Optic atrophy-associated TMEM126A is an assembly factor for the ND4-module of mitochondrial complex I

Abstract

Mitochondrial disease is a debilitating condition with a diverse genetic etiology. Here, we report that TMEM126A, a protein that is mutated in patients with autosomal-recessive optic atrophy, participates directly in the assembly of mitochondrial complex I. Using a combination of genome editing, interaction studies, and quantitative proteomics, we find that loss of TMEM126A results in an isolated complex I deficiency and that TMEM126A interacts with a number of complex I subunits and assembly factors. Pulse-labeling interaction studies reveal that TMEM126A associates with the newly synthesized mitochondrial DNA (mtDNA)-encoded ND4 subunit of complex I. Our findings indicate that TMEM126A is involved in the assembly of the ND4 distal membrane module of complex I. In addition, we find that the function of TMEM126A is distinct from its paralogue TMEM126B, which acts in assembly of the ND2-module of complex I.

Keywords: complex I; membrane protein; mitochondria; optic atrophy; oxidative phosphorylation.

Fig. 1.

Complex I subunits reduced in T126A^KO mitochondria. (A) Isolated mitochondria from control and T126A^KO cell lines were subjected to SDS-PAGE and immunoblot analysis. SDHA was used as a loading control. (B) Volcano plot of mitochondrial protein abundance in T126A^KO cells relative to control. SILAC ratios were log₂ transformed and the mean (n = 3) plotted against -log₁₀(P value) from single-sample, two-sided Student’s t test. The horizontal line indicates P = 0.05; the vertical lines indicate 1.5-fold change. The blue dots represent complex I subunits with a fold change less than 1.5. (C) Topographical heatmap of subunit ratios were fitted to complex I (PDB: 5XTH) (27). The gray regions represent subunits that were not reliably detected (n.d.).

Fig. 2.

T126A^KO cells have an isolated complex I deficiency. (A) Mitochondria were isolated from control and T126A^KO cell and subjected to BN-PAGE and Western blotting for complex I (NDUFB8) and complex II (SDHA) used as a control. *, distal membrane arm subcomplex. (B) The relative abundance of fully assembled supercomplex and complex I was quantified (mean ± SEM, n = 3). (C) In-gel complex I activity assay using isolated mitochondria. ◊, Dihydrolipoamide dehydrogenase activity (65). (D) Isolated mitochondria subjected to BN-PAGE and Western blotting for complexes III (Core 1), IV (COX4), and V (ATP5A). †, residual COX4 signal following reprobe.

Fig. 3.

TMEM126A interacts with the ND4-module during complex I assembly. (A) Mitochondria isolated from control, T126A^KO-1, and T126A^KO-1 +TMEM126A^Flag cells and were subjected to BN-PAGE and Western blot analysis. Complex II (SDHA) was used as a loading control. (B) Digitonin-solubilized mitochondria from control and T126A^KO-1 +TMEM126A^Flag–expressing cells were subjected to affinity enrichment using anti-Flag agarose beads. Elutions were subjected to LFQ proteomics. Significance was determined through a two-sided, two-sample t test using permutation-based FDR statistics. The curved represents FDR < 1% using an s0 parameter of 1.5. (C) Mitochondria from control and T126A^KO cells were solubilized in digitonin and analyzed by BN-PAGE and immunoblotting using antibodies as indicated. ◊, ∼450 kDa MCIA complex; §, 400/440 kDa TIMMDC1 complex; #, 720 kDa complex. (D) Mitochondria from control and complex I accessory subunit KO cells were isolated and analyzed by BN-PAGE and Western blotting using TMEM126A antibodies. Complex II (SDHA) was used as a loading control. Lower TMEM126A signal is saturated to detect higher-molecular–weight complexes. †, Accumulated TMEM126A complex; ^, nonspecific signal. (E) Pulse-labeled mtDNA-encoded subunits from control and T126A^KO-1 +TMEM126A^Flag–expressing cells were chased for the indicated times, Isolated mitochondria were solubilized in digitonin and subjected to affinity enrichment, SDS-PAGE analysis, and phosphorimaging. Immunoblotting for TMEM126A and SDHA served as controls. (F) Pulse SILAC analysis of newly translated MT-ND4 in control and T126A^KO cells. Log₂ transformed heavy peptide–derived intensities were plotted relative to control intensities. The data are represented as mean ± SD n = 3. (G) As for F but heavy media exchanged with light media after a 4 h pulse and cells harvested after 3 and 24 h. The data are represented as mean ± SD n = 3. *FDR < 0.05 and **FDR < 0.01.

Fig. 4.

TMEM126A functions independently of TMEM126B. (A) Mitochondria isolated from control, T126A^KO-1, and T126A^KO-1 expressing TMEM126A^Flag or TMEM126B^Flag cells were analyzed by BN-PAGE after solubilization in 1% TX100 and immunoblotted for the complex I subunit NDUFB8. SDHA/CII was used as a loading control (B) The relative abundance of fully assembled complex I (from A) was quantified in control and T126A^KO cells expressing TMEM126A^Flag and TMEM126B^Flag as indicated (mean ± SEM, n = 3). (C) Mitochondria from MCIA^KO and T126A^KO cells were analyzed by BN-PAGE following 1% digitonin solubilization and immunoblotting using TMEM126A antibodies. CII (SDHA) was used as a loading control. Lower TMEM126A signal is saturated to detect higher-molecular–weight complexes. †, accumulated TMEM126A complex; *, nonspecific signal. (D) Mitochondria were isolated from control, T126A^KO, T126B^KO, and T126A/B^DKO cells and analyzed by SDS-PAGE and Western blotting with antibodies as shown. SDHA was used as a loading control. (E) Mitochondria were isolated from control, T126A^KO, T126B^KO, and T126A/B^DKO cells and analyzed by SDS-PAGE and Western blotting with antibodies as shown. ATP5A was used as a loading control. (F) Mitochondria from control, T126A^KO, T126B^KO, and T126A/B^DKO cells were analyzed by BN-PAGE following 1% digitonin solubilization and immunoblotting using ND4 antibodies. CV (ATP5A) was used as a loading control. †, accumulated TMEM126A complex; ‡, accumulated ND4-module. (G) Mitochondria were isolated from cells expressing TMEM126A^Flag or TMEM126B^Flag as indicated, solubilized in 1% digitonin, and subjected to Flag affinity enrichment prior to SDS-PAGE analysis. Immunoblotting was performed with antibodies as indicated.