ATG9A vesicles are a subtype of intracellular nanovesicle.

Cells are filled with thousands of vesicles, which mediate protein transport and ensure homeostasis of the endomembrane system. Distinguishing these vesicles functionally and molecularly represents a major challenge. Intracellular nanovesicles (INVs) are a large class of transport vesicles that likely comprise multiple subtypes. Here, we define the INV proteome and find that it is molecularly heterogeneous and enriched for transmembrane cargo molecules, including integrins, transporters and ATG9A, a lipid scramblase associated with autophagy. ATG9A is known to reside in 'ATG9A vesicles' - small vesicles that contribute to autophagosome formation. Here, using in-cell vesicle capture assays, we found that ATG9A, as well as other ATG9A vesicle cargoes, are in INVs. Quantitative analysis showed that virtually all ATG9A vesicles are INVs, but that only ∼20% of INVs are ATG9A vesicles, suggesting that ATG9A vesicles are in fact a subtype of INV, which we term ATG9A-flavor INVs. Finally, we show that perturbing ATG9A-flavor INVs impairs the autophagy response induced by starvation.