Lipidation of the LC3/GABARAP family of autophagy proteins relies on a membrane-curvature-sensing domain in Atg3

Abstract

The protein biochemistry supporting autophagosome growth on the cup-like isolation membrane is likely different from the biochemistry on the closed and maturing autophagosome. Thus, the highly curved rim of the cup may serve as a functionally-required surface for transiently-associated components of the early-acting autophagic machinery. Here we demonstrate that the E2-like enzyme, Atg3, facilitates LC3/GABARAPlipidation only on membranes exhibiting local lipid-packing defects. This activity requires an amino-terminal amphipathic helix similar to motifs found on proteins targeting highly curved intracellular membranes. By tuning the hydrophobicity of this motif, we can promote or inhibit lipidation in vitro and in rescue experiments in Atg3 knockout cells, implying a physiologic role for this stress detection. The need for extensive lipid-packing defects suggests that Atg3 is designed to work at highly-curved membranes perhaps including the limiting edge of the growing phagophore.

A LRRK2-Dependent EndophilinA Phosphoswitch Is Critical for Macroautophagy at Presynaptic Terminals

“…While it is very challenging to locate EndoA at the preautophagosome edge in vivo, highly curved membrane domains serve as docking sites for autophagic factors including Atg3, Atg14 and Atg1 (Fan et al, 2011;Nath et al, 2014;Ragusa et al, 2012 Macroautophagy mutants atg5 and atg7 display neurodegeneration and both genes are risk factors in PD (Chen et al, 2013a(Chen et al, , 2013bHara et al, 2006;Juhász et al, 2007;Komatsu et al, 2006). PD is often characterized by misfolded, dysfunctional proteins and synaptic dysfunction is recognized in PD models as well (Burke and O'Malley, 2013).…”

“…While it is very challenging to locate EndoA at the preautophagosome edge in vivo, highly curved membrane domains serve as docking sites for autophagic factors including Atg3, Atg14 and Atg1 (Fan et al, 2011;Nath et al, 2014;Ragusa et al, 2012 . This suggests that Atg3 detects lipid packing defects in highly curved membranes, possibly by inserting an helix into the bent membrane (Nath et al, 2014;Lauwers et al, 2016 The concept of synaptic dysfunction and proteopathic stress as a consequence of failing protein quality control in neurodegenerative disease is emerging, but how quality control systems operate at synapses is poorly understood.…”

“…The curved edges on nascent preautophagosomes and PI(3)P are docking sites for essential autophagic factors, including Atg3 (Fan et al, 2011;Nath et al, 2014). EndoA, a protein mostly studied for its role in synaptic vesicle endocytosis , may be a good candidate 8 to be involved in this process because the phosphorylation of EndoA by LRRK/LRRK2 at S75 in helix H1 alters the ability of EndoA to interact with membranes (Ambroso et al, 2014;.…”

“…These edges serve as protein docking sites, attracting specific autophagic factors such as Atg3, Atg14/Barkor and Atg1 that insert into such zones, recognizing specific lipids (phosphatidylinositol-3-phosphate (PI(3)P)) and lipid packing defects (Fan et al, 2011;Nath et al, 2014;Ragusa et al, 2012). The recruitment of these factors then promotes the further steps of autophagosome formation; in particular the E2-like protein Atg3 itself recruits the autophagic marker LC3/Atg8 (Nath et al, 2014). However, how these highly curved edges are formed and maintained is very poorly understood.…”

Role of actin in shaping autophagosomes

“…Both roles are highly probable. It is known that one of the E2-like enzymes engaged in ubiquitination-type conjugation reactions, ATG3, facilitates LC3/GABARAP lipidation only on membranes exhibiting local lipid-packing defects, 19 for example at highly-curved membranes existing at the limiting edge of the growing phagophore (Fig. 1Cvi).…”