ATG9B is a tissue-specific homotrimeric lipid scramblase that can compensate for ATG9A

ABSTRACT

Macroautophagy/autophagy is a fundamental aspect of eukaryotic biology, and the autophagy-related protein ATG9A is part of the core machinery facilitating this process. In addition to ATG9A vertebrates encode ATG9B, a poorly characterized paralog expressed in a subset of tissues. Herein, we characterize the structure of human ATG9B revealing the conserved homotrimeric quaternary structure and explore the conformational dynamics of the protein. Consistent with the experimental structure and computational chemistry, we establish that ATG9B is a functional lipid scramblase. We show that ATG9B can compensate for the absence of ATG9A in starvation-induced autophagy displaying similar subcellular trafficking and steady-state localization. Finally, we demonstrate that ATG9B can form a heteromeric complex with ATG2A. By establishing the molecular structure and function of ATG9B, our results inform the exploration of niche roles for autophagy machinery in more complex eukaryotes and reveal insights relevant across species.

Abbreviation: ATG: autophagy related; CHS: cholesteryl hemisuccinate; cryo-EM: single-particle cryogenic electron microscopy; CTF: contrast transfer function: CTH: C- terminal α helix; FSC: fourier shell correlation; HDIR: HORMA domain interacting region; LMNG: lauryl maltose neopentyl glycol; MD: molecular dynamics simulations; MSA: multiple sequence alignment; NBD-PE: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2–1,3-benzoxadiazol-4-yl ammonium salt); POPC: palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; RBG: repeating beta groove domain; RMSD: root mean square deviation; SEC: size-exclusion chromatography; TMH: transmembrane helix

KEYWORDS:

• ATG9A
• autophagy
• cryo-EM
• membrane protein dynamics
• phagophore
• scramblase

Acknowledgements

We thank the Structural Biology Science Technology Platform at the Francis Crick Institute, particularly Andrea Nans, for assistance with cryo-EM data collection and computing. We also acknowledge Danny Lang, John Roche, and Mike Gavrielides for technical assistance with data storage and processing as well Matthew Butt for assistance with molecular movie preparation. Finally, we thank members of the Molecular Cell Biology of Autophagy lab for helpful discussions of the work as it was being undertaken.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/15548627.2023.2275905

Additional information

Funding

The work was supported by the Cancer Research UK [CC2058]; Cancer Research UK [CC2134]; European Research Council [788708]; Medical Research Council [CC2058]; Medical Research Council [CC2134]; Wellcome Trust [CC2134]; Wellcome Trust [CC2058].