FACS-mediated isolation of native autophagic vesicles

Autophagy. 2023 Jul;19(7):2146-2147. doi: 10.1080/15548627.2022.2151188. Epub 2022 Nov 29.


Autophagosome isolation enables the thorough investigation of structural components and engulfed materials. Recently, we introduced a novel antibody-based FACS-mediated method for isolation of native macroautophagic/autophagic vesicles and confirmed the quality of the preparations. We performed phospholipidomic and proteomic analyses to characterize autophagic vesicle-associated phospholipids and protein cargoes under different autophagy conditions. Lipidomic analyses identified phosphoglycerides and sphingomyelins within autophagic vesicles and revealed that the lipid composition was unaffected by different rates of autophagosome formation. Proteomic analyses identified more than 4500 potential autophagy substrates and showed that in comparison to autophagic vesicles isolated under basal autophagy conditions, starvation only marginally affected the cargo profile. Proteasome inhibition, however, resulted in the enhanced degradation of ubiquitin-proteasome system components. Taken together, the novel isolation method enriched large quantities of autophagic vesicles and enabled detailed analyses of their lipid and cargo composition.

Keywords: Autophagic vesicles; autophagy; cargo profiling; lipid profiling; vesicle isolation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Autophagosomes / metabolism
  • Autophagy* / physiology
  • Lipids
  • Proteasome Endopeptidase Complex* / metabolism
  • Proteomics


  • Proteasome Endopeptidase Complex
  • Lipids

Grants and funding

This work was funded by the Deutsche Forschungsgemeinschaft (German Research Foundation) – Project-ID 259130777-SFB1177.