Isolation and mass spectrometry analysis of ATG9A vesicles from HAP1 cells.

ABSTRACT: Autophagy is a conserved cellular degradation pathway involving the formation of double-membrane autophagosomes that engulf cytoplasmic material for delivery to the lysosome. Autophagy-related (ATG) proteins orchestrate the steps of autophagosome formation and maturation, among which ATG9A is unique as the only integral membrane protein essential for autophagy progression. ATG9A localizes to Golgi-derived vesicles/compartments that constitute a seed for membrane nucleation. In this dataset we show proteomics analysis of mammalian ATG9A vesicles. To isolate and characterize native mammalian ATG9A-containing compartments, we generated a CRISPR/Cas9-engineered HAP1 knock-in cell line expressing ATG9A endogenously tagged ATG9A with a C-terminal GFP and FLAG tag. The FLAG-tag enabled the purification of native ATG9A vesicles via FLAG-based affinity isolation, while GFP-tag was used to submit the vesicles for mass spectrometry analysis. Both WT and ATG9A-GFP-FLAG expressing HAP1 cell lines were cultured under nutrient-rich conditions to prevent the induction of starvation-mediated autophagy.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Markus Hartl  

LAB HEAD: Sascha Martens

PROVIDER: PXD066668 | Pride | 2026-08-04

REPOSITORIES: Pride