Autophagy. 2022 Jan 24. 1-16
Macroautophagy/autophagy is an evolutionarily conserved intracellular degradation pathway that maintains cellular homeostasis. Over the past two decades, a series of scientific breakthroughs have helped explain autophagy-related molecular mechanisms and physiological functions. This tremendous progress continues to depend largely on powerful research methods, specifically, various autophagy marker Atg8-PE protein-based methods for studying membrane dynamics and monitoring autophagic activity. Recently, several biochemical approaches have been successfully developed to produce the lipidated protein Atg8-PE or its mimics in vitro, including enzyme-mediated reconstitution systems, chemically defined reconstitution systems, cell-free lipidation systems and protein chemical synthesis. These approaches have contributed important insights into the mechanisms underlying Atg8-mediated membrane dynamics and protein-protein interactions, creating a new perspective in autophagy studies. In this review, we comprehensively summarize Atg8-PE protein-based in vitro biochemical approaches and recent advances to facilitate a better understanding of autophagy mechanisms. In addition, we highlight the advantages and disadvantages of various Atg8-PE protein-based approaches to provide general guidance for their use in studying autophagy.
Keywords: Atg8–PE; LC3–PE; autophagy; biochemical approaches; cell-free lipidation system; chemically defined reconstitution systems; enzyme-mediated reconstitution system; protein chemical synthesis