Fig. 1: Post-translational modifications of Arabidopsis ATG8 during autophagy.

A During autophagosome formation, ATG8 undergoes reversible conjugation with lipids present on the autophagosomal membranes. Lipidation of ATG8 onto phagophore membranes is essential for phagophore elongation. This process begins with the truncation of ATG8 through proteolytic processing by the ATG4 protease followed by lipidation aided by other core ATG proteins. Later, lipidated ATG8 is removed from the outer autophagosomal membrane during the maturation of the vesicle. B ATG8 is constitutively processed by ATG4 to remove the C-terminal peptide. The obtained truncated ATG8 (ATG8ΔC) has the C-terminal Gly residue exposed for the lipidation reaction. Lipidation of ATG8, i.e., its covalent conjugation to the phosphatidylethanolamine (PE) lipid present in the phagophore membranes (formation of ATG8–PE), is executed by ATG8-lipidation complex, best known as two ubiquitin-like conjugation systems, comprising several autophagy-related proteins, including ATG5 and ATG7. Delipidation of ATG8–PE on the outer membrane of autophagosome is performed by the ATG4 protease, which cleaves the amide bond between PE and ATG8, releasing the ATG8ΔC back into the pool of cytoplasmic ATG8 available for autophagosome biogenesis. C Diagram showing C-terminal sequences of the three artificially created variants of the Arabidopsis isoform ATG8E: ATG8E, full-length protein sequence including the C-terminal peptide masking the critical Gly residue; ATG8E G/A, full-length protein in which the Gly residue was replaced with Ala; ATG8EΔC, artificially truncated ATG8 protein lacking the C-terminal peptide that was masking the Gly residue. D Detection of three fluorescently labeled ATG8E variants (shown in C) in Arabidopsis thaliana epidermal root cells of 7-day-old seedlings treated with AZD8055 and concanamycin A. WT wild-type, atg4a/b atg4a-2/b-2 double knockout of ATG4A and ATG4B genes. Arrowheads indicate autophagic bodies inside the vacuoles. The experiment was repeated twice. Scale bars, 20 μm.