RESUMO
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.
Assuntos
Mitocôndrias , Doenças Mitocondriais , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Fosforilação Oxidativa , Transporte Proteico , Humanos , Fibroblastos/metabolismo , Células HEK293 , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/genética , Mitocôndrias/metabolismo , Doenças Mitocondriais/metabolismo , Doenças Mitocondriais/patologia , Doenças Mitocondriais/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Membranas Mitocondriais/metabolismo , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Mutação/genética , Proteômica/métodosRESUMO
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.