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.

Human Tim8a, Tim8b and Tim13 are auxiliary assembly factors of mature Complex IV.

Human Tim8a and Tim8b are paralogous intermembrane space proteins of the small TIM chaperone family. Yeast small TIMs function in the trafficking of proteins to the outer and inner mitochondrial membranes. This putative import function for hTim8a and hTim8b has been challenged in human models, but their precise molecular function(s) remains undefined. Likewise, the necessity for human cells to encode two Tim8 proteins and whether any potential redundancy exists is unclear. We demonstrate that hTim8a and hTim8b function in the assembly of cytochrome c oxidase (Complex IV). Using affinity enrichment mass spectrometry, we define the interaction network of hTim8a, hTim8b and hTim13, identifying subunits and assembly factors of the Complex IV COX2 module. hTim8-deficient cells have a COX2 and COX3 module defect and exhibit an accumulation of the Complex IV S2 subcomplex. These data suggest that hTim8a and hTim8b function in assembly of Complex IV via interactions with intermediate-assembly subcomplexes. We propose that hTim8-hTim13 complexes are auxiliary assembly factors involved in the formation of the Complex IV S3 subcomplex during assembly of mature Complex IV.

Loss of mitochondrial fatty acid ß-oxidation protein short-chain Enoyl-CoA hydratase disrupts oxidative phosphorylation protein complex stability and function.

Short-chain enoyl-CoA hydratase 1 (ECHS1) is involved in the second step of mitochondrial fatty acid ß-oxidation (FAO), catalysing the hydration of short-chain enoyl-CoA esters to short-chain 3-hyroxyl-CoA esters. Genetic deficiency in ECHS1 (ECHS1D) is associated with a specific subset of Leigh Syndrome, a disease typically caused by defects in oxidative phosphorylation (OXPHOS). Here, we examined the molecular pathogenesis of ECHS1D using a CRISPR/Cas9 edited human cell 'knockout' model and fibroblasts from ECHS1D patients. Transcriptome analysis of ECHS1 'knockout' cells showed reductions in key mitochondrial pathways, including the tricarboxylic acid cycle, receptor-mediated mitophagy and nucleotide biosynthesis. Subsequent proteomic analyses confirmed these reductions and revealed additional defects in mitochondrial oxidoreductase activity and fatty acid ß-oxidation. Functional analysis of ECHS1 'knockout' cells showed reduced mitochondrial oxygen consumption rates when metabolising glucose or OXPHOS complex I-linked substrates, as well as decreased complex I and complex IV enzyme activities. ECHS1 'knockout' cells also exhibited decreased OXPHOS protein complex steady-state levels (complex I, complex III2 , complex IV, complex V and supercomplexes CIII2 /CIV and CI/CIII2 /CIV), which were associated with a defect in complex I assembly. Patient fibroblasts exhibit varied reduction of mature OXPHOS complex steady-state levels, with defects detected in CIII2 , CIV, CV and the CI/CIII2 /CIV supercomplex. Overall, these findings highlight the contribution of defective OXPHOS function, in particular complex I deficiency, to the molecular pathogenesis of ECHS1D.

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.

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.