Figure 3. MCL-1 mutants restrict mitochondrial localization.
(a) Schematic illustration of MCL-1 mutants. Yellow boxes indicate BCL-2 homology (BH) domains and the red box indicates the C-terminal hydrophobic domain. Point mutations are indicated with the original amino acid, site-position and mutated residue. N. crassa ATP-synthase MTS is indicated in green. (b) Confocal immunofluorescence of Mcl-1-deficient MEFs transiently-expressing indicated Mcl-1 constructs were fixed and stained for MCL-1 (green), mitochondria (Mitotracker, in red), and nucleus (DAPI, in blue). Co-localization of green and red channels is shown in yellow overlay. Size bars represent 10 μm. (c & e) Whole cell lysates (WCL), isolated heavy membranes (HM) enriched for mitochondria or cytosolic (cyto) sub-cellular fractions were isolated from Mcl-1-deleted MEFs stably-expressing indicated MCL-1 constructs. Fractions were western blotted for MCL-1, BAX (cytosolic marker) or MnSOD (mitochondrial marker). (d) Mitochondria from Mcl-1-deficient MEFs stably-expressing human BCL-2 (hBCL-2) or a chimeric fusion of the first 50 amino acids of MCL-1 to hBCL-2 (mMCL-150-hBCL-2) were subjected to protease treatment as described in Fig. 2d. Both the fusion protein and hBCL-2 were detected with anti-hBCL-2 antibody. (f) Mitochondria from Mcl-1-deficient MEFs stably-expressing indicated constructs were subjected to protease treatment as described in Fig. 2d.