Parkin-dependent mitophagy occurs via proteasome-dependent steps sequentially targeting separate mitochondrial sub-compartments for autophagy.

PINK1/parkin-dependent mitophagy initially involves (phospho)ubiquitin-directed proteasome-dependent degradation of certain outer mitochondrial membrane (OMM) proteins (e.g. mitofusins) and the recruitment of autophagy adaptors to a group of ubiquitinated OMM proteins, eventually leading to autophagic removal of damaged mitochondria in stressed cells. Here we provide evidence that mitochondrial degradation occurs via stepwise delivery of separate mitochondrial sub-compartments for autophagic degradation. OMM and inner mitochondrial material appears to become separately isolated for autophagolysosomal degradation, not only in parkin-overexpressing HeLa cells but also in cells that express endogenous parkin (human embryonic kidney cells and neural progenitor cells) with slower mitophagy kinetics. The remaining inner mitochondrial material becomes degraded only after much prolonged membrane depolarization, potentially involving another proteasome-sensitive step. The present combined microscopy and proteomics analyses support the idea that cell stress-induced parkin-dependent mitophagy is a complex multi-step process with distinct mitochondrial sub-compartments being separately targeted for autophagic degradation. Abbreviations: BafA, Bafilomycin A; CCCP, carbonyl cyanide 3-chlorophenylhydrazone; COX IV, cytochrome c oxidase subunit IV; CS, citrate synthase; DMEM, Dulbecco's modified Eagle's medium; EGFP, enhanced green fluorescent protein; FBS, fetal bovine serum; IF, immunofluorescence; IMM, inner mitochondrial membrane; KO, knock-out; LC3, microtubule-associated protein 1 light chain 3; MDVs, mitochondria-derived vesicles; MFN, mitofusin; NPCs, neural progenitor cells; OMM, outer mitochondrial membrane; p62/SQSTM, 62kDa protein sequestosome-1; PBS, phosphate-buffered saline; PINK1, phosphatase and tensin homolog (PTEN)-induced putative kinase protein 1; RT, room temperature; SSBP1, single-stranded DNA binding protein 1; TAX1BP1, Tax1-binding protein 1; TEM, transmission electron microscopy, TOM20, translocase of outer mitochondrial membrane 20kDa subunit; TOM70, translocase of outer mitochondrial membrane 70kDa subunit; Ub, ubiquitin; UPS, ubiquitin proteasome system; VDAC, voltage-dependent anion-selective channel protein; WB, Western blot; WT, wild-type.