ATG9A Knockout Cell Line (HEK293)

The integral membrane protein ATG9A plays a key role in autophagy. It displays a broad intracellular distribution and is present in numerous compartments, including the plasma membrane (PM). The reasons for the distribution of ATG9A to the PM and its role at the PM are not understood. Here, we show that ATG9A organizes, in concert with IQGAP1, components of the ESCRT system and uncover cooperation between ATG9A, IQGAP1 and ESCRTs in protection from PM damage. ESCRTs and ATG9A phenocopied each other in protection against PM injury. ATG9A knockouts sensitized the PM to permeabilization by a broad spectrum of microbial and endogenous agents, including gasdermin, MLKL and the MLKL-like action of coronavirus ORF3a. Thus, ATG9A engages IQGAP1 and the ESCRT system to maintain PM integrity.

The multispanning membrane protein ATG9A is a scramblase that flips phospholipids between the two membrane leaflets, thus contributing to the expansion of the phagophore membrane in the early stages of autophagy. Herein, we show that depletion of ATG9A does not only inhibit autophagy but also increases the size and/or number of lipid droplets in human cell lines and C. elegans. Moreover, ATG9A depletion blocks transfer of fatty acids from lipid droplets to mitochondria and, consequently, utilization of fatty acids in mitochondrial respiration. ATG9A localizes to vesicular-tubular clusters (VTCs) that are tightly associated with an ER subdomain enriched in another multispanning membrane scramblase, TMEM41B, and also in close proximity to phagophores, lipid droplets and mitochondria. These findings indicate that ATG9A plays a critical role in lipid mobilization from lipid droplets to autophagosomes and mitochondria, highlighting the importance of ATG9A in both autophagic and non-autophagic processes.

ABBREVIATION: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 A: bafilomycin A; CRISPR: clustered regularly interspaced short palindromic repeats; CTD: C-terminal domain; Cys: cysteine; DAB: 3,3'-diaminobenzidine; EV: empty vector; HO: 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.