(A–C) Blue native electrophoresis followed by immunoblot analysis of MIC60, MIC10 and ATP5A in control neonatal and patients’ mitochondria from skin fibroblasts. In Panel D, mitochondrial extracts were subjected to SDS-PAGE and immunoblotting with the indicated antibodies. (E,F) Cells from control neonatal fibroblasts were labeled with light lysine (K0) while fibroblasts obtained from patient 2 were labelled with heavy lysine (K8). Cells were mixed at a 1:1 ratio and mitochondria were subsequently isolated, lysed in 1% digitonin and native protein complexes were separated in a blue native gel. Gel bands from >1 MDa to ~66 kDa were excised and subjected to mass spectrometry analysis. Heavy: Light ratios were calculated for the sum of all peptides quantified for each MICOS subunit and represented in a heatmap where ratios below 1 are represented in green and ratios above 1 are represented in red. (G,H) Blue-native gel analysis of MIC10 and ATP5A containing complexes from neonatal control fibroblasts or fibroblasts from patients 1 and 2 with or without rescue by stable expression of QIL1-HA-FLAG. Panel G, anti-MIC10. Panel H, anti-ATP5A. (I) Mitochondrial lysates from panels G–H were examined by SDS-PAGE and immunoblotting using the indicated antibodies. (J) Schematic representation of the effect of QIL1 loss on MICOS assembly in patients’ fibroblasts. In the mature MICOS complex in control cells, multiple transmembrane components of MICOS (MIC60, MIC10, MIC26, MIC27 and QIL1) associate with MIC25 and MIC19 to promote the formation of cristae membrane structures. In the mitochondria of patient cells lacking QIL1, the abundance of MIC10, MIC26, and MIC27 is greatly reduced, leading to loss of the MICOS complex and the absence of normal cristae structures within mitochondria. Blue-native gel analysis of MICOS from QIL1-deficient cells revealed a ~500 kDa complex containing reduced levels of MIC60 and MIC19, which appears to be unable to maintain cristae structure morphology within mitochondria.