Figure 4.

The PINK1 R98F mutant resistant to PARL-mediated cleavage is located inside mitochondria. (a) Mitochondria isolated from HeLa cells transfected with YFP-tagged PINK1 R98F were incubated with various concentrations of PK for 30 min on ice and immunoblotted for PINK1, Tom20, Cyt c, AIF, and Hsp70. Green arrowhead, FL and ΔMTS-PINK1; red arrowhead, 52-kD PINK1. (b) HeLa cells transfected with mito-YFP were treated with DMSO or 10 µM CCCP for 3 h followed by incubation in either PBS alone or PBS containing 0.005% digitonin or 0.25% Triton X-100 (TX-100). Cells were immunostained using antibodies against Tom20 and Cyt c and analyzed by confocal microscopy. (c) HeLa cells were transfected with YFP-tagged WT PINK1 or PINK1 R98F mutant for 18 h. Cells were then treated with DMSO or CCCP for 3 h, permeabilized, and immunostained with the indicated antibodies. Images were taken by confocal microscopy. (d) HeLa cells (≥150/condition) stained in c were counted for GFP immunofluorescence. Counting results were represented as mean ± SEM from four replicates. (e) HeLa cells cotransfected with YFP-tagged PINK1 R98F mutant and mCherry-Parkin were incubated with either DMSO or 10 µM CCCP for 1 h followed by confocal imaging. (f) PINK1 KO MEFs transfected with YFP-tagged WT PINK1 or PINK1 R98F mutant together with mCherry-Pax were treated with DMSO or 10 µM CCCP for 3 h, and cells were counted for mitochondrial translocation of Parkin (≥50 cell counts for each sample). Counting results were represented as mean ± SEM from four replicates. Bars, 20 µm.