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bioRxiv · 10.1101/2024.07.03.601942

ATG5 is dispensable for ATG8ylation of cellular proteins

Ketteler, R.·Kiso, K.·von Chamier, L.·Agrotis, A.
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Abstract

Protein ATG8ylation refers to a post-translational modification involving covalent attachment of ubiquitin-like autophagy-related protein ATG8 (LC3/GABARAP) to other cellular proteins, with reversal mediated by ATG4 proteases. While lipid ATG8ylation is important for autophagosome formation and mechanistically well-characterized, little is known about the mechanism of protein ATG8ylation. Here, we investigated the conjugation machinery of protein ATG8ylation in CRISPR/Cas9-engineered knockout human cell lines, utilizing a deconjugation-resistant (Q116P G120) form of MAP1LC3B. We report that protein ATG8ylation requires the E1-like activating enzyme ATG7 and E2-like conjugating enzyme ATG3, in common with ATG8 lipidation. However, in contrast, the E3-like ATG12-ATG5-ATG16L1 complex involved in lipidation is dispensable for protein ATG8ylation, since ATG5 knockout cells can form ATG8ylated protein conjugates. Further, we uncover that ATG7 itself is a target of ATG8ylation. Overall, our work provides crucial insight into the mechanism of protein ATG8ylation, distinguishing it from ATG8 lipidation, which will aid investigating its functional role.

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Ketteler, R., Kiso, K., von Chamier, L., Agrotis, A.. 2024-07-05. ATG5 is dispensable for ATG8ylation of cellular proteins. https://doi.org/10.1101/2024.07.03.601942

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