S-palmitoylation coordinates the trafficking of ATG9A to mediate autophagy initiation

Macroautophagy (hereafter autophagy), a major intracellular catabolic process, is evolutionarily conserved from yeast to mammals and is associated with a broad range of human diseases. Autophagy is morphologically characterized by the formation of double-membrane autophagosomes. ATG9A, a multi-spanning transmembrane protein and lipid scramblase, is a core component of the autophagy machinery that complements membrane sources and equilibrates lipids across membrane bilayers. Here, we report that palmitoyltransferase ZDHHC5 is indispensable for autophagosome nucleation and subsequent autophagosome formation. Upon autophagy induction, ZDHHC5 is internalized from the plasma membrane into intracellular compartments via clathrin-mediated endocytosis. This enzyme activates ATG9A S-palmitoylation at cysteine 155/156, which orchestrates the interaction of ATG9A with the heterotetrameric adaptor protein complex family member AP4E1/AP-4ε and subsequent trafficking from the trans-Golgi network to endosomal compartments. Functionally, impairment of ATG9A S-palmitoylation results in defects in autophagy initiation and autophagosome formation. These findings identify a regulatory mechanism that coordinates ATG9A-binding with AP4E1 and vesicular trafficking events through ATG9A S-palmitoylation by ZDHHC5, thereby ensuring the spatiotemporal fidelity of membrane trafficking and maintenance of autophagic homeostasis. 17-ODYA: 17-octadecynoic acid; 293T: HEK293T; 2-BP: 2-bromopalmitate; 2CS: Cys155Ser and Cys156Ser; ABE: acyl-biotin exchange; AP: adaptor protein; APEX2: ascorbate peroxidase 2; ATG: autophagy related; baf A₁: bafilomycin A₁; CRISPR: clustered regularly interspaced short palindromic repeats; CTD: C-terminal domain; Cys: cysteine; DAB: 3,3’-diaminobenzidine; EV: empty vector; H₂O₂: hydrogen peroxide; IF: immunofluorescence; IP: immunoprecipitation; KO: knockout; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; NTD: N-terminal domain; PAS: phagophore assembly site; PBS: phosphate-buffered saline; PtdIns3K-CI: class III phosphatidylinositol 3-kinase complex I; PM: plasma membrane; PTM: post-translational modifications; Ser: serine; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; TGN: trans-Golgi network; ULK1: unc-51 like autophagy activating kinase 1; WCL, whole cell lysates; WDR45/WIPI4: WD repeat domain 45; WT: wild-type; ZFYVE1/DFCP1: zinc finger FYVE-type containing 1.