Differential centrifugation coupled to affinity purification-mass spectrometry of mcherry-ATG8E

Autophagosomes are double-membraned vesicles that traffic harmful or unwanted cellular macromolecules to the vacuole for recycling. Although autophagosome biogenesis has been studied extensively, autophagosome maturation, i.e., delivery and fusion with the vacuole, remains largely unknown in plants. To start filling these gaps, we devised a differential centrifugation set up, where we enriched intact autophagosomes and performed affinity purification-mass spectrometry (AP-MS), using ATG8 as a bait. This method enabled the identification of an autophagy adaptor protein.