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• Reversible one-way lipid transfer at ER-autophagosome membrane contact sites via Atg2
• Posted by 東京大学·新領域 先端生命科学専攻　HAO Li
• 投稿日 2026/03/12

I would like to share our latest paper published in the Journal of Cell Biology.

Phospholipids are essential components of organelle membranes and are maintained through vesicular and nonvesicular transport pathways. Nonvesicular lipid trafficking largely occurs at membrane contact sites (MCSs), where lipid transfer proteins (LTPs) mediate lipid exchange between organelles. Recent studies have identified a class of bridge-like LTPs containing repeating β-groove (RBG) domains that form extended hydrophobic channels capable of bridging two membranes and facilitating efficient lipid transfer.

Among these proteins, Atg2 is essential for autophagosome biogenesis. Although structural and biochemical studies have suggested that Atg2 mediates lipid transfer at ER–IM contact sites, direct evidence of phospholipid transfer in living cells has been limited due to the lack of suitable probes.

In this study, we characterized the lipophilic dye octadecyl rhodamine B (R18), which internalizes and labels the ER in a manner that requires flippases and oxysterol-binding protein–related proteins. We showed that R18 labels each autophagy-related membrane structure in yeast. We also confirmed the applicability of R18 in mammalian cells using both fluorescence microscopy and freeze-fracture replica electron microscopy.

Because R18 stains both the ER and IM, we used it as a probe to visualize phospholipid transfer from the ER to the IM in vivo. Time-lapse imaging and FRAP analysis revealed rapid lipid supply accompanied by IM expansion. Importantly, using Atg2 mutants predicted or shown to be defective in bridge-type lipid transfer, we found that the transfer of R18 from the ER to the IM requires a broad and continuous hydrophobic groove within the bridge-like architecture of Atg2, providing direct in vivo evidence that the ER serves as the primary source of lipids for IM expansion.

Finally, we asked whether lipid flow changes when autophagy is terminated. Upon nutrient replenishment, which terminates autophagy, our data suggested reversed phospholipid flow from the IM to the ER, consistent with the persistence of the Atg2–Atg18 complex at the ER–IM MCS during termination. Together, these observations indicate reversible one-way lipid transfer mediated by Atg2 at ER–IM membrane contact sites in response to environmental changes.

Overall, our work identified a traceable probe for phospholipids undergoing nonvesicular transport and provided direct in vivo evidence for reversible, one-way lipid transfer via Atg2 at the ER–IM MCS. These findings highlight the critical role of bridge-like LTPs in MCS-mediated phospholipid homeostasis in response to environmental changes.

We thank Dr. Nobuo N. Noda for collaboration. We thank Drs. Yuta Ogasawara and Yutaro Hama for experiments in mammalian cells, Dr. Takuma Tsuji for freeze-fracture replica electron microscopy, Dr. Takuma Kishimoto for the super-tether mutant, and Dr. Kazuaki Matoba for performing the lipid transfer assay of the atg2LT protein. We also thank Drs. Christopher T. Beh, Hitoshi Nakatogawa, Jasper Rine, Ryouichi Fukuda, and Kazuma Tanaka for providing plasmids and strains.

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