Reversible One-way Lipid Transfer at ER–Autophagosome Membrane Contact Sites via Bridge-like Lipid Transfer Protein Atg2

Bridge-like lipid transfer proteins (LTPs) contain a repeating β-groove domain and long hydrophobic grooves that act as bridges at membrane contact sites (MCSs) to efficiently transfer lipids. Atg2 is one such bridge-like LTP essential for autophagosome formation, during which a newly synthesized isolation membrane (IM) emerges and expands through lipid supply. However, studies on Atg2-mediated lipid transfer are limited toin vitrostudies due to the lack of a suitable probe for monitoring phospholipid dynamicsin vivo. Here, we characterized the lipophilic dye octadecyl rhodamine B (R18), which reached the endoplasmic reticulum (ER) through the actions of flippases and oxysterol-binding protein-related proteins. Using R18, we demonstrated phospholipid transfer from the ER to the IM during autophagyin vivo. Upon autophagy termination, we observed reversible phospholipid flow from the IM to the ER in response to environmental changes. Our findings highlight the critical role of bridge-like LTPs in MCS-mediated phospholipid homeostasis.