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

Macroautophagy (hereafter autophagy), a major intracellular catabolic process, is evolutionarily conserved from yeasts 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.