PRKN activation for mitophagy requires an NME3-regulated phosphatidic acid signal that separates mitochondria from endoplasmic reticulum tethering

PINK1-dependent activation of PRKN/parkin on depolarized mitochondria causes mitophagy. The deficiency of NME3, a nucleoside diphosphate kinase/NDPK on the outer mitochondria membrane (OMM), is associated with a fatal neurodegenerative disorder. Here, we report that NME3 deficiency impairs p-S65-ubiquitin (Ub)-dependent PRKN binding on depolarized mitochondria without involving the loss of Ub phosphorylation by PINK1. Our mechanistic investigation revealed that NME3 interacts with PLD6/MitoPLD to generate phosphatidic acid (PA) from cardiolipin on the OMM of damaged mitochondria after depolarization. This lipid signal is essential for positioning MFN2 nearby PINK1 for phosphorylation of Ub conjugates on MFN2, thus enabling the subsequent amplification of PRKN binding to mitochondria. We provide further evidence that mitochondria-endoplasmic reticulum (Mito-ER) tethering prohibits the proximity of MFN2 with PINK1 and PRKN amplification on mitochondria. Importantly, the loss of NME3-regulated PA signal causes Mito-ER tethering. Overall, our findings suggest that NME3 cooperates with PLD6 to generate PA as a critical step in Mito-ER untethering, allowing MFN2 access to PINK1 for p-S65-poly-Ub-dependent feedforward activation of PRKN.<b>Abbreviation</b> ACTB: actin beta; BDNF brain derived neurotrophic factor; CL: cardiolipin; CRISPR: clustered regularly interspaced short palindromic repeats; DAG: diacylglycerol; ER: endoplasmic reticulum; FCCP: carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone; FRET: Förster resonance energy transfer; IF: immunofluorescence; KO: knockout; KD: knockdown; LPIN1: lipin 1; MERCS: mitochondria-endoplasmic reticulum contact sites; MFN2: mitofusin 2; Mito: mitochondria; OMM: outer mitochondrial membrane; p-Ub: phosphorylated ubiquitin; PA: phosphatidic acid; PD: Parkinson disease; PINK1: PTEN induced kinase 1; PLA: proximity ligation assay; PLD6/MitoPLD: phospholipase D family member 6; PRKN: parkin RBR E3 ubiquitin protein ligase; RA: retinoic acid; RT-qPCR: reverse transcription-quantitative polymerase chain reaction; TEM: transmission electron microscopy; TN-NME3: TOMM20-NΔ-NME3; TOMM20: translocase of outer mitochondrial membrane 20; TUBB: tubulin beta class I; Ub: ubiquitin; VDAC: voltage dependent anion channel; WB: western blot.