ATG9 resides on a unique population of small vesicles in presynaptic nerve terminals

ABSTRACT

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
• autophagy
• RAB26
• synapse
• synaptic vesicles
• vesicle proteome

Acknowledgements

We thank Dr. Cornelius Schneider for the useful suggestions and constructive discussion. We thank Dr. Antonio Politi and Dr. Jan Keller-Findeison (MPI) for writing the initial code for the Minflux data analysis. We also thank Isabelle Jansen (Abberior Instruments) for the assistance in Minflux data acquisition. We thank Sabine König, Monika Raabe, Ralf Pflanz and Uwe Plessmann for help in mass spectrometry. We thank Brigitte Barg-Kues for the technical assistance and Sigrid Schmidt for the preparation of neuronal cultures.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The python code used in this study for Minflux imaging data analysis is available in the GitHub link https://github.com/ssamban1/Minflux. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [98] partner repository with the dataset identifier PXD041702

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/15548627.2023.2274204

Additional information

Funding

The work was supported by the Deutsche Forschungsgemeinschaft [SFB1286].