Figure 3. PARL cleaves PINK1F104M-YFP between A103 and M104. (A) HCT116 cells stably expressing wild-type (F104F) and the F104M mutant of PINK1-YFP-IRES-DsRed were immunostained with anti-TOMM20. Scale bars: 10 μm. (B) HCT116 cells stably expressing PINK1F104M-YFP were solubilized with 0.5% Triton X-100 and subjected to immunoprecipitation with GFP-Trap or control beads. Proteins were analyzed by SDS-PAGE followed by coomassie brilliant blue staining. Red arrowhead represents PARL-cleaved F104M mutant. Mass spectrometry identified HSP90AA1 and CDC37 as interacting partners of cleaved F104M mutant. (C) The protein band described by arrowhead in (B) was analyzed by LC/MS/MS, and the identified sequences of PINK1F104M moiety are underlined. Identifying the peptide corresponding to MGLGLGLIEEK indicates that PARL cleaves the PINK1F104M mutant between A103 and M104.