Membrane curvature sensing and stabilization by the autophagic LC3 lipidation machinery

Abstract

How the highly curved phagophore membrane is stabilized during autophagy initiation is a major open question in autophagosome biogenesis. Here, we use in vitro reconstitution on membrane nanotubes and molecular dynamics simulations to investigate how core autophagy proteins in the LC3 (Microtubule-associated proteins 1A/1B light chain 3) lipidation cascade interact with curved membranes, providing insight into their possible roles in regulating membrane shape during autophagosome biogenesis. ATG12(Autophagy-related 12)–ATG5-ATG16L1 was up to 100-fold enriched on highly curved nanotubes relative to flat membranes. At high surface density, ATG12–ATG5-ATG16L1 binding increased the curvature of the nanotubes. While WIPI2 (WD repeat domain phosphoinositide-interacting protein 2) binding directs membrane recruitment, the amphipathic helix α2 of ATG16L1 is responsible for curvature sensitivity. Molecular dynamics simulations revealed that helix α2 of ATG16L1 inserts shallowly into the membrane, explaining its curvature-sensitive binding to the membrane. These observations show how the binding of the ATG12–ATG5-ATG16L1 complex to the early phagophore rim could stabilize membrane curvature and facilitate autophagosome growth.

Unique Amphipathicα-helix Drives Membrane Insertion and Enzymatic Activity of ATG3

“…Consistently, the lipidated LC3 N-terminus has been shown to interact with liposomes in vitro and in silico ( 14 ). These weak membrane associations might work in a concerted manner, in tandem with the ATG16L1 and WIPI2 proteins ( 33 ), to facilitate LC3 lipidation and/or autophagosome biogenesis.…”

“…Whether membrane binding and curvature sensing are the exclusive functions of AH ATG3 in autophagy is still unresolved. In fact, ATG16L1, a component of the E3-like ATG12–5-16L1 complex, is a dominant factor for curvature sensing among the components of LC3 conjugation machinery ( 19, 33 ), implying that AH ATG3 has another pivotal function in addition to membrane anchoring ( 34 ).…”

Membrane Curvature: The Inseparable Companion of Autophagy

“…The specific motif in the C-terminal of ATG16L1 also binds directly to membranes [ 92 ]. WIPI2 is required to recruit ATG12-ATG5-ATG16L1 to flat membranes [ 101 ], and the ATG12-ATG5-ATG16L1 complex induces membrane curvature at a high surface density [ 102 ] ( Figure 2 d). It was demonstrated that ATG16L1 and WIPI2 curvature sensing was independent of ATG3 [ 102 ].…”

“…WIPI2 is required to recruit ATG12-ATG5-ATG16L1 to flat membranes [ 101 ], and the ATG12-ATG5-ATG16L1 complex induces membrane curvature at a high surface density [ 102 ] ( Figure 2 d). It was demonstrated that ATG16L1 and WIPI2 curvature sensing was independent of ATG3 [ 102 ]. However, the properties of yeast Atg16 have been reported to differ from mammalian ATG16L1, so the model may be not suitable for yeast Atg16 [ 102 ].…”

Membrane free-energy landscapes derived from atomistic dynamics explain nonuniversal cholesterol-induced stiffening

“…A membrane-bound protein may however reshape the surrounding bilayer, sometimes strikingly, because the free-energy cost of membrane bending can be offset by free-energy gains resulting from adequate solvation of the protein surface (see [1] and references therein). In other words, lipid bilayers change shape around proteins to avoid large energetic penalties due to dehydration of ionized surface residues and/or exposure of large hydrophobic clusters to water [2, 3, 4, 5, 6, 7, 8]. This kind of balance between competing energetic contributions is not uncommon, and governs many other processes in molecular membrane physiology, including ligand- induced allostery, ion permeation, and so on.…”