Structural basis for membrane recruitment of ATG16L1 by WIPI2 in autophagy

Abstract

Autophagy is a cellular process that degrades cytoplasmic cargo by engulfing it in a double membrane vesicle, known as the autophagosome, and delivering it to the lysosome. The ATG12-5-16L1 complex is responsible for conjugating members of the ubiquitin-like ATG8 protein family to phosphatidylethanolamine in the growing autophagosomal membrane, known as the phagophore. ATG12-5-16L1 is recruited to the phagophore by a subset of the phosphatidylinositol 3-phosphate-binding seven bladed â-propeller WIPI proteins. We determined the crystal structure of WIPI2d in complex with the WIPI2 interacting region (W2IR) of ATG16L1 comprising residues 207-230 at 1.85 Å resolution. The structure shows that the ATG16L1 W2IR adopts an alpha helical conformation and binds in an electropositive and hydrophobic groove between WIPI2 â-propeller blades 2 and 3. Mutation of residues at the interface reduces or blocks the recruitment of ATG12-5-16L1 and the conjugation of the ATG8 protein LC3B to synthetic membranes. Interface mutants show a decrease in starvation-induced autophagy. Comparisons across the four human WIPIs suggest that WIPI1 and 2 belong to a W2IR-binding subclass responsible for localizing ATG12-5-16L1 and driving ATG8 lipidation, whilst WIPI3 and 4 belong to a second W34IR-binding subclass responsible for localizing ATG2, and so directing lipid supply to the nascent phagophore. The structure provides a framework for understanding the regulatory node connecting two central events in autophagy initiation, the action of the autophagic PI 3-kinase complex on the one hand, and ATG8 lipidation on the other.

Membrane curvature sensing and stabilization by the autophagic LC3 lipidation machinery

“…Residues 272-296 within the 6CD loop of WIPI2d 23 were identified to constitute a candidate curvature-sensing element based on secondary structure prediction 36 , analysis of physicochemical properties 37 , and atomistic molecular dynamics simulations. The prediction of two short amphipathic α-helices, respectively consisting of residues 272-284 and 290-296 (Extended Data Fig.…”

“…Atomistic models of the ATG12–5-16L1 and WIPI2d-ATG16L1 complexes were based on crystal structures with PDB IDs 4NAW 54 and 7MU2 23 , respectively. The ATG16L1 N-terminal helix in the former complex was replaced by a more complete structure (PDB ID: 4TQ0 55 ) and residues 1-9 were added using the DEMO server 56 to give a model of residues 1-50.…”

“…The E1 ATG7 binds LC3, handing it off to the E2 ATG3, which works together with the E3 ATG12–5-16L1 to covalently conjugate LC3 to PE headgroups on the phagophore. The ATG12–5-16L1 is recruited to PI(3)P-positive autophagic membranes by the PROPPIN WIPI2 through its binding to a WIPI2 interacting region in ATG16L1 22,23 . These reactions have been reconstituted in vitro on sonicated liposomes 17,24 and flat membranes 25 .…”

Specific microRNAs for Modulation of Autophagy in Spinal Cord Injury

“…WIPI1 and WIPI2 are the first to associate with the phagophore, and while not strictly necessary for autophagy, WIPI1 has been shown to associate with and enhance the action of WIPI2 [ 61 ]. WIPI2 subsequently recruits the ATG12–ATG5–ATG16L1 complex, which is necessary for the lipidation of LC3 (microtubule-associated protein Light Chain 3)/GABARAP (GABA type A Receptor-Associated Protein) family proteins to the inner and outer autophagosomal membranes [ 62 , 63 , 64 ]. LC3 lipidation is accomplished through a ubiquitin-like conjugation system.…”

Multilayered regulation of autophagy by the Atg1 kinase orchestrates spatial and temporal control of autophagosome formation

“…The dimeric architecture of the Atg1 complex ( Ragusa et al., 2012 ) and its arrangement into a higher-order mesh-like structure ( Yamamoto et al., 2016 ) is thought to tether Atg9-containing vesicles, thus initiating autophagosomal membrane formation at the pre-autophagosomal structure (PAS) ( Kishi-Itakura et al., 2014 ; Mari et al., 2010 ; Rao et al., 2016 ). Phosphatidylinositol 3-phosphate (PI3P) synthesis in the growing phagophore catalyzed by the Vps34 Atg14/Atg38 complex recruits the Atg18-Atg2 complex ( Obara et al., 2008 ), which in turn contributes to autophagosome formation by tethering membranes and transfering lipids ( Kotani et al., 2018 ; Maeda et al., 2019 ; Osawa et al., 2019 ; Valverde et al., 2019 ) and by recruiting part of the Atg8 lipidation machinery ( Dooley et al., 2014 ; Strong et al., 2021 ).…”