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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. Abbreviations:  ATG: autophagy related; ATP: adenosine triphosphate; COPII: coat protein complex II; DGS-NTA: 1,2-dioleoyl- sn -glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt); DPPE: 1,2-dipalmitoyl- sn -glycero-3-phosphoethanolamine; DSPE: 1,2-distearoyl- sn -glycero-3-phosphoethanolamine;  E. coli: Escherichia coli ; EPL: expressed protein ligation; ERGIC: ER-Golgi intermediate compartment; GABARAP: GABA type A receptor-associated protein; GABARAPL1: GABA type A receptor associated protein like 1; GABARAPL2: GABA type A receptor associated protein like 2; GFP: green fluorescent protein; GUVs: giant unilamellar vesicles; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MBP: maltose binding protein; MEFs: mouse embryonic fibroblasts; MESNa: 2-mercaptoethanesulfonic acid sodium salt; NCL: native chemical ligation; NTA: nitrilotriacetic acid; PE: phosphatidylethanolamine; PS: phosphatidylserine; PtdIns3K: class III phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol-3-phosphate; SPPS: solid-phase peptide synthesis; TEV: tobacco etch virus; WT: wild-type.

Atg8–PE protein-based in vitro biochemical approaches to autophagy studies