Abstract
Specific protein-lipid interactions lead to a gradual recruitment of AuTophaGy-related (ATG) proteins to the nascent membrane during autophagosome (AP) formation. ATG3, a key protein in the movement of LC3 towards the isolation membrane, has been proposed to facilitate LC3/GABARAP lipidation in highly curved membranes. In this work we have performed a biophysical study of human ATG3 interaction with membranes containing phosphatidylethanolamine, phosphatidylcholine and anionic phospholipids. We have found that ATG3 interacts more strongly with negatively-charged phospholipid vesicles or nanotubes than with electrically neutral model membranes, cone-shaped anionic phospholipids (cardiolipin and phosphatidic acid) being particularly active in promoting binding. Moreover, an increase in membrane curvature facilitates ATG3 recruitment to membranes although addition of anionic lipid molecules makes the curvature factor relatively less important. The predicted N-terminus amphipathic α-helix of ATG3 would be responsible for membrane curvature detection, the positive residues Lys 9 and 11 being essential in the recognition of phospholipid negative moieties. We have also observed membrane aggregation induced by ATG3 in vitro, which could point to a more complex function of this protein in AP biogenesis. Moreover, in vitro GABARAP lipidation assays suggest that ATG3-membrane interaction could facilitate the lipidation of ATG8 homologues.
Highlights
Macroautophagy is a bulk degradation pathway conserved among eukaryotic cells[1]
In order to assess the direct implication of the N-terminal region of ATG3 in membrane binding and curvature sensing, we produced the ATG3 K9D/K11D mutant, in which the sequence predicted to fit into the bilayer-water interface is modified chemically and electrically
We first assayed the ability of ATG3 and its mutant to interact with and insert into lipid monolayers formed at the air-water interface in a Langmuir balance
Summary
Macroautophagy is a bulk degradation pathway conserved among eukaryotic cells[1]. This process is characterized by the generation of a double membrane structure called autophagosome (AP) which engulfs organelles or cytoplasmic portions and subsequently delivers the material into the lysosome for degradation[2]. AP formation requires more than 30 autophagy-related (ATG) proteins acting in a hierarchical way[3]. Among these proteins an ubiquitin-like (UBL) system, composed by Atg[7], Atg[3] and Atg[8], triggers the covalent attachment of Atg[8] (LC3 and GABARAP subfamilies in mammals) to phosphatidylethanolamine (PE), a lipid found in the AP membrane[4,5]. Human ATG3, the E2-like enzyme for Atg[8] conjugation, contains some disordered regions that allow its classification among the intrinsically disordered proteins[11] This property is found in proteins that participate in processes required for quick cellular responses, such as autophagy. We have observed the effect of membrane tethering induced by the ATG3 in vitro, which might point to a more complex role of this protein in the process of AP formation
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