In neurons, autophagosome biogenesis occurs mainly in distal axons, followed by maturation during retrograde transport. Autophagosomal growth depends on the supply of membrane lipids which requires small vesicles containing ATG9, a lipid scramblase essential for macroautophagy/autophagy. Here, we show that ATG9-containing vesicles are enriched in synapses and resemble synaptic vesicles in size and density. The proteome of ATG9-containing vesicles immuno-isolated from nerve terminals showed conspicuously low levels of trafficking proteins except of the AP2-complex and some enzymes involved in endosomal phosphatidylinositol metabolism. Super resolution microscopy of nerve terminals and isolated vesicles revealed that ATG9-containing vesicles represent a distinct vesicle population with limited overlap not only with synaptic vesicles but also other membranes of the secretory pathway, uncovering a surprising heterogeneity in their membrane composition. Our results are compatible with the view that ATG9-containing vesicles function as lipid shuttles that scavenge membrane lipids from various intracellular membranes to support autophagosome biogenesis.Abbreviations: AP: adaptor related protein complex: ATG2: autophagy related 2; ATG9: autophagy related 9; DNA PAINT: DNA-based point accumulation for imaging in nanoscale topography; DyMIN STED: dynamic minimum stimulated emission depletion; EL: endosome and lysosome; ER: endoplasmic reticulum; GA: Golgi apparatus; iBAQ: intensity based absolute quantification; LAMP: lysosomal-associated membrane protein; M6PR: mannose-6-phosphate receptor, cation dependent; Minflux: minimal photon fluxes; Mito: mitochondria; MS: mass spectrometry; PAS: phagophore assembly site; PM: plasma membrane; Px: peroxisome; RAB26: RAB26, member RAS oncogene family; RAB3A: RAB3A, member RAS oncogene family; RAB5A: RAB5A, member RAS oncogene family; SNARE: soluble N-ethylmaleimide-sensitive-factor attachment receptor; SVs: synaptic vesicles; SYP: synaptophysin; TGN: trans-Golgi network; TRAPP: transport protein particle; VTI1: vesicle transport through interaction with t-SNAREs.
Keywords: ATG9; RAB26; autophagy; synapse; synaptic vesicles; vesicle proteome.