ABSTRACT

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.