Membrane atg8ylation in Canonical and Noncanonical Autophagy

Abstract

Membrane atg8ylation is a homeostatic process responding to membrane remodeling and stress signals. Membranes are atg8ylated by mammalian ATG8 ubiquitin-like proteins through a ubiquitylation-like cascade. A model has recently been put forward which posits that atg8ylation of membranes is conceptually equivalent to ubiquitylation of proteins. Like ubiquitylation, membrane atg8ylation involves E1, E2 and E3 enzymes. The E3 ligases catalyze the final step of atg8ylation of aminophospholipids in membranes. Until recently, the only known E3 ligase for membrane atg8ylation was ATG16L1 in a noncovalent complex with the ATG12-ATG5 conjugate. ATG16L1 was first identified as a factor in canonical autophagy. During canonical autophagy, the ATG16L1-based E3 ligase complex includes WIPI2, which in turn recognizes phosphatidylinositiol 3-phosphate and directs atg8ylation of autophagic phagophores. As an alternative to WIPIs, binding of ATG16L1 to the proton pump V-ATPase guides atg8ylation of endolysosomal and phagosomal membranes in response to lumenal pH changes. Recently, a new E3 complex containing TECPR1 instead of ATG16L1, has been identified that responds to sphingomyelin's presence on the cytofacial side of perturbed endolysosomal membranes. In present review, we cover the principles of membrane atg8ylation, catalog its various presentations, and provide a perspective on the growing repertoire of E3 ligase complexes directing membrane atg8ylation at diverse locations.

Keywords: Autophagy; CASM; GABARAP; LAP; LC3; atg8ylation; lipids; ubiquitination.

Figure 1.. Membrane atg8ylation.

A,B. Membrane atg8ylation is covalent membrane modification by ubiquitin-like proteins of the mammalian ATG8 family (mATG8s) which includes LC3A, LC3B, LC3C, GABARAP, GABARAPL1, and GABARAPL2. It is a response to membrane stress, damage and remodeling signals and participates in various atg8ylation-dependent processes including canonical autophagy and a range of non-autophagic phenomena. The membrane atg8ylation and de-atg8ylation cycle is similar to the process of protein ubiquitylation and deubiquitylation. C. The Gly residues at the C-termini of mATG8s, exposed through proteolytic processing by ATG4 peptidases, are activated by ATP and conjugated via a Gly-Cys thioester bond with the E1 enzyme ATG7, transferred to the E2 enzyme ATG3, and finally conjugated to the headgroups of aminophospholipids (phosphatidylethanolamine/PE or phosphatidylserine/PS). The final step is catalyzed by multi-subunit E3 ligases, with obligatory catalytic component, the ATG12–ATG5 covalent conjugate that is generated in its own conjugation cascade. The ATG12–ATG5 catalytic components is brought to the membrane sites via ATG16L1 or TECPR1. Modified after Kumar et al.

Figure 2.

Structural similarities and differences between ubiquitin and mATG8s.

Figure 3.. Membrane atg8ylation outputs.

A. Atg8ylation cycle core components. B. Atg8ylation outputs. Canonical autophagy depends on the formation of autophagosomes that sequester and digest diverse cytoplasmic cargo, with membrane atg8ylation playing a role in cargo capture, membrane remodeling and closure, and in the maintenance of autophagosomal membrane integrity. Membrane atg8ylation participates in membrane repair following damage of or stress imposed on various organelles. LAP, LC3-associated phagocytosis; LAM, LC3-associated micropinocytosis; LANDO, LC3-associated endocytosis; VAIL, V-ATPase-ATG16L1-induced LC3 lipidation; CASM, conjugation of ATG8 to single membranes; SMAC, single membrane ATG8 conjugation. Secretory autophagy is a collection of diverse processes that lead to exocytosis and extracellular release of cargo. All of the above phenomena depend on atg8ylation of single membranes (phospholipid bilayer) and only upon closure of canonical autophagosomes (not depicted) do these organelles appear as ‘double membrane’ Lipid droplets atg8ylation requires only a monolayer/hemilayer of phospholipids. C. Classes of mammalian ATG8s and representative structures.

Figure 4.. E3 ligases directing membrane atg8ylation.

A. ATG12–ATG5-ATG16L1-WIPI2 atg8ylation E3 ligase, well-known for its role in canonical autophagy. B. ATG12–ATG5-ATG16L1-V-ATPase atg8ylation E3 ligase, responding to perturbances in V-ATPase containing compartments including neutralization or permeabilization leading to increase in lumenal pH. C. ATG12–ATG5-TECPR1 atg8ylation E3 ligase, responding to lysosome stress or damage. Models are based on structural information (PDB, Electron Microscopy, or Alphafold databases). Gray line, membrane bilayer. SM, sphingomyelin (recognized by TECPR1) on stressed membranes presented on the cytofacial leaflet. PI3P, phosphatidylinositol 3-phosphate, recognized by WIPI2, generated by phosphatidylinositol 3-kinase (PI3K) as a part of canonical autophagy. pH↑, neutralization/proton leak from the lumen of V-ATPase containing organelles. Modified after Deretic and Klionsky.