Human ATG3 Binding to Lipid Bilayers. Role of Lipid Geometry and Electric Charge

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.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 Atg7, Atg3 and Atg8, triggers the covalent attachment of Atg8 (LC3 and GABARAP subfamilies in mammals) to phosphatidylethanolamine (PE), a lipid found in the AP membrane 4,5 . It has been shown that Atg3 is responsible for transferring Atg8 to the membrane 6,7 , where Atg8 participates in AP formation, maturation and closure. In fact, it has been described that mammalian Atg8-like proteins (LC3, GATE-16 and GABARAP) induce membrane tethering and fusion in vitro [8][9][10] , which could be related to the AP growth process. The correct equilibrium of these processes is vital for the maintenance of cell homeostasis, and their imbalance is related to human disorders e.g. neurodegenerative diseases and cancer.Human ATG3, the E2-like enzyme for Atg8 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. Atg3 handle region (HR) and flexible region (FR) have been found to interact with Atg7 and Atg8, respectively 12 . These two domains ac...

Protein Amphipathic Helix Insertion: A Mechanism to Induce Membrane Fission

“…Among 16 AHs shown in the Table 1 of Drin and Antonny (2010), this motif is present only in α-synuclein, a protein that binds cardiolipin (Nakamura et al, 2008;Robotta et al, 2014;Ryan et al, 2018). However, except proteins listed in the Table 3, this motif is present in the AHs of at least two proteins that interact with cardiolipin but, to the best of our knowledge, are not known to be involved in membrane fission: PmtA from Bradyrhizobium japonicum (Danne et al, 2017a) and ATG3 (Nath et al, 2014;Hervás et al, 2017). Arg14 and Phe21 belong to TABLE 3 | Amphipathic helices of fission-inducing proteins that use cardiolipin as a lipid cofactor.…”

“…Amphipathic helix References the (K/R)x 6 (F/Y) motif in the AH of PmtA from B. japonicum (Danne et al, 2017a), whereas Lys11 and Tyr18 belong to the (K/R)x 6 (F/Y) motif in the AH of Atg3 (Hervás et al, 2017). In the AH of cardiolipin-binding fission-inducing protein PmtA from A. tumefaciens (Danne et al, 2017b), both Lys12 and Phe19 belonging to the Kx 6 F motif are critical for membrane binding (Danne et al, 2015).…”

Atg3 promotes Atg8 lipidation via altering lipid diffusion and rearrangement

“…Previous studies have identified two ubiquitin‐like modification systems, Atg7–Atg3–Atg8 and Atg5–Atg12–Atg16, which synergistically conjugate Atg8 to PE 15 . It is also well known that Atg3, the E2‐like enzyme, transfers Atg8 to phagophore via binding of its NH domain to the highly curved membrane structures 21,23 . In this study, our results suggest that in addition to the membrane interaction, Atg3 attenuates lipid diffusion while simultaneously enriches local PE concentration to promote Atg8 conjugation to PE.…”

“…Hence, the increased concentration of PE caused by Atg3 may account for the ability of Atg3 in promoting Atg8 lipidation. Although direct evidence for the interaction between Atg3 and PE is currently lacking, we assume that Atg3 functions via deforming the membranes, 21,43 which leads to lipid arrangement and PE enrichment.…”

“…The rate and efficiency of the conjugation reaction rely on the enzyme activity of Atg3 and the concentrations of the reaction components (i.e., the Atg3–Atg8 complex and PE). The Atg3 NH domain has been implicated in membrane binding and membrane curvature sensing, both essential for increasing local Atg3 concentration at specific sites on the autophagosome membrane 20–23 . In addition, in vitro studies showed that high PE concentration leads to efficient Atg8‐PE conjugation.…”