(A–C) Immunogold labeling of 293T cells overexpressing C-terminally tagged QIL1 (A), MIC25 (B), or NDUFA13 (C) using an α-HA antibody coupled to 10-nm gold particles. Bars, 100 nm. Representative mitochondria are shown. The arrows point to the position of a gold particle. The distance in nanometers between the gold particles and the nearest CJ was measured using the ImageJ software. The histograms show the fraction of gold particles within the indicated distance to the crista junction in nanometers in QIL1-HA (n = 231 gold particles), MIC25-HA (n = 192 gold particles), and NDUFA13-HA (n = 309 gold particles) expressing cells. (D) Mitochondria isolated from stable 293T cell lines expressing C-terminally tagged QIL1, MIC60, MIC19, or MIC25 were lysed in 1% digitonin, subjected to BN-PAGE followed by immunotransfer to nitrocellulose membranes and probing with α-HA antibody. The mature ∼700 kDa MICOS complex is highlighted with an asterisk. MIC60, MIC19, and MIC25 were also detected in a sub-complex of ∼500 kDa (two asterisks). (E) Two-dimensional blue native electrophoresis of 293T mitochondrial lysates. Endogenous QIL1 is present in the mature ∼700 kDa MICOS complex (asterisk). MIC60, MIC19, and MIC25 were also present in a smaller sub-complex (two asterisks). (F–G) C-terminally tagged (F) or endogenous QIL (G) was immunopurified from mitochondria lysed in 1% digitonin or 1% Triton X-100 (TX100). Immunoblot analysis was performed to detect interaction with other MICOS subunits.