ABSTRACT
During the evolution from yeast to mammals the Mia40 protein, the regulator of the redox-sensitive mitochondrial intermembrane space import machinery, has lost its membrane-anchorage segment to become CHCHD4, which interacts with the flavoprotein apoptosis-inducing factor (AIF). Our results establish CHCHD4 as the missing link between AIF deficiency and dysfunctional biogenesis of respiratory chain complexes.
KEYWORDS: Disulfide relay, mitochondrial import, mitochondriopathy, respiratory chain complexes
Mitochondrial diseases are a group of heterogeneous inherited metabolic disorders that affect 1 in 5,000 individuals and are essentially caused by defects in the mitochondrial oxidative phosphorylation machinery.1 Our poor understanding of these diseases and the lack of efficient therapeutic solutions reflect the uniqueness of the respiratory chain, which is the only metabolic machinery under the dual control of nuclear and mitochondrial genes. Among the 90 subunits that constitute the 5 respiratory chain complexes (CI to CV), 13 subunits are encoded by the mitochondrial DNA whereas the additional 77 are nuclear encoded and imported into the organelle.1 Moreover, optimal performance of the organelle requires the import of more than 1,000 additional nuclear-encoded proteins that are indispensible for the biogenesis and/or the assembly of respiratory chain complexes, expression of the mitochondrial genome, regulation of mitochondrial ultrastructure, the finely tuned movement of mitochondria, and for crosstalk between the organelle and the nuclear compartment.1,2
Approximately 30% of human mitochondriopathies affect the complex CI (nicotinamide adenine dinucleotide (NADH): ubiquinone oxidoreductase; EC 1.6.5.3), a multiprotein complex that is composed of 7 mitochondrial- and 38 nuclear-encoded subunits and requires a series of “assembly factors” that are not contained in the mature complex for its assembly and maturation. One of the nuclear-encoded mitochondrial proteins that has been listed in the category of CI assembly factors is the redox-active flavoprotein apoptosis-inducing factor (AIF) that was initially discovered as a proapoptotic protein.3 In mitochondria from healthy cells AIF is confined to the intermembrane space (IMS). In all investigated species, the loss or downregulation of AIF provokes severe CI-related respiratory defects that are caused by a post-translational loss of CI protein subunits.3 Although complex CI remains the primary target of AIF dysfunction, losses of complex CIII (ubiquinol cytochrome c reductase; EC 1.10.2.2) and CIV (cytochrome c oxidase; EC 1.9.3.1) subunits were also observed in specific cell types.3 In humans, mutations in AIF have been associated with severe X-linked pediatric mitochondriopathies.4-6
The recent isolation of the first mitochondrial interactor of AIF, a protein called coiled-coil-helix-coiled-coil-helix domain containing 4 (CHCHD4), has shed new light on the mitochondrial activity of AIF.7 CHCHD4, which is the human homolog of yeast mitochondrial intermembrane space import and assembly protein 40 (Mia40),8 plays a central role in the import and oxidative folding of a group of small nuclear-encoded proteins (substrates) that essentially carry 2 cysteine-x3-cysteine (Cx3C)2 or 2 cysteine-x9-cysteine (Cx9C)2 motifs and participate in a large panel of heterogeneous activities in the IMS8-10 (Table 1). Given the vast number of mitochondrial processes that are covered by potential CHCHD4 substrates8-10 (Table 1), AIF cannot be solely considered as a complex CI assembly factor but rather as a central component of the redox-active CHCHD4-dependent import machinery that, in addition to the biogenesis of respiratory chain subunits,7 has the capacity to regulate additional processes ranging from protein import to intramitochondrial lipid homeostasis, antioxidant response, mitochondrial translation, or mitochondrial membrane organization.
Table 1.
Human proteins containing 2 (Cx9C) and 2 (Cx3C) motifs*.
| Symbol | Synonyms | Accession number | Cysteine motif | Function# | Chromosomal location |
|---|---|---|---|---|---|
| CHCHD1 | FLJ25854 | NP_976043 | Cx9C-Cx9C | Mitochondrial translation | 10q22.3 |
| CHCHD3 | MINOS3; Mic19 | NP_060282.1 | Cx9C-Cx9C | MICOS complex | 7q33 |
| CHCHD6 | CHCM1; Mic25 | NP_115719.1 | Cx9C-Cx9C | MICOS complex | 3q21.3 |
| CHCHD4 | MIA40; TIMM40 | NP_001091972.1 | Cx9C-Cx9C | IMS redox-regulated import | 3p25.1 |
| CHCHD10 | N27C7–4 | NP_001288268.1 | Cx9C-Cx9C | Cristae morphology | 22q11.23 |
| NDUFA8 | PGIV | NP_055037.1 | Cx9C-Cx9C | CI subunit | 9q33.2 |
| NDUFB7 | B18 | NP_004137.2 | Cx9C-Cx9C | CI subunit | 19p13.12 |
| NDUFS5 | CI-15k | NP_001171908.1 | Cx9C-Cx9C | CI subunit | 1p34.3 |
| UQCRH | QCR6 | AAH93060 | Cx10C-Cx9C | CIII subunit | 1p34.1 |
| COX6B1 | COXG | NP_001854.1 | Cx9C-Cx10C | CIV subunit | 19q13.12 |
| COX6B2 | CT59 | NP_653214.2 | Cx9C-Cx10C | CIV subunit | 19q13.42 |
| COX17 | NP_005685.1 | Cx9C-Cx9C | CIV copper chaperone | 3q13.33 | |
| COX19 | MGC104475 | NP_001026788 | Cx9C-Cx9C | CIV copper chaperone | 7p22.3 |
| CHCHD7 | COX23 | NP_001011668 | Cx9C-Cx9C | CIV copper chaperone | 8q11.23 |
| COA4 | CMC3; E2IG2 | NP_057649.2 | Cx9C-Cx9C | CIV assembly factor | 11q13.4 |
| COA5 | FLJ27524; Pet191 | NP_001008216 | Cx9C-Cx10C | CIV assembly factor | 2q11.2 |
| COA6 | NP_001013003.1 | Cx9C-Cx10C | CIV assembly factor | 1q42.2 | |
| CMC1 | MGC61571 | NP_872329 | Cx9C-Cx9C | CIV biogenesis | 3p24–1 |
| CMC2 | MGC45036; DC13 | NP_064573 | Cx9C-Cx9C | CIV biogenesis | 16q23.2 |
| CHCHD2 | NP_057223 | CX9C-CX9C | CIV subunit expression and assembly | 7p11.2 | |
| TRIAP1 | MDM35 | NP_057483 | Cx9C-Cx9C | Mitochondrial lipid homeostasis | 12q24.31 |
| CHCHD5 | MIC14 | NP_115685.1 | Cx9C-Cx9C | Unknown | 2q14.1 |
| CMC4 | P8MTCP1 | NP_001018024.1 | Cx9C-Cx9C | Unknown | Xq28 |
| UPF0545 | NP_776154 | Cx9C-Cx9C | Unknown | 22q11.21 | |
| C17orf89 | NP_001079990.1 | Cx9C-Cx9C | Unknown | 17q25.3 | |
| TIMM8A | DDP1 | NP_001139423.1 | Cx3C-Cx3C | Protein import | Xq22.1 |
| TIMM8B | DDP2 | NP_036591.2 | Cx3C-Cx3C | Protein import | 11q23.1 |
| TIMM9 | TIM9A | NP_001291414.1 | Cx3C-Cx3C | Protein import | 14q23.1 |
| TIMM10 | TIM10A | NP_036588.1 | Cx3C-Cx3C | Protein import | 11q12.1 |
| TIMM10B | TIM10B; Tim9B | NP_036324.1 | Cx3C-Cx3C | Protein import | 11p15.4 |
| TIMM13 | Tim13 | NP_036590.1 | Cx3C-Cx3C | Protein import | 19p13.3 |
Data have been extracted from References 8 to 10, as well as from public databases.
CI, respiratory chain complex CI; CIII, respiratory chain complex CIII; CIV, respiratory chain complex CIV; IMS, intermembrane space; MICOS, mitochondrial contact site.
Obviously, for a better understanding of the molecular impact of AIF activity on the CHCHD4-dependent import pathway, several questions need to be answered in the future. For instance, co-crystalization of AIF and CHCHD4 will reveal how AIF affects the structural maturation of CHCHD4 and its interaction with cofactors, its substrates, and its redox recycling partner, the sulfhydryl oxidase ERV1/ALR.8 The second question concerns the organo-specific impact of AIF deficiency on the biogenesis of specific respiratory chain complexes. Such tissue specificity is well described in the case of the AIF hypomorphic Harlequin (Hq) mouse model.3 CI deficiency was detected only in the brain and retina of Hq mice, but not in other organs, correlating with the tissue-specific degeneration that characterizes the phenotype of this mouse model.3 Is this peculiarity explainable by tissue-specific expression of certain CHCHD4 substrates? During the evolution from yeast to mammals, (Cx9C)2 motif-containing proteins have almost doubled in number. Beyond the evolutionary-conserved substrates implicated in the biogenesis of complex CIV (conserved between yeast and mammals), substrates corresponding to the subunits of complex CI (absent from yeast) have made their appearance (Table 1). An in-depth characterization of CHCHD4 substrates and their redox-regulated mitochondrial import in mammals should help us better understand the tissue-specific effect of AIF dysfunction on CI. Is this effect caused by the loss of CI subunits that are directly imported in a CHCHD4-dependent manner or is it an indirect effect provoked by the loss of other substrates? As recent progress in the field tends to support the notion that the assembly of complex CI is dependent on the formation of supercomplexes with CIII and CIV, an open alternative question concerns the potential crosstalk between complexes CI and CIV to explain the phenotype of AIF-deficient cells.
The molecular characterization of the physical and function interaction between AIF and CHCHD4 may spur new therapeutic strategies for the correction of respiratory defects that are caused by the loss or downregulation of AIF. As our results show that in vitro transfection of cells with a modified CHCHD4 whose mitochondrial import does not rely on AIF can repair the respiratory defect of AIF-deficient cells,7 we believe that strategies for the correction of CHCHD4-dependent import pathway should be considered as a potential therapeutic strategy for the treatment of mitochondriopathies that are caused by the loss or downregulation of AIF.
Disclosure of potential conflicts of interest
No potential conflicts of interest were disclosed.
Funding
This research was supported by French National Research Agency.
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