PARK2 Knockout Cell Line-HeLa

The E3 ubiquitin ligase PARK2 and the mitochondrial protein kinase PINK1 are required for the initiation of mitochondrial damage-induced mitophagy. Together, PARK2 and PINK1 generate a phospho-ubiquitin signal on mitochondrial outer membrane proteins, triggering the recruitment of the autophagic machinery. Here, researchers describe the detection of a specific 500 kDa phospho-ubiquitin-enriched PARK2 complex that accumulates on mitochondria upon treatment with the membrane uncoupler CCCP. The formation of this complex is dependent on the presence of PINK1, which is absent in mutant forms of PARK2 and thus prevents mitophagy. These results suggest that a functional signaling complex is essential for the progression of mitophagy. Visualization of the PARK2 signaling complex represents a new marker for this critical step in mitophagy and can be used to monitor the progression of mitophagy in PARK2 mutants and reveal additional upstream factors required for PARK2-mediated mitophagy signaling.

The results presented here demonstrate that formation of the PARK2 signaling complex is sensitive to PARK2 mutations and correlates with blockade of mitophagy, thus demonstrating its utility as a tool for analyzing signaling defects in PARK2 mutants (Figure 1A and B). To eliminate any interference by endogenous PARK2, knockout strains were created using CRISPR/Cas9, and elimination of PARK2 was confirmed by immunoblotting (Figure 1C) and sequencing. Recruitment of PARK2 to mitochondria was assessed by cell fractionation (Figure 1D). Both wild-type FLAG-PARK2 and the R275W mutant were efficiently recruited to mitochondria. Consistent with previous reports, the C431S mutant did not translocate to mitochondria, nor did the T240R mutant (Figure 1D). In PARK2 knockout cells, all mutants, including the C431S mutant, failed to form complexes (Figure 1E), suggesting that endogenous PARK2, even at low levels, can interfere with functional analysis of PARK2 mutants.

Figure 1. PARK2 mutants blocked in mitophagy have impaired complex formation. (A) Steady-state levels of PARK2 substrates in wild-type FLAG-PARK2 and the indicated variants. (B) Isolated mitochondria were solubilized and protein complexes were separated using BN-PAGE. Complexes containing PARK2 were detected by immunoblotting for PARK2. (C) Confirmation of PARK2 knockout. (D) PARK2 knockout cell lines were transiently transfected with wild-type FLAG-PARK2 and the corresponding mutant versions. Two days after transfection, cells were treated with 10 µM CCCP or DMSO for 3 hours. Cell lysates were separated into cytosol and mitochondria-enriched fractions and analyzed by SDS-PAGE and western blotting. (E) Mitochondria-enriched fractions from (D) were solubilized and separated by BN-PAGE or SDS-PAGE analysis followed by immunoblotting. (Callegari S, et al., 2017)