Formosa Luke E, Mimaki Masakazu, Frazier Ann E, McKenzie Matthew, Stait Tegan L, Thorburn David R, Stroud David A, Ryan Michael T
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.
Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC 3086, Australia.
Hum Mol Genet. 2015 May 15;24(10):2952-65. doi: 10.1093/hmg/ddv058. Epub 2015 Feb 12.
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.
人类线粒体复合物I是呼吸链中最大的酶，由44个不同的亚基组成。复合物I的亚基由核DNA和线粒体（mt）DNA共同编码，其组装需要许多其他蛋白质。含FAD依赖氧化还原酶结构域蛋白1（FOXRED1）最近被鉴定为一种假定的组装因子，患者中的FOXRED1突变会导致复合物I缺乏；然而，其在组装中的作用尚不清楚。在这里，我们证明FOXRED1参与复合物I组装的中后期阶段。在一名患有FOXRED1突变的患者中，成熟复合物I的水平显著降低，并检测到一个较小的约475 kDa亚复合物。在没有FOXRED1的情况下，mtDNA编码的复合物I亚基仍可翻译并短暂组装成一个晚期约815 kDa中间体；但该中间体没有进一步转变为成熟的复合物I，而是分解为一个约475 kDa的复合物。由于患者细胞含有残余组装的复合物I，我们通过TALEN介导的基因编辑在HEK293T细胞中破坏了FOXRED1基因。缺乏FOXRED1的细胞复合物I水平约为10%，复合物I活性降低，并且无法在半乳糖培养基上生长。有趣的是，含有患者突变的FOXRED1过表达能够挽救复合物I的组装。此外，发现FOXRED1与许多复合物I亚基共免疫沉淀。我们的研究表明，FOXRED1是复合物I有效组装的关键成分，FOXRED1中的突变导致功能部分丧失会引起复合物I生物合成缺陷。
