Abstract
De novo formation of the double-membrane compartment autophagosome is seeded by small vesicles carrying membrane protein autophagy-related 9 (ATG9), the function of which remains unknown. Here we find that ATG9A scrambles phospholipids of membranes in vitro. Cryo-EM structures of human ATG9A reveal a trimer with a solvated central pore, which is connected laterally to the cytosol through the cavity within each protomer. Similarities to ABC exporters suggest that ATG9A could be a transporter that uses the central pore to function. Moreover, molecular dynamics simulation suggests that the central pore opens laterally to accommodate lipid headgroups, thereby enabling lipids to flip. Mutations in the pore reduce scrambling activity and yield markedly smaller autophagosomes, indicating that lipid scrambling by ATG9A is essential for membrane expansion. We propose ATG9A acts as a membrane-embedded funnel to facilitate lipid flipping and to redistribute lipids added to the outer leaflet of ATG9 vesicles, thereby enabling growth into autophagosomes.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Autophagosomes / chemistry*
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Autophagosomes / metabolism
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Autophagy-Related Proteins / chemistry*
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Autophagy-Related Proteins / genetics
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Autophagy-Related Proteins / metabolism
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Binding Sites
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Biological Transport
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Cell Line
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Cryoelectron Microscopy
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Fibroblasts / metabolism
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Fibroblasts / ultrastructure
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Gene Expression
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Green Fluorescent Proteins / chemistry
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Green Fluorescent Proteins / genetics
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Green Fluorescent Proteins / metabolism
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HEK293 Cells
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HeLa Cells
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Humans
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Lipid Bilayers / chemistry
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Lipid Bilayers / metabolism
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Luminescent Proteins / genetics
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Luminescent Proteins / metabolism
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Membrane Proteins / chemistry*
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Membrane Proteins / genetics
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Membrane Proteins / metabolism
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Mice
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Molecular Dynamics Simulation
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Phospholipids / chemistry*
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Phospholipids / metabolism
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Protein Binding
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Protein Conformation, alpha-Helical
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Protein Conformation, beta-Strand
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Protein Interaction Domains and Motifs
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Protein Multimerization
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Proteolipids / chemistry*
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Proteolipids / metabolism
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Recombinant Fusion Proteins / chemistry
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism
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Red Fluorescent Protein
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Vesicular Transport Proteins / chemistry*
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Vesicular Transport Proteins / genetics
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Vesicular Transport Proteins / metabolism
Substances
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ATG9A protein, human
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Autophagy-Related Proteins
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Lipid Bilayers
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Luminescent Proteins
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Membrane Proteins
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Phospholipids
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Proteolipids
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Recombinant Fusion Proteins
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Vesicular Transport Proteins
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proteoliposomes
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Green Fluorescent Proteins