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

  • 2271-Pos Board B287 Ionization and Dynamic Properties of Single and Multiple Histidine Residues on a Transmembrane Helical Backbone Fahmida Afrose, Denise V

  • 2269-Pos Board B285 Human ATG3 Binding to Lipid Bilayers

  • Solid-state 2H NMR was performed on the peptide in the lipid-bilayers of DOPC, DMPC, and DLPC

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Summary

Introduction

2271-Pos Board B287 Ionization and Dynamic Properties of Single and Multiple Histidine Residues on a Transmembrane Helical Backbone Fahmida Afrose, Denise V. To allow analysis of a wide range of lipid interactions, we employ lipoprotein nanodiscs to deliver membrane proteins for native MS. We can measure the mass of the intact nanodisc, which allows direct measurement of the oligomeric state of the membrane protein within the lipid bilayer of the nanodisc. Our ultimate goal is to develop a general method for characterizing protein-lipid interactions of varying strengths and specificities.

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