Fig. 1: CI-deficiency disease-mutants populate surfaces of IMM-arm subunits.
a Schematic representing structural framework of metazoan CI. 44 subunits associate to form 4 functional modules namely NADH dehydrogenase module (N-module; pink), ubiquinone reduction module (Q-module; blue), proton pumping proximal and distal modules (PP and PD modules; purple and black). Curved arrow: reaction catalyzed by N-module; straight arrows: flow of protons (H+). NAD+ - Nicotinamide adenine dinucleotide; NADH - Nicotinamide adenine dinucleotide hydrogen. Nuclear encoded and mitochondria encoded subunits prefixed as Nduf and Mt. b Pie-chart indicating different types of mutations in CI-deficiency diseases including MELAS, Leigh and LHON syndrome as obtained from OMIM database (omim.org). Details in Supplementary Data S1a. c Left: Cartoon explaining buried and exposed amino acids. Involvement in PPI or otherwise (non-PPI) indicated. Amino acids with no neighboring atom in 3 Å2 space considered as surface exposed. PPIs include H-bonds, salt bridges, cation-π interactions. Right: Stacked-bars indicating distribution of 79 missense CI-deficiency mutations on CI-structure (PDB: 5XTD). Details in Supplementary Data S2a. d Stick representation of PPI and non-PPI amino acids in Ndufa2 and Ndufa1 as visualized by PyMol. Ndufa2 and Ndufa1: purple; interacting subunits: teal. Dashed black lines indicating PPI with bond length in Å. e Schematic representing workflow used for Figs. f–i. f Microscopy showing EGFP-tagged wild type (WT) and mutant subunits. Each image represents single Z-section of 0.3 μm. DAPI (blue): nucleus. Tom20 (red): mitochondrial marker. Scale bar: 10 μm. Colocalization coefficients for EGFP tagged subunits and mitochondria included in Supplementary Table 1. Experiments repeated thrice. g SDS-PAGE followed by immunoblots indicating WT and mutant proteins in mitochondrial fraction probed with EGFP antibody. Hsp60 as loading control. h in-gel EGFP fluorescence (IGF) assays of overexpressed wild type and mutant CI-subunits and western blots (WBs) of digitonin solubilised mitochondrial fractions of same probed for CI and CII using anti-Ndufs1 and anti-Sdha. SC- supercomplex; HC- holocomplex; SubC- subcomplex. Sdha*- HC; Sdha**- SubC. i in-gel CI activity assays. Top: Reaction scheme. Bottom: BN-PAGE gels processed for CI activity. All uncropped gels and blots in Source Data. f–i: Data were verified in three independent experiments.
