Real-time measurement of the ATG8 lipidation reaction by fluorescence spectroscopy

Autophagy is a highly conserved intracellular degradation pathway, in which damaged organelles and/or dysfunctional cytosolic components are enveloped via double-membraned autophagosomes and subsequently delivered to lysosomes for degradation. The ubiquitin-like ATG8 family proteins (LC3s and GABARAPs) are covalently conjugated to phosphatidylethanolamine (PE) on autophagic membranes via ubiquitin-like conjugation systems, a process known as ATG8 lipidation. Lipidated ATG8 is the most widely used membrane marker for autophagosomes, and its flux is commonly used as a readout for autophagy activity. In vitro reconstitution of the ATG8 lipidation reaction is well-established, and the end-point reaction is typically resolved by SDS-PAGE. This endpoint readout is not suitable to monitor the kinetics of this reaction, and tools to study this process have been lacking. Here, we describe a real-time assay to measure the ATG8 lipidation reaction. This approach not only reveals the subsequential formation of covalently bound intermediates of ATG8 with the E1 (ATG7) and E2 (ATG3) enzymes, as well as ATG8-PE itself, but also provides insights into the interaction interface of ATG8 with proteins and membranes during the conjugation reaction.