ATG9 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 comprises of 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 ATG9 vesicles: small vesicles that contribute to autophagosome formation. Using in-cell vesicle capture assays we found that ATG9A, as well as other ATG9 vesicle cargos, were in INVs. Quantitative analysis showed that virtually all ATG9 vesicles are INVs, but that only [~]20% of INVs are ATG9 vesicles, suggesting that ATG9 vesicles are in fact a subtype of INV, which we term ATG9A-flavor INVs. Finally, we show that perturbing ATG9A-flavor INVs impaired the autophagy response induced by starvation.