Figure 5. QIL1 deficiency impairs MICOS assembly.
(A) Light-labeled (K0) shFF2 and heavy (K8)-labeled shQIL1 stable cell lines were mixed at a 1:1 ratio and mitochondria subsequently isolated and lysed with 1% Digitonin. Mitochondrial protein complexes were separated using BN-PAGE and sliced in 20 gel pieces ranging from >1 MDa to <60 kDa. Proteins were subjected to tryptic digestion and analyzed by quantitative mass spectrometry. Heavy to light (H:L) ratios were calculated for MICOS components. The ratios were plotted in heatmaps where values <1 are represented in green and values >1 are represented in red. (B) Ratios from the summed heavy and light intensities for each peptide separately across all BN-PAGE fractions from Figure 4A. (C) BN-PAGE followed by immunotransfer to nitrocellulose membranes. QIL1 knockdown lead to a decrease in MIC60, MIC19, and MIC10 in the ∼700 kDa mature MICOS complex (asterisk) and accumulation of MIC60 and MIC19 in a smaller ∼500 kDa sub-complex (two asterisks). (D) Immunoblot analysis of MICOS subunits. (E) Densitometry analysis was performed using ImageJ. (F) qPCR analysis. (G) Endogenous MIC60 was immunopurified from crude mitochondria isolated from HCT116 cells stably expressing FF2 or QIL1 shRNA. Endogenous interactions with MIC19, MIC25, SAMM50, MIC26, MIC10, MIC27, AFG3L2, or QIL1 were assessed. (H) Densitometry analysis was performed using ImageJ.