Fig 1: MOK is a mitochondrial intermembrane space (IMS)-localized protein.(A) Sub-mitochondrial localizations of new mitochondrial kinases. Transfected HeLa cells were stained and imaged by 3D structured illumination microscopy (3D-SIM). (B) MOK2 is imported into mitochondria. Transfected HeLa cells were fixed and permeabilized with digitonin or Triton X-100 before staining. (C) MOK is not a mitochondrial membrane protein. Mitochondria purified from transfected HeLa cells were incubated in indicated buffers, and then centrifuged. Supernatants (S) and pellets (P) were analyzed by western blotting. Equal loading was achieved by normalizing to the same number of cells. (D) MOK is an IMS protein. COS-7 cells transfected with MOK2-GFP were imaged by Hessian SIM. MitoTracker Red marked inner membrane (IMM) and co-transfected HSP60-mCherry marked matrix. (E) MOK2 has better mitochondrial localization than MOK1. HeLa cells were transfected with full-length (FL) or kinase domain (KD) of MOK1 and MOK2. Cells with mitochondrial MOK were quantified. Data is represented as mean ± SD; n = 3 independent experiments, two-tailed Student’s t test; n = 100 cells were analyzed in each experiment. (F) Mitochondrial localization of MOK mutants. Transfected HeLa cells were stained with anti-Myc and anti-TOM20 antibodies. (G) TOM20-TIM23 complex is required for mitochondrial importing of MOK. HeLa cells expressing specific shRNAs were transfected with MOK2-Myc and stained (Figure 6—figure supplement 1).Figure 6—source data 1.Uncropped western blot for Figure 6.Figure 6—source data 2.Raw data for Figure 6 and Figure 6—figure supplement 1.