Journal of Molecular Biology
Mutations in the Gene Encoding C8orf38 Block Complex I Assembly by Inhibiting Production of the Mitochondria-Encoded Subunit ND1
Graphical Abstract
Research Highlights
► C8orf38 is a mitochondrial protein involved in complex I biogenesis. ► Mutations in C8orf38 result in Leigh disease with isolated complex I deficiency. ► C8orf38 is involved in the biogenesis of the complex I subunit ND1. ► Defects in C8orf38 disrupt the assembly of complex I.
Introduction
NADH–ubiquinone oxidoreductase (complex I) is the first enzyme complex of the mitochondrial respiratory chain, oxidizing NADH to liberate electrons that facilitate the translocation of protons across the inner membrane to generate a proton gradient (Δψm).1 Mammalian complex I is ∼ 1 MDa in size and has an intricate structure composed of 45 subunits,2 38 of which are encoded by nuclear genes and 7 of which are encoded by mitochondrial DNA (mtDNA). Electron microscopy and structural analysis have revealed that complex I has an L-shaped structure and consists of a hydrophobic membrane arm and a peripheral arm that protrudes into the mitochondrial matrix.3, 4, 5, 6 The coordinated assembly of 45 different subunits to generate complex I in mammals is a complicated process. Analyses of mitochondria from complex-I-deficient patient cells using blue native-PAGE (BN-PAGE) have identified stalled assembly intermediates that correspond to discrete stages in the assembly pathway.7, 8 Assembly intermediates have also been identified by analysis of de novo assembly after depletion of complex I9, 10 or by tracking the progression of individual nuclear-encoded subunits into the mature holoenzyme.10, 11, 12 These studies suggest that complex I is assembled by a mechanism involving subunit entry into modular intermediates and the dynamic exchange of newly imported subunits with preexisting components of the mature complex.13
Isolated defects in complex I are the most common of the respiratory chain disorders and frequently result in decreased levels of the mature holo-complex.8, 14, 15 Only about one-third of these cases can be attributed to mutations in genes encoding structural subunits of complex I, suggesting that defects in other proteins, such as assembly factors/chaperones that are involved in complex I biogenesis, account for the remaining cases.16 To date, mutations in nine nuclear genes that cause complex I enzymatic deficiency due to impaired assembly have been described; these are CIA30/NDUFAF1,17, 18, 19 B17.2L/NDUFAF2,20, 21, 22 C6orf66/NDUFAF4,23 C20orf7,24, 25, 26 C3orf60/NDUFAF3,27 FOXRED1,16, 28 NUBPL,16 C8orf38,29 and ACAD9.30, 31, 32 In addition, ECSIT (an evolutionary conserved signaling intermediate in Toll pathways), which is found in intermediates with NDUFAF1,33 and MidA, which interacts with the complex I subunit NDUFS2,34 are also involved in various aspects of complex I biogenesis; however, human pathogenic gene mutations have not been identified.
Phylogenetic profiling has also been used to identify putative complex I assembly factors by comparing genes that have coevolved with complex I.29 The translation products of some of these complex I phylogenetic profile (COPP) genes are yet to be fully characterized, but future biochemical analyses may confirm these proteins as bona fide complex I assembly factors. Indeed, one of these COPP genes, C8orf38, was subsequently identified by homozygosity mapping to harbor mutations in two siblings presenting with Leigh syndrome and isolated complex I deficiency (although the pathogenicity of these mutations was not established).29 RNA interference studies of C8orf38 in control human fibroblasts were also shown to reduce complex I activity by 80%,29 providing compelling evidence that C8orf38 is required for normal complex I activity. However, it is not yet known if (and how) C8orf38 is involved in the biogenesis of complex I.
In this report, we find that C8orf38 is localized to mitochondria where it is found peripherally associated with the matrix face of the inner membrane. Patient fibroblasts harboring a mutation in the C8orf38 gene contained reduced steady-state complex I levels and a defect in the translation of the mtDNA-encoded subunit ND1. The loss of this subunit resulted in the stalled assembly of other subunits in early-stage assembly intermediates. Complementation in C8orf38 mutant patient cells with wild-type C8orf38 restored both the amount and the activity of complex I to normal levels, confirming the C8orf38 mutation as the cause of the complex I deficiency. We conclude that C8orf38 is involved in complex I assembly, specifically acting at the level of ND1 biogenesis.
Section snippets
C8orf38 is associated with the matrix face of the inner membrane
C8orf38 was initially categorized as a mitochondrial protein by mass spectrometric analysis of mitochondria purified from various mouse tissues.29 Three alternatively spliced isoforms are predicted for human C8orf38† (Fig. 1a). The “canonical” reference sequence (NP_689629) translates a 38-kDa precursor protein of 333 amino acids (isoform 1, Q330K2-1), which has a predicted mitochondrial localization (92%, Mitopred; 72%, Targetp v1.1). This isoform of human
Discussion
C8orf38 was initially identified as a protein involved in complex I biogenesis using two separate methods: (1) phylogenetic profiling, which categorized it as part of the COPP gene set, and (2) homozygosity mapping in two siblings with Leigh syndrome and complex I deficiency.29 While this information validated the phylogenetic approach used by Pagliarini et al., the functional role of C8orf38 in complex I biogenesis was not determined. Using patient fibroblasts, we found that the pathogenic
Cell lines and culture conditions
Cells were grown at 37 °C and 5% CO2 in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 5–10% (v/v) fetal bovine serum (Invitrogen) and 50 μg/ml uridine.
Mitochondrial isolation
Mitochondria were isolated according to Johnston et al.46 Cell pellets were resuspended in isolation solution [20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.6), 220 mM mannitol, 70 mM sucrose, 1 mM ethylenediaminetetraacetic acid, 0.5 mM PMSF, and 2 mg/ml bovine serum albumin] and
Acknowledgements
This work was supported by grants and fellowships from the National Health and Medical Research Council (433028 and 541911 to M.M.; 436901 and 436906 to D.T.), the Ramaciotti Foundation, the James and Vera Lawson Trust, an Australian Postgraduate Award (E.T.), and the Victorian Government's Operational Infrastructure Support Program.
References (49)
- et al.
Bovine complex I is a complex of 45 different subunits
J. Biol. Chem.
(2006) Three-dimensional structure of bovine NADH:ubiquinone oxidoreductase (complex I) at 22 Å in ice
J. Mol. Biol.
(1998)- et al.
A role for native lipids in the stabilization and two-dimensional crystallization of the Escherichia coli NADH–ubiquinone oxidoreductase (complex I)
J. Biol. Chem.
(2003) - et al.
The structure of eukaryotic and prokaryotic complex I
J. Struct. Biol.
(2010) - et al.
Identification and characterization of a common set of complex I assembly intermediates in mitochondria from patients with complex I deficiency
J. Biol. Chem.
(2003) - et al.
Subunits of mitochondrial complex I exist as part of matrix- and membrane-associated subcomplexes in living cells
J. Biol. Chem.
(2008) - et al.
Identification of mitochondrial complex I assembly intermediates by tracing tagged NDUFS3 demonstrates the entry point of mitochondrial subunits
J. Biol. Chem.
(2007) - et al.
Assembly of mitochondrial complex I and defects in disease
Biochim. Biophys. Acta
(2009) - et al.
The unique neuroradiology of complex I deficiency due to NDUFA12L defect
Mol. Genet. Metab.
(2008) - et al.
C6ORF66 is an assembly factor of mitochondrial complex I
Am. J. Hum. Genet.
(2008)