Figure 4.

Complexome Profiling of Fibroblasts from Subject 1 Confirms Complex I Assembly Defect

Enriched mitochondrial membranes derived from control (A) and subject 1 (B) fibroblasts were solubilized with digitonin, separated on native gradient gels, cut into 60 pieces and digested with trypsin. Peptides were analyzed by quantitative mass spectrometry. Intensity-based absolute quantification (IBAQ) values of subject set were normalized to the sum of all values from control dataset. For visualization in a heatmap, values were normalized to maximum appearance between both samples. There is a reduction in the functional respirasome (complex I/III₂/IV) (purple box) in subject fibroblasts and stalled assembly intermediates corresponding to P_D-a (blue box) and a partly-assembled Q module (orange box), compared to control fibroblast data. There is also an increase in “free” complex III (gray box). Complex I subunits are presented according to their ascribed module, N-module (N), Q-module (Q), proximal P-module (P_P-a containing ND1, P_P-b containing ND2/3/6), distal P-module (P_D-a containing ND4, P_D-b containing ND5) and assembly factors (A). Color-matched dashed boxes are used to denote the corresponding regions of interest in the control data (Panel A). Subcomplex denotations are according to Guerrero-Castillo and colleagues²⁸ C. Complexome data schematic summarizes the consequence of defective NDUFAF8 on complex I assembly. Complex I subcomplexes are color-coded and named consistent with Panels A and B.