Fig. 1: ETFDH–CIII interaction is required for OXPHOS efficiency.
a, Scheme depicting how the iron–sulfur flavoprotein ETFDH canalizes electrons from FAO, BCAA catabolism and choline metabolism to OXPHOS. The FAD (orange) and Q (blue) redox reactions are illustrated, with the proteins of the ETFDH pathway highlighted in red. ACADs, acyl-CoA dehydrogenases; IVD, isovaleryl-CoA dehydrogenase; DMGDH, dimethylglycine dehydrogenase; SARDH, sarcosine dehydrogenase; RFK, riboflavin kinase; FLAD1, FAD synthetase 1. b, Representative western blot of ETFDH protein levels in CRL, ETFDH-ko and cDNA-rescued ETFDH-ko (+ETFDH) myoblasts. Two samples per condition. Tubulin is shown as a loading control. c, FAO fluxes in CRL, ETFDH-ko and cDNA-rescued ETFDH-ko myoblasts. n = 3. d, [14C(u)]leucine (Leu) oxidation to CO2 in myoblasts. n = 4 replicates per condition. e–g, Cell growth in CRL and ETFDH-ko myoblasts cultured in substrate-free medium supplemented with palmitate (e), oleate (f) or glucose (g). n = 3, seven replicates per condition. h, Left, representative respiratory profile of CRL (grey trace), ETFDH-ko (red trace) and cDNA-rescued ETFDH-ko (lavender trace) myoblasts. Right, quantification histogram. n = 3. i, NAD/NADH levels in CRL, ETFDH-ko and cDNA-rescued ETFDH-ko myoblasts. n = 3. j, Immunocapture (IP) of all subunits of CIII blotted with anti-ETF antibody (ETFA) in Skm from mouse hindlimb. Tubulin is shown as a loading control. IgG, immunoglobulin G. k, PyMOL representation of a ClusPro docking study for CIII–ETFDH interaction. l, Immunocapture (IP) of UQCRC2 blotted with anti-ETFDH antibody in mouse brain, heart and Skm extracts. Tubulin is shown as a loading control. m, Left, representative images of PLA between CIII (UQCRC2) and ETFDH in CRL, ETFDH-ko and cDNA-rescued ETFDH-ko myoblasts. Right, quantification histogram. Seven images per condition, n = 3. n, Representative 1D BN-PAGE and 2D SDS–PAGE of mitochondrial membrane proteins from wt mice, followed by proteomics. Migration in 1D BN-PAGE of respiratory CI, CIII and CIV; ETF complex subunits ETFDH, ETFA and ETFB; and COQ2 (in blue) are shown. Migration in 2D SDS–PAGE of CIII, ETFDH, the FAO enzyme HADHA, and COQ2 (in orange) are shown. The upper image shows the proteomic analysis of 12 bands from 1D BN-PAGE. White to red scale indicates protein abundance within a band. ETFDH was found to comigrate with CIII in two bands. Results are shown as the mean ± s.e.m. of the indicated n. *, **, ***, ****P < 0.05, 0.01, 0.001 and 0.0001 when compared with CRL by one-way ANOVA with Tukey’s test (c, d, h, i, m) or two-way ANOVA with Šidák’s test (e, f, g).
