysiological mechanisms at the level of biochemistry and cell biology are less clear. Progress in this area has been hampered by the limited accessibility of neurologically relevant material for analysis. SCOPE OF REVIEW: Here we discuss the recent development of a variety of model systems that have greatly extended our capacity to understand the biochemical features associated with mitochondrial neuropathology. These include animal and cell based models, with mutations in both nuclear and mitochondrial DNA encoded genes, which aim to recapitulate the neuropathology and cellular biochemistry of mitochondrial diseases. MAJOR CONCLUSIONS: Analysis of neurological tissue and cells from these models suggests that although there is no unifying mode of pathogenesis, dysfunction of the oxidative phosphorylation (OXPHOS) system is often central. This can be associated with altered reactive oxygen species (ROS) generation, disruption of the mitochondrial membrane potential (ΔΨm) and inadequate ATP synthesis. Thus, other cellular processes such as calcium (Ca(2+)) homeostasis, cellular signaling and mitochondrial morphology could be altered, ultimately compromising viability of neuronal cells. GENERAL SIGNIFICANCE: Mechanisms of neuronal dysfunction in mitochondrial disease are only just beginning to be characterised, are system dependent and complex, and not merely driven by energy deficiency. The diversity of pathogenic mechanisms emphasises the need for characterisation in a wide range of models, as different therapeutic strategies are likely to be needed for different diseases. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research.

Reduced Protein Import via TIM23 SORT Drives Disease Pathology in TIMM50-Associated Mitochondrial Disease.

TIMM50 is a core subunit of the TIM23 complex, the mitochondrial inner membrane translocase responsible for the import of pre-sequence-containing precursors into the mitochondrial matrix and inner membrane. Here we describe a mitochondrial disease patient who is homozygous for a novel variant in TIMM50 and establish the first proteomic map of mitochondrial disease associated with TIMM50 dysfunction. We demonstrate that TIMM50 pathogenic variants reduce the levels and activity of endogenous TIM23 complex, which significantly impacts the mitochondrial proteome, resulting in a combined oxidative phosphorylation (OXPHOS) defect and changes to mitochondrial ultrastructure. Using proteomic data sets from TIMM50 patient fibroblasts and a TIMM50 HEK293 cell model of disease, we reveal that laterally released substrates imported via the TIM23SORT complex pathway are most sensitive to loss of TIMM50. Proteins involved in OXPHOS and mitochondrial ultrastructure are enriched in the TIM23SORT substrate pool, providing a biochemical mechanism for the specific defects in TIMM50-associated mitochondrial disease patients. These results highlight the power of using proteomics to elucidate molecular mechanisms of disease and uncovering novel features of fundamental biology, with the implication that human TIMM50 may have a more pronounced role in lateral insertion than previously understood.

CLPB disaggregase dysfunction impacts the functional integrity of the proteolytic SPY complex.

CLPB is a mitochondrial intermembrane space AAA+ domain-containing disaggregase. CLPB mutations are associated with 3-methylglutaconic aciduria and neutropenia; however, the molecular mechanism underscoring disease and the contribution of CLPB substrates to disease pathology remains unknown. Interactions between CLPB and mitochondrial quality control (QC) factors, including PARL and OPA1, have been reported, hinting at dysregulation of organelle QC in disease. Utilizing proteomic and biochemical approaches, we show a stress-specific aggregation phenotype in a CLPB-null environment and define the CLPB substrate profile. We illustrate an interplay between intermembrane space proteins including CLPB, HAX1, HTRA2, and the inner membrane quality control proteins (STOML2, PARL, YME1L1; SPY complex), with CLPB deficiency impeding SPY complex function by virtue of protein aggregation in the intermembrane space. We conclude that there is an interdependency of mitochondrial QC components at the intermembrane space/inner membrane interface, and perturbations to this network may underscore CLPB disease pathology.

MiD49 and MiD51, new components of the mitochondrial fission machinery.

Mitochondria form intricate networks through fission and fusion events. Here, we identify mitochondrial dynamics proteins of 49 and 51 kDa (MiD49 and MiD51, respectively) anchored in the mitochondrial outer membrane. MiD49/51 form foci and rings around mitochondria similar to the fission mediator dynamin-related protein 1 (Drp1). MiD49/51 directly recruit Drp1 to the mitochondrial surface, whereas their knockdown reduces Drp1 association, leading to unopposed fusion. Overexpression of MiD49/51 seems to sequester Drp1 from functioning at mitochondria and cause fused tubules to associate with actin. Thus, MiD49/51 are new mediators of mitochondrial division affecting Drp1 action at mitochondria.

Mitochondrial biology and dysfunction in secondary mitochondrial disease.

Mitochondrial diseases are a broad, genetically heterogeneous class of metabolic disorders characterized by deficits in oxidative phosphorylation (OXPHOS). Primary mitochondrial disease (PMD) defines pathologies resulting from mutation of mitochondrial DNA (mtDNA) or nuclear genes affecting either mtDNA expression or the biogenesis and function of the respiratory chain. Secondary mitochondrial disease (SMD) arises due to mutation of nuclear-encoded genes independent of, or indirectly influencing OXPHOS assembly and operation. Despite instances of novel SMD increasing year-on-year, PMD is much more widely discussed in the literature. Indeed, since the implementation of next generation sequencing (NGS) techniques in 2010, many novel mitochondrial disease genes have been identified, approximately half of which are linked to SMD. This review will consolidate existing knowledge of SMDs and outline discrete categories within which to better understand the diversity of SMD phenotypes. By providing context to the biochemical and molecular pathways perturbed in SMD, we hope to further demonstrate the intricacies of SMD pathologies outside of their indirect contribution to mitochondrial energy generation.

Inhibition of Bak activation by VDAC2 is dependent on the Bak transmembrane anchor.

Bax and Bak are pro-apoptotic factors that are required for cell death by the mitochondrial or intrinsic pathway. Bax is found in an inactive state in the cytosol and upon activation is targeted to the mitochondrial outer membrane where it releases cytochrome c and other factors that cause caspase activation. Although Bak functions in the same way as Bax, it is constitutively localized to the mitochondrial outer membrane. In the membrane, Bak activation is inhibited by the voltage-dependent anion channel isoform 2 (VDAC2) by an unknown mechanism. Using blue native gel electrophoresis, we show that in healthy cells endogenous inactive Bak exists in a 400-kDa complex that is dependent on the presence of VDAC2. Activation of Bak is concomitant with its release from the 400-kDa complex and the formation of lower molecular weight species. Furthermore, substitution of the Bak transmembrane anchor with that of the mitochondrial outer membrane tail-anchored protein hFis1 prevents association of Bak with the VDAC2 complex and increases the sensitivity of cells to an apoptotic stimulus. Our results suggest that VDAC2 interacts with the hydrophobic tail of Bak to sequester it in an inactive state in the mitochondrial outer membrane, thereby raising the stimulation threshold necessary for permeabilization of the mitochondrial outer membrane and cell death.

Human Miltons associate with mitochondria and induce microtubule-dependent remodeling of mitochondrial networks.

Proper mitochondrial distribution is crucial for cell function. In Drosophila, mitochondrial transport is facilitated by Miro and Milton, which regulate mitochondrial attachment to microtubules via kinesin heavy chain. Mammals contain two sequence orthologs of Milton however, they have been ascribed various functions in intracellular transport. In this report, we show that the human Miltons target to mitochondria irrespective of whether they are linked to GFP at their C- or N-termini. Their ectopic expression induces the formation of extended mitochondrial tubules as well as large bulbous-like mitochondria with narrow tubular membrane necks that connect them to the mitochondrial mass. The mitochondrial extensions appear highly dynamic and their formation relies on the presence of microtubules. Using the photoswitchable fluorescent protein Dendra2 targeted to the mitochondrial matrix, we found that the mitochondrial extensions and bulbous mitochondria are fused with neighboring regions of the network. Truncation analysis of huMilton1 revealed that the N-terminal region, inclusive of the coiled-coil segment could localize to microtubules, suggesting that Milton attachment to kinesin occurs independent of Miro or mitochondrial attachment. In addition, we show that the huMiltons have the capacity to self-interact and can also facilitate mitochondrial recruitment of a cytosolic Miro mutant. We conclude that the human Miltons are important mediators of the mitochondrial trafficking machinery.

Shy1 couples Cox1 translational regulation to cytochrome c oxidase assembly.

Cytochrome c oxidase (complex IV) of the respiratory chain is assembled from nuclear and mitochondrially-encoded subunits. Defects in the assembly process lead to severe human disorders such as Leigh syndrome. Shy1 is an assembly factor for complex IV in Saccharomyces cerevisiae and mutations of its human homolog, SURF1, are the most frequent cause for Leigh syndrome. We report that Shy1 promotes complex IV biogenesis through association with different protein modules; Shy1 interacts with Mss51 and Cox14, translational regulators of Cox1. Additionally, Shy1 associates with the subcomplexes of complex IV that are potential assembly intermediates. Formation of these subcomplexes depends on Coa1 (YIL157c), a novel assembly factor that cooperates with Shy1. Moreover, partially assembled forms of complex IV bound to Shy1 and Cox14 can associate with the bc1 complex to form transitional supercomplexes. We suggest that Shy1 links Cox1 translational regulation to complex IV assembly and supercomplex formation.

Mdm38 interacts with ribosomes and is a component of the mitochondrial protein export machinery.

Saccharomyces cerevisiae Mdm38 and Ylh47 are homologues of human Letm1, a protein implicated in Wolf-Hirschhorn syndrome. We analyzed the function of Mdm38 and Ylh47 in yeast mitochondria to gain insight into the role of Letm1. We find that mdm38Delta mitochondria have reduced amounts of certain mitochondrially encoded proteins and low levels of complex III and IV and accumulate unassembled Atp6 of complex V of the respiratory chain. Mdm38 is especially required for efficient transport of Atp6 and cytochrome b across the inner membrane, whereas Ylh47 plays a minor role in this process. Both Mdm38 and Ylh47 form stable complexes with mitochondrial ribosomes, similar to what has been reported for Oxa1, a central component of the mitochondrial export machinery. Our results indicate that Mdm38 functions as a component of an Oxa1-independent insertion machinery in the inner membrane and that Mdm38 plays a critical role in the biogenesis of the respiratory chain by coupling ribosome function to protein transport across the inner membrane.

Rothmund-Thomson Syndrome-Like RECQL4 Truncating Mutations Cause a Haploinsufficient Low-Bone-Mass Phenotype in Mice.

Rothmund-Thomson syndrome (RTS) is an autosomal recessive disorder characterized by defects in the skeletal system, such as bone hypoplasia, short stature, low bone mass, and an increased incidence of osteosarcoma. RTS type 2 patients have germ line compound biallelic protein-truncating mutations of RECQL4. As existing murine models employ Recql4 null alleles, we have attempted to more accurately model RTS by generating mice with patient-mimicking truncating Recql4 mutations. Truncating mutations impaired the stability and subcellular localization of RECQL4 and resulted in homozygous embryonic lethality and a haploinsufficient low-bone mass phenotype. Combination of a truncating mutation with a conditional Recql4 null allele demonstrated that the skeletal defects were intrinsic to the osteoblast lineage. However, the truncating mutations did not promote tumorigenesis. We utilized murine Recql4 null cells to assess the impact of human RECQL4 mutations using an in vitro complementation assay. While some mutations created unstable protein products, others altered subcellular localization of the protein. Interestingly, the severity of the phenotypes correlated with the extent of protein truncation. Collectively, our results reveal that truncating RECQL4 mutations in mice lead to an osteoporosis-like phenotype through defects in early osteoblast progenitors and identify RECQL4 gene dosage as a novel regulator of bone mass.