Protein import motor complex reacts to mitochondrial misfolding by reducing protein import and activating mitophagy

Mitophagy is essential to maintain mitochondrial function and prevent diseases. It activates upon mitochondria depolarization, which causes PINK1 stabilization on the mitochondrial outer membrane. Strikingly, a number of conditions, including mitochondrial protein misfolding, can induce mitophagy without a loss in membrane potential. The underlying molecular details remain unclear. Here, we report that a loss of mitochondrial protein import, mediated by the pre-sequence translocase-associated motor complex PAM, is sufficient to induce mitophagy in polarized mitochondria. A genome-wide CRISPR/Cas9 screen for mitophagy inducers identifies components of the PAM complex. Protein import defects are able to induce mitophagy without a need for depolarization. Upon mitochondrial protein misfolding, PAM dissociates from the import machinery resulting in decreased protein import and mitophagy induction. Our findings extend the current mitophagy model to explain mitophagy induction upon conditions that do not affect membrane polarization, such as mitochondrial protein misfolding. Overall design: Recombinant mt-mKEIMA expressing human HeLa FlpIn Trex cells with Parkin expression were transfected with viral Brunello Cas9 library and an expression cassette for Cas9 enzyme. Starting two days after transduction, cells were selected by maintaining 2 µg/ml Puromycin. After seven days cells were treated with 0.1% DMSO for 16h, collected by 0.25% trypsin/EDTA-assisted detachment and sorted as mitophagy-positive cells (DMSO1_plus) by increased 561 nm excited mt-mKEIMA fluorescence compared to untransduced cells. As a reference the same amount of Brunello Cas9 library transducd cells were collected and pooled without sorting (DMSO1_complete). The collected cells were lysed and genomic DNA extracted by GeneJet DNA purification kit (Thermo Scientific, K0721).