RAB1 mediates OPTN-dependent mitophagy via ATG9A recruitment.

Damaged mitochondria are selectively eliminated through mitophagy, a critical quality control process. A kinase PINK1 and an E3 ubiquitin ligase PRKN/Parkin, both of which are mutated in familial Parkinson disease, amplify ubiquitin signals on the damaged mitochondria. The autophagy receptor OPTN plays a pivotal role in mitophagy by bridging ubiquitinated mitochondria with autophagy components. Although OPTN is known to recruit ATG9A-positive vesicles to facilitate mitophagy progression, the precise molecular mechanisms governing this recruitment remain poorly understood. In this study, we identify the small RAB GTPases RAB1A and RAB1B as direct binding partners of the OPTN leucine zipper (LZ) domain. We demonstrate that RAB1A/1B is required for the recruitment of ATG9A vesicles to mitochondria during the initial stages of mitophagy. Knockdown of RAB1A and RAB1B significantly impaired the assembly of OPTN at phagophore formation sites, leading to a profound inhibition of mitophagy progression. Mechanistically, we found that RAB1A/1B associate with ATG9A-positive vesicles via their C-terminal prenylation, thereby tethering these vesicles to the OPTN-bound mitochondria. Our findings establish a novel OPTN-RAB1-ATG9A axis that drives the de novo synthesis of phagophore membranes in close proximity to damaged mitochondria. This work clarifies how selective autophagy receptors spatially coordinate membrane trafficking to ensure the efficient clearance of dysfunctional organelles.Abbreviations: AO: antimycin A and oligomycin; ASH: assembly helper; ATG: autophagy related; CALCOCO2: calcium-binding and coiled-coil domain-containing protein 2; CC: coiled coil; ER: endoplasmic reticulum; Fluoppi: fluorescence-based protein-protein interaction; GABARAP: GABA type A receptor-associated protein; GAP: GTPase-activating protein; GEF: guanine nucleotide exchange factor; HAG: humanized azami-green; IB: immunoblotting; ipTM: interface predicted template modeling score; IQR: interquartile range; KO: knockout; LIR: LC3-interacting region; LRRK2: leucine rich repeat kinase 2; LZ: leucine zipper; MAP1LC3: microtubule associated protein 1 light chain 3; N.D.:not detectable; OPTN: optineurin; PBS: phosphate-buffered saline; PINK1: PTEN-induced kinase 1; PLDDT: predicted local distance difference test; PRKN: Parkin RBR E3 ubiquitin protein ligase; PtdIns3K: phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol 3-phosphate; pTM: predicted template modeling score; PVDF: polyvinylidene difluoride; RAB: RAB, member RAS oncogene family; RB1CC1: RB1 inducible coiled-coil protein 1; SDS: sodium dodecyl sulfate; siRNA: small interfering RNA; TAX1BP1: Tax1 binding protein 1; TBC1D17: TBC1 domain family member 17; TBK1: TANK-binding kinase 1; TKO: triple knockout; TOMM20: translocase of outer mitochondrial membrane 20; Ub: ubiquitin; UBAN: ubiquitin binding in ABIN proteins and NEMO; WIPI: WD-repeat domain phosphoinositide-interacting; WT: wild-type.