Subnanometer resolution cryo-EM structure of Arabidopsis thaliana ATG9

Autophagy. 2020 Mar;16(3):575-583. doi: 10.1080/15548627.2019.1639300. Epub 2019 Jul 16.

Abstract

Macroautophagy/autophagy is an essential process for the maintenance of cellular homeostasis by recycling macromolecules under normal and stress conditions. ATG9 (autophagy related 9) is the only integral membrane protein in the autophagy core machinery and has a central role in mediating autophagosome formation. In cells, ATG9 exists on mobile vesicles that traffic to the growing phagophore, providing an essential membrane source for the formation of autophagosomes. Here we report the three-dimensional structure of ATG9 from Arabidopsis thaliana at 7.8 Å resolution, determined by single particle cryo-electron microscopy. ATG9 organizes into a homotrimer, with each protomer contributing at least six transmembrane α-helices. At the center of the trimer, the protomers interact via their membrane-embedded and C-terminal cytoplasmic regions. Combined with prediction of protein contacts using sequence co-evolutionary information, the structure provides molecular insights into the ATG9 architecture and testable hypotheses for the molecular mechanism of autophagy progression regulated by ATG9.Abbreviations: 2D: 2-dimensional; 3D: 3-dimensional; AtATG9: Arabidopsis ATG9; Atg: autophagy-related; ATG9: autophagy-related protein 9; cryo-EM: cryo-electron microscopy; DDM: dodecyl maltoside; GraDeR: gradient-based detergent removal; LMNG: lauryl maltose-neopentyl glycol; PAS: phagophore assembly site; PtdIns3K: phosphatidylinositol 3-kinase.

Keywords: ATG9; autophagosome; autophagy; cryo-electron microscopy; single particle.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Arabidopsis / metabolism*
  • Arabidopsis / ultrastructure*
  • Arabidopsis Proteins / metabolism*
  • Arabidopsis Proteins / ultrastructure
  • Autophagy-Related Proteins / metabolism*
  • Autophagy-Related Proteins / ultrastructure
  • Cryoelectron Microscopy*
  • Membrane Proteins / metabolism*
  • Membrane Proteins / ultrastructure
  • Models, Molecular
  • Nanotechnology*
  • Protein Multimerization
  • Protein Structure, Secondary
  • Structural Homology, Protein

Substances

  • APG9 protein, Arabidopsis
  • Arabidopsis Proteins
  • Autophagy-Related Proteins
  • Membrane Proteins

Grants and funding

This work was supported by grants from the Research Grants Council of Hong Kong (14105517 to W.C.Y.L.) and (C4011-14R, C4012-16E, C4002-17G and AoE/M-05/12 to LJ.), National Natural Science Foundation of China (31670179 and 91854201 to L.J.), CUHK Faculty Strategic Development funding (to L.J.) and Research Committee of CUHK Direct Grant for Research (4053182 to W.C.Y.L.).