A unified mechanism for mitochondrial damage sensing in PINK1-Parkin-mediated mitophagy
Damaged mitochondria can be cleared from the cell by mitophagy, using a pathway formed by the recessive Parkinson's disease genes PINK1 and Parkin. Whether the pathway senses diverse forms of mitochondrial damage by a common mechanism, however, remains uncertain. Here, using a novel Parkin reporter in genome-wide screens, we identified that diverse forms of mitochondrial damage converge on loss of mitochondrial membrane potential (MMP) to activate PINK1. Loss of MMP, but not the PAM import motor, blocked progression of PINK1 import through the translocase of the inner membrane (TIM23), causing it to remain bound to the translocase of the outer membrane (TOM). Ablation of TIM23 was sufficient to arrest PINK1 in TOM, irrespective of MMP. Meanwhile, TOM (including subunit TOMM5) was required for PINK1 retention on the mitochondrial surface. The energy-state outside of the mitochondria further modulated the pathway by controlling the rate of new PINK1 synthesis. Together, our findings point to a convergent mechanism of PINK1-Parkin activation by mitochondrial damage: loss of MMP stalls PINK1 import during its transfer from TOM to TIM23. Overall design: Genome-wide CRISPRi screen utilizing a dual sgRNA library in HeLa dCas9 BFP KRAB MFN2 Halo mCherry Parkin, HeLa dCas9 BFP KRAB MFN2 Halo, or HEK293 dCas9 BFP ZIM3 cells. Cells were sorted on d7 post transduction via FACS into low (bottom 30% MFN2 Halo expressing cells) and high bins (top 30% MFN2 Halo expressing cells). Each screen was done in duplicate. HeLa cells were treated with antimycin/oligomycin and HEK293 cells were treated with CCCP.