Cargo and lipid profiling of isolated autophagosomes upon basal autophagy, starvation, and proteasome inhibition

Version 1

Autophagy is responsible for degradation of an extensive portfolio of cytosolic cargoes that are engulfed in autophagosomes to facilitate their transport to lysosomes. Besides basal autophagy, which constantly degrades cellular material, the pathway is dynamically altered by different conditions, resulting in enhanced autophagosome formation and cargo turnover. The extensive profile of autophagosome content as well as the phospholipid composition of human autophagosome membranes remains elusive. Here, we introduce a novel FACS-based approach for isolation of native autophagosomes and confirm the quality and specificity of the preparations. Employing quantitative proteomics and lipidomics, we obtained a profound cargo and lipid profile of autophagosomes isolated upon basal autophagy conditions, nutrient deprivation, and proteasome inhibition. This resource will allow to identify the impact of different autophagy conditions on the degradation of distinct proteins and to dissect the phospholipid composition of the human autophagosome membrane in detail.