Mechanistic studies of autophagic cargo recruitment and membrane shaping through in vitro reconstitution

Macroautophagy/autophagy, a conserved intracellular catabolic pathway, removes deleterious cytosolic material to maintain homeostasis and survival. Upon autophagy induction, a unique double-membraned structure, the phagophore, forms and engulfs cytosolic material, the cargo, as it closes to become an autophagosome. Mammalian Atg8-family proteins (ATG8s) are ubiquitin-like proteins which are essential for engulfment of the cargo and membrane closure. ATG8s are recruited to the phagophore by ATG12–ATG5-ATG16L1, an E3-like ligase which is recruited by PtdIns3P-binding WIPI proteins. Covalent lipidation of the ATG8s to phosphatidylethanolamine by the E3 ligase occurs specifically on the phagophore membrane allowing recruitment of cytosolic cargo and cargo receptors, such as SQSTM1/p62. While ATG8-cargo receptor interactions are well established, how the ATG8s bind cargo and cargo receptors on the inner membrane of the phagophore has not been studied. To recapitulate these events, we use giant unilamellar vesicles (GUVs) and encapsulate protein machinery and cargo, generating a membrane platform to which ATG8 proteins can be recruited. Inside the GUVs we reconstituted WIPI2B-directed and cargo-directed ATG8 lipidation revealing distinct roles of WIPI2B and SQSTM1 in initiating ATG8 conjugation. We show that SQSTM1 and SQSTM1 droplets are recruited to the GUV inner membrane through interaction with membrane bound ATG8s. Through the development of a bead-based membrane deformation assay, we show redistribution and local enrichment of membrane-bound ATG8s occurs upon binding to SQSTM1 droplets. Our work demonstrates fundamental molecular mechanisms into phagophore-ATG8-cargo interactions providing novel model systems to investigate ATG8-cargo interactions on the inner phagophore membrane.Abbreviations:ATG: autophagy related; cDICE: continuous droplet interface crossing encapsulation; DOPE: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; GABARAP: GABA type A receptor-associated protein; GUV: giant unilamellar vesicle; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; LIR: LC3-interacting region; LUV: large unilamellar vesicle; NBD: 7-nitrobenz-2-oxa-1,3-diazol-4-yl; PE: phosphatidylethanolamine; PtdIns: phosphatidylinositol; PtdIns3P: phosphatidylinositol-3-phosphate; PolyUb: K63-linked polyubiquitin; POPC: 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine; POPE: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; Rh-PE: 18:1 Liss Rhod PE; SQSTM1/p62: sequestosome 1; WIPI2B: WD repeat domain, phosphoinositide interacting 2B