title: Characterization of mitochondrial FOXRED1 in the assembly of respiratory chain complex I.
abstract: 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.
authors: Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC 3086, Australia, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Melbourne 3800, Australia.
authors: Luke E
authors: LE
authors: Formosa
authors: Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC 3086, Australia.
authors: Masakazu
authors: M
authors: Mimaki
authors: Murdoch Childrens Research Institute, Royal Children's Hospital and University of Melbourne Department of Paediatrics, University of Melbourne, Melbourne, VIC 3052, Australia.
authors: Ann E
authors: AE
authors: Frazier
authors: Centre for Genetic Diseases, MIMR-PHI Institute of Medical Research, Clayton, Melbourne 3168, Australia and.
authors: Matthew
authors: M
authors: McKenzie
authors: Murdoch Childrens Research Institute, Royal Children's Hospital and University of Melbourne Department of Paediatrics, University of Melbourne, Melbourne, VIC 3052, Australia.
authors: Tegan L
authors: TL
authors: Stait
authors: Murdoch Childrens Research Institute, Royal Children's Hospital and University of Melbourne Department of Paediatrics, University of Melbourne, Melbourne, VIC 3052, Australia, Victorian Clinical Genetics Services, Royal Children's Hospital, Melbourne, VIC 3052, Australia.
authors: David R
authors: DR
authors: Thorburn
authors: Department of Biochemistry and Molecular Biology, Monash University, Clayton, Melbourne 3800, Australia.
authors: David A
authors: DA
authors: Stroud
authors: Department of Biochemistry and Molecular Biology, Monash University, Clayton, Melbourne 3800, Australia, Michael.Ryan@monash.edu.
authors: Michael T
authors: MT
authors: Ryan
mesh_terms: D042967:Electron Transport Complex I / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000378:metabolism* / Q000502:physiology; D018832:Molecular Chaperones / Q000235:genetics / Q000502:physiology*; D009154:Mutation; D055503:Protein Multimerization
chemical_list: C556294:FOXRED1 protein, human; D024101:Mitochondrial Proteins; D018832:Molecular Chaperones; D042967:Electron Transport Complex I