S-palmitoylation of ATG4B facilitates the deconjugation of LC3-II to promote autophagy

Macroautophagy/Autophagy plays a critical role in maintaining cellular homeostasis. A defining characteristic of Autophagy is the formation of the autophagosomes, which is regulated by a series of ATG (Autophagy related) proteins. ATG4B serves as a pivotal protein responsible for the cleavage of the precursor form of MAP1LC3/LC3 and the deconjugation of LC3-II, which is a prerequisite for the formation and expansion of phagophores, the precursors to autophagosomes. In the present study, we demonstrated that ATG4B undergoes S-palmitoylation, in which palmitic acid is attached to the side chain of a cysteine residue. S-palmitoylation of ATG4B is catalyzed by ZDHHC9 (zDHHC palmitoyltransferase 9), and reversed by ABHD17B (abhydrolase domain containing 17B, depalmitoylase). Interestingly, S-palmitoylation of ATG4B at Cys89 is crucial for LC3-II deconjugation in vitro and in vivo, but not for proLC3 cleavage. In conclusion, our study reveals a crucial role of ATG4B S-palmitoylation in the deconjugation of LC3-II in Autophagy.Abbreviations: ABE: acyl-biotin exchange assay; ABHD17A: abhydrolase domain containing17A, depalmitoylase; Alk-C16: alkynyl palmitic acid; Alk-C18: alkynyl stearic acid; ATG: Autophagy related; BafA₁: bafilomycin A₁; 2-BP: 2-bromopalmitate; co-IP: co-immunoprecipitation; ER: endoplasmic reticulum; GABARAP: GABA type A receptor-associated protein; HAM: hydroxylamine; H₂O₂: hydrogen peroxide; KO: knockout; LYPLA1/APT1: lysophospholipase 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NP-40: Nonidet P-40; PBS: phosphate-buffered saline; PE: phosphatidylethanolamine; proLC3: the precursor form of LC3; PTM: posttranslational modification; SDS: sodium dodecyl sulfate; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; WIPI2B: WD repeat domain, phosphoinositide interacting 2B; WT: wild-type; ZDHHC: zDHHC palmitoyltransferase.