FIGURE 6.

Import, processing, and submitochondrial localization of ³⁵S-labeled C-terminal Pink1 deletion constructs. A, schematic representation of the wild-type Pink1 domain structure, of a C-terminal Pink1 deletion construct lacking the C-terminal 66 residues (Pink1_Δ516–581), and of the PD-related Pink1_W437X mutant. For wild-type Pink1, previously suggested processing sites consistent with the apparent molecular weight of Pink1_f60 and Pink1_f53, respectively (positions 35 and 104), and the proposed TMD (residues 93–111) are indicated. B, import of radiolabeled wild-type Pink1 and C-terminal deletion mutants into isolated HeLa mitochondria as described for Fig. 1 with subsequent separation of samples by SDS-PAGE and analysis by digital autoradiography (p, full-length precursor protein; PF, processing fragment corresponding to Pink1_f53). C, protease titration of mitochondria following import of wild-type Pink1 (upper panel), Pink1_Δ516–581 (middle panel), and Pink1_W437X (lower panel). After the import reaction, mitochondria were incubated with trypsin in the indicated concentrations. As a control, immunodecorations against the endogenous proteins Tom70, Tim23, and Trap1 were carried out (p, full-length precursor protein; f1, intermediate processing fragment corresponding to Pink1_f60; PF, processing fragment corresponding to Pink1_f53). D, quantification of the trypsin resistance of radiolabeled full-length forms and the main cleavage fragments (processing fragment) of WT-Pink1 and the C-terminal deletion constructs after import in intact mitochondria. Autoradiography signals for the indicated Pink1 fragments in the presence of 40 μg/ml trypsin were quantified and depicted as percentage of the imported amounts without protease treatment. Mean values and S.E. were determined for n = 4 independent experiments. WB, Western blot.