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bioRxiv · 10.1101/2025.05.24.655882

Mechanism of bridge-type phospholipid transfer by Atg2 for autophagosome biogenesis

Sakai, Y.·Matoba, K.·Kotani, T.·Hao, L.·Suzuki, K.·Kakuta, C.·Sugita, Y.·Osawa, T.·Nakatogawa, H.·Noda, N. N.
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Abstract

Autophagosome biogenesis requires delivery of millions of phospholipids. In budding yeast, Atg2 and Atg18 couple the ER to the isolation membrane for lipid transport, yet the mechanism is poorly understood. Combining molecular dynamics simulations, in vitro imaging-based lipid transfer assays, and in vivo analyses, we show that Atg1-mediated phosphorylation activates Atg2 as a lipid-transfer bridge. Simulations reveal that Atg2 contains a water-excluding hydrophobic cavity that accommodates [~]25 phospholipids with bilayer-like fluidity. Atg1 phosphorylates an N-terminal helix of Atg2 in vitro and in vivo, thereby opening the ER-facing entrance to the Atg2 cavity and enabling lipid flow between membranes in silico and in vitro. Consistently, phosphodeficient but not phosphomimetic mutations impair autophagy. Cavity narrowing along the conduit disrupts lipid alignment in silico, phosphorylation-activated bridge-type lipid transfer in vitro, and ER-to-isolation-membrane lipophilic dye flow and autophagosome expansion in vivo. These data suggest that Atg1 gates Atg2-mediated bridge-type lipid transport to build autophagosomes.

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Sakai, Y., Matoba, K., Kotani, T., Hao, L., Suzuki, K., Kakuta, C., Sugita, Y., Osawa, T., Nakatogawa, H., Noda, N. N.. 2025-05-26. Mechanism of bridge-type phospholipid transfer by Atg2 for autophagosome biogenesis. https://doi.org/10.1101/2025.05.24.655882

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