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

• Published in: Human molecular genetics 2015-05, Vol.24 (10), p.2952-2965
• Main Authors: Formosa, Luke E, Mimaki, Masakazu, Frazier, Ann E, McKenzie, Matthew, Stait, Tegan L, Thorburn, David R, Stroud, David A, Ryan, Michael T
• Format: Article
• Language: English
• Subjects: Electron Transport Complex I - metabolism; HEK293 Cells; Humans; Mitochondria - metabolism; Mitochondrial Proteins - metabolism; Mitochondrial Proteins - physiology; Molecular Chaperones - genetics; Molecular Chaperones - physiology; Mutation; Protein Multimerization
• ISSN: 0964-6906; 1460-2083
• DOI: 10.1093/hmg/ddv058

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.