Fig. 3

The A’ helix targets CYP11A1 into the membrane and remodels mitochondria cristae. A Immunohistochemistry images of EGFP-fused CYP11A1 fragments transfected in COS1 cells. B Immunoblotting of EGFP-fused CYP11A1 fragments after alkaline buffer extraction and fractionation by ultracentrifugation. The EGFP and 39-EGFP (abbreviated as 39) proteins were in the supernatant, while the 85- and 521-EGFP proteins were in the pellet. Asterisk indicates non-specific binding of antibody used (T: total; P: pellet; S: supernatant). TOM20 is a control for membrane protein, and cytochrome c (Cyt C) is a control for soluble protein. C Immunohistochemistry images of EGFP-fused CYP11A1 signal peptide (AA #1–39) and the anchoring region A’-helix (39 + A’-EGFP) showing its localization in the mitochondria. TOM20 (red) is a marker for mitochondria, and DAPI (blue) stains the nucleus. D Partitioning of EGFP-fused CYP11A1 fragments with the membrane-anchoring region A’-helix in transient transfection (39 + A’) or in a stable clone (SC39 + A’) detected by Western blots. The EGFP protein fused to AA #1–85 in a stable clone (SC85) also goes to the pellet. Asterisks indicate non-specific band (T: total; P: pellet; S: supernatant). E TEM images of mitochondria of COS1 cells transfected with various fragments of human CYP11A1 fused to EGFP-APEX2. COS1 cells transfected with EGFP-APEX2 only are denoted with a “- “ sign. F The A’-helix is sufficient to change cristae into tubulovesicular as quantified after TEM observation. All protein fragments are fused to EGFP-APEX (G-A). More than 50 cells were counted for quantification of each sample. Unpaired t-test was used, and data shown are mean with standard deviation