Dominant optic atrophy (DOA)1,2 and axonal peripheral neuropathy (Charcot-Marie-Tooth Type 2 or CMT2)3 are hereditary neurodegenerative disorders most commonly caused by mutations in the canonical mitochondrial fusion genes OPA1 and MFN2, respectively4. In yeast, homologs of OPA1(Mgm1) and MFN2(Fzo1) work in concert with Ugo15,6, which has no human equivalent to date7. By whole exome sequencing patients with optic atrophy and CMT2, we identified four families with recessive mutations in SLC25A46. We demonstrate that SLC25A46, like Ugo1, is a modified carrier protein that has been recruited to the outer mitochondrial membrane and interacts with the inner membrane remodeling protein, mitofilin(Fcj1). Loss-of-function in cultured cells and in zebrafish unexpectedly leads to increased mitochondrial connectivity, while severely affecting the development and maintenance of neurons in the fish. The discovery of SLC25A46 strengthens the genetic overlap between optic atrophy and CMT2, while exemplifying a novel class of modified solute transporters linked to mitochondrial dynamics.

SUMMARY The assembly and function of the mitochondrial respiratory chain (RC) involve the organization of RC enzyme complexes in supercomplexes or respirasomes through an unknown biosynthetic process. This leads to structural interdependences between RC complexes, which are highly relevant from biological and biomedical perspectives, because RC defects lead to severe human disorders. We show that in human cells, respirasome biogenesis involves a complex I assembly intermediate acting as a scaffold for the combined incorporation of complexes III and IV subunits, rather than originating from the association of preassembled individual holoenzymes. The process ends with the incorporation of complex I NADH dehydrogenase catalytic module, which leads to the respirasome activation. While complexes III and IV assemble either as free holoenzymes or by incorporation of free subunits into supercomplexes, the respirasomes constitute the structural units where complex I is assembled and activated, thus explaining the functional significance of the respirasomes for RC function.

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.

Cytochrome c-oxidase (COX), the terminal enzyme of the mitochondrial respiratory chain, plays a key role in the regulation of aerobic production of energy. Biogenesis of eukaryotic COX involves the coordinated action of two genomes. Three mitochondrial DNA-encoded subunits form the catalytic core of the enzyme, which contains metal prosthetic groups. Another 10 subunits encoded in the nuclear DNA act as a protective shield surrounding the core. COX biogenesis requires the assistance of >20 additional nuclear-encoded factors acting at all levels of the process. Expression of the mitochondrial-encoded subunits, expression and import of the nuclear-encoded subunits, insertion of the structural subunits into the mitochondrial inner membrane, addition of prosthetic groups, assembly of the holoenzyme, further maturation to form a dimer, and additional assembly into supercomplexes are all tightly regulated processes in a nuclear-mitochondrial-coordinated fashion. Such regulation ensures the building of a highly efficient machine able to catalyze the safe transfer of electrons from cytochrome c to molecular oxygen and ultimately facilitate the aerobic production of ATP. In this review, we will focus on describing and analyzing the present knowledge about the different regulatory checkpoints in COX assembly and the dynamic relationships between the different factors involved in the process. We have used information mostly obtained from the suitable yeast model, but also from bacterial and animal systems, by means of large-scale genetic, molecular biology, and physiological approaches and by integrating information concerning individual elements into a cellular system network.

Multiple mitochondrial DNA deletions are associated with clinically heterogeneous disorders transmitted as mendelian traits. Dominant missense mutations were found in the gene encoding the heart and skeletal muscle-specific isoform of the adenine nucleotide translocator (ANT1) in families with autosomal dominant progressive external opthalmoplegia and in a sporadic patient. We herein report on a sporadic patient who presented with hypertrophic cardiomyopathy, mild myopathy with exercise intolerance and lactic acidosis but no ophthalmoplegia. A muscle biopsy showed the presence of numerous ragged-red fibers, and Southern blot analysis disclosed multiple deletions of muscle mitochondrial DNA. Molecular analysis revealed a C to A homozygous mutation at nucleotide 368 of the ANT1 gene. The mutation converted a highly conserved alanine into an aspartic acid at codon 123 and was absent in 500 control individuals. This is the first report of a recessive mutation in the ANT1 gene. The clinical and biochemical features are different from those found in dominant ANT1 mutations, resembling those described in ANT1 knockout mice. No ATP uptake was measured in proteoliposomes reconstituted with protein extracts from the patient's muscle. The equivalent mutation in AAC2, the yeast ortholog of human ANT1, resulted in a complete loss of transport activity and in the inability to rescue the severe Oxidative Phosphorylation phenotype displayed by WB-12, an AAC1/AAC2 defective strain. Interestingly, exposure to reactive oxygen species (ROS) scavengers dramatically increased the viability of the WB-12 transformant, suggesting that increased redox stress is involved in the pathogenesis of the disease and that anti-ROS therapy may be beneficial to patients.

The oxidative phosphorylation (OXPHOS) system consists of five multimeric complexes embedded in the mitochondrial inner membrane. They work in concert to drive the aerobic synthesis of ATP. Mitochondrial and nuclear DNA mutations affecting the accumulation and function of these enzymes are the most common cause of mitochondrial diseases and have also been associated with neurodegeneration and aging. For this reason, several approaches for the assessment of the OXPHOS system enzymes have been developed. Based on the methods described elsewhere, we present here assays for a biochemical characterization of the OXPHOS system in cells and mitochondria isolated from cultured cells or tissues.

The ribosome is a structurally and functionally conserved macromolecular machine universally responsible for catalyzing protein synthesis. Within eukaryotic cells, mitochondria contain their own ribosomes (mitoribosomes), which synthesize a handful of proteins, all essential for the biogenesis of the oxidative phosphorylation system. High-resolution cryo-EM structures of the yeast, porcine and human mitoribosomal subunits and of the entire human mitoribosome have uncovered a wealth of new information to illustrate their evolutionary divergence from their bacterial ancestors and their adaptation to synthesis of highly hydrophobic membrane proteins. With such structural data becoming available, one of the most important remaining questions is that of the mitoribosome assembly pathway and factors involved. The regulation of mitoribosome biogenesis is paramount to mitochondrial respiration, and thus to cell viability, growth and differentiation. Moreover, mutations affecting the rRNA and protein components produce severe human mitochondrial disorders. Despite its biological and biomedical significance, knowledge on mitoribosome biogenesis and its deviations from the much-studied bacterial ribosome assembly processes is scarce, especially the order of rRNA processing and assembly events and the regulatory factors required to achieve fully functional particles. This article focuses on summarizing the current available information on mitoribosome assembly pathway, factors that form the mitoribosome assembly machinery, and the effect of defective mitoribosome assembly on human health.

Conflict of interest: GWB, AF, and SM are inventors on pending or issued patents (US 10,183,038 and US 10,052,345) aimed at diagnosing or treating proteinuric kidney diseases. They stand to gain royalties from the future commercialization of these patents. AF is Chief Scientific Officer of L&F Health LLC and is a consultant for Variant Pharmaceuticals. Variant Pharmaceuticals has licensed worldwide rights from L&F Research to develop and commercialize hydroxypropyl-beta-cyclodextrin for the treatment of kidney disease. AF is Chief Medical Officer of LipoNexT LLC. SM holds equity interest in a company presently commercializing the form of cyclodextrin referenced in this paper. AF and SM are supported by Hoffman-La Roche. AF is supported by Boehringer Ingelheim.