Figure 3.

Submitochondrial topology of CHCHD2 and CHCHD10. (A) Proteinase K protection assay in mitochondria (M) and mitoplasts (Mp) prepared by hypotonic swelling of mitochondria. As a control, a mitochondrial sample was sonicated (Son) to give full access to the protease. In this assay, the controls used were TIMM50 (inner transmembrane protein facing the intermembrane space) and HSP60 (matrix protein). (B) CHCHD2 (D2) and CHCHD10 (D10) solubilization by sonication and alkaline carbonate (pH 11.5) extraction. Mitochondria (M) isolated from HEK293 cells were sonicated, and the soluble (S), and membrane-bound fractions were separated by centrifugation. The pellet was subsequently extracted with alkaline sodium carbonate and fractionated into supernatant (CS) and pellet (CP). The different fractions were analysed by immunoblotting using antibodies that recognize CHCHD2, CHCHD10, p32, and the controls COA3 and SCO1 (membrane proteins), SDHA and CMC1 (loosely bound to IM), and LON-P (soluble protein) (C) Isolated mitochondria were fractionated into inner and outer membranes by sonication and centrifugation. The crude membrane fractions were analysed by sucrose gradient sedimentation in a linear 30–60% sucrose gradient. In each case, following ultracentrifugation, 15 gradient fractions were collected, separated by SDS-PAGE and analysed by immunoblotting using antibodies against VDAC (OM marker), COX1 (IM marker), CHCHD2 and CHCHD10. (D) Digitonin solubilization of mitochondria using increasing detergent: protein ratios. Following solubilization, soluble (S) and insoluble material (P) were separated by centrifugation and analysed by immunoblotting using the indicated antibodies.