Autophagosome formation is a fundamental cellular process involving the de novo generation and expansion of a double-membraned structure from a precursor known as the isolation membrane (IM). While key protein factors driving this pathway have been elucidated, the mechanisms governing lipid supply to the growing autophagic membrane remain incompletely understood. Recent studies suggest that lipid transfer from the endoplasmic reticulum (ER) to the IM is facilitated by a protein complex comprising the lipid transport protein Atg2, the IM-localized Atg18, and the lipid scramblase Atg9. However, direct in vivo evidence supporting this lipid flow model is limited.
We demonstrate that the unique lipid composition of the autophagic membrane is critical for proper autophagosome maturation. Our data reveal that ER-localized enzymes involved in phosphatidylcholine (PC) synthesis—specifically Cho2 and Opi3 of the CDP-DAG pathway—relocate to an ER subdomain adjacent to the IM in an autophagy- and Atg2- dependent manner. Furthermore, under conditions promoting the CDP-choline pathway, the rate-limiting enzyme Pct1 is similarly recruited to the vicinity of the IM upon autophagy induction. These findings support a model in which local PC synthesis at ER–IM contact sites regulates membrane composition and facilitates lipid delivery, thereby driving autophagosome biogenesis.