The formation of autophagosomes requires the rapid expansion of a membrane structure around cytoplasmic cargo. In mammalian cells, phagophore assembly and expansion depend on ATG9A-positive vesicular seed compartments, the lipid transfer protein ATG2A, and the class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1). PI3KC3-C1 generates phosphatidylinositol 3-phosphate (PI3P), which facilitates downstream events critical for phagophore maturation, including ATG8 protein lipidation. While many components of this pathway are known, the mechanisms coordinating their activities remain incompletely understood.Â
Here, we demonstrate that ATG9A vesicles lack detectable levels of phosphatidylinositol (PI), the substrate required for PI3P synthesis. Nevertheless, ATG2A is recruited to ATG9A vesicles, where it transfers lipids from donor membranes - including PI - into these compartments. Notably, ATG9A vesicles can support ATG8 lipidation even in the absence of detectable PI3P, and ATG8 proteins themselves enhance ATG2A-mediated lipid transfer. In cells, the presence of PI3P on ATG9A-positive compartments is dependent on ATG2A, suggesting that PI must first be delivered to these membranes to enable PI3P synthesis.
Our findings reveal a lipid transfer–dependent feedback loop that activates ATG9A compartments for phagophore expansion. We propose that PI transfer from donor membranes by ATG2A is a prerequisite for PI3KC3-C1 activity and subsequent PI3P production. This, in turn, promotes recruitment of ATG8 and WIPI proteins, which further stabilize ATG2A at the membrane and enhance phospholipid transfer.