An N-terminal conserved region in human Atg3 couples membrane curvature sensitivity to conjugase activity during autophagy

Yan Shen,Yansheng Ye,Xuejun Jiang,Erin R Tyndall,Fang Tian,Hong-Gang Wang,Zhenyuan Tang,John M Flanagan,Van Bui

doi:10.1038/s41467-020-20607-0

Abstract

During autophagy the enzyme Atg3 catalyzes the covalent conjugation of LC3 to the amino group of phosphatidylethanolamine (PE) lipids, which is one of the key steps in autophagosome formation. Here, we have demonstrated that an N-terminal conserved region of human Atg3 (hAtg3) communicates information from the N-terminal membrane curvature-sensitive amphipathic helix (AH), which presumably targets the enzyme to the tip of phagophore, to the C-terminally located catalytic core for LC3–PE conjugation. Mutations in the putative communication region greatly reduce or abolish the ability of hAtg3 to catalyze this conjugation in vitro and in vivo, and alter the membrane-bound conformation of the wild-type protein, as reported by NMR. Collectively, our results demonstrate that the N-terminal conserved region of hAtg3 works in concert with its geometry-selective AH to promote LC3–PE conjugation only on the target membrane, and substantiate the concept that highly curved membranes drive spatial regulation of the autophagosome biogenesis during autophagy.

Highlights

During autophagy the enzyme Atg[3] catalyzes the covalent conjugation of LC3 to the amino group of phosphatidylethanolamine (PE) lipids, which is one of the key steps in autophagosome formation
Using LC3B, a mammalian homolog of yeast Atg[8], we show that mutations in this region greatly reduce or abolish LC3B–PE conjugations in the in vitro and in vivo assays, despite retaining normal liposome binding and formation of the thioester LC3B-hAtg[3] intermediate
Despite the fact that the NT is essential for protein activity, this region sharply diverges in sequence identity and length, with fungi tending to have shorter helices and more large hydrophobic residues compared to other kingdoms

Summary

Introduction

During autophagy the enzyme Atg[3] catalyzes the covalent conjugation of LC3 to the amino group of phosphatidylethanolamine (PE) lipids, which is one of the key steps in autophagosome formation. The corresponding putative AH region of mouse Atg[3] (mAtg3) has recently been shown to be responsible for its membrane curvature-dependent conjugation activity; the catalytic activity of mAtg[3] for LC3–PE formation markedly increases as the size of liposomes decreases below 50 nm[15,16] This has led to the suggestion that the N-terminal AH guides the enzyme to the leading edge of an expanding phagophore, where the membrane is highly curved with a radius possibly as small as 10 nm[17]. Sequence analysis of the Atg[3] N-terminus from multiple species reveals a highly conserved region extending from residues 17 to 26 of hAtg[3] (Fig. 1a) This region includes the last three resides of the N-terminal AH (residues 3–19), and the corresponding region in the yeast and A. thaliana homologs is not observed in existing crystal structures. Non-polar and aromatic residues are in yellow, polar residues in green, positively charged and histidine residues in blue, negatively charged residues in red, alanine and glycine residues in black, and proline residue in purple. b Diagrams of hAtg[3] NT constructs prepared in this study

Methods

Results

Conclusion