Abstract
Microtubule-associated protein 1 light chain 3 α (LC3)/GABA type A receptor-associated protein (GABARAP) comprises a family of ubiquitin-like proteins involved in (macro)autophagy, an important intracellular degradation pathway that delivers cytoplasmic material to lysosomes via double-membrane vesicles called autophagosomes. The only currently known cellular molecules covalently modified by LC3/GABARAP are membrane phospholipids such as phosphatidylethanolamine in the autophagosome membrane. Autophagy-related 4 cysteine peptidase (ATG4) proteases process inactive pro-LC3/GABARAP before lipidation, and the same proteases can also deconjugate LC3/GABARAP from lipids. To determine whether LC3/GABARAP has other molecular targets, here we generated a pre-processed LC3B mutant (Q116P) that is resistant to ATG4-mediated deconjugation. Upon expression in human cells and when assessed by immunoblotting under reducing and denaturing conditions, deconjugation-resistant LC3B accumulated in multiple forms and at much higher molecular weights than free LC3B. We observed a similar accumulation when pre-processed versions of all mammalian LC3/GABARAP isoforms were expressed in ATG4-deficient cell lines, suggesting that LC3/GABARAP can attach also to other larger molecules. We identified ATG3, the E2-like enzyme involved in LC3/GABARAP lipidation, as one target of conjugation with multiple copies of LC3/GABARAP. We show that LC3B–ATG3 conjugates are distinct from the LC3B–ATG3 thioester intermediate formed before lipidation, and we biochemically demonstrate that ATG4B can cleave LC3B–ATG3 conjugates. Finally, we determined ATG3 residue Lys-243 as an LC3B modification site. Overall, we provide the first cellular evidence that mammalian LC3/GABARAP post-translationally modifies proteins akin to ubiquitination (“LC3ylation”), with ATG4 proteases acting like deubiquitinating enzymes to counteract this modification (“deLC3ylation”).
Highlights
Microtubule-associated protein 1 light chain 3 ␣ (LC3)/ GABA type A receptor-associated protein (GABARAP) comprises a family of ubiquitin-like proteins involved inautophagy, an important intracellular degradation pathway that delivers cytoplasmic material to lysosomes via doublemembrane vesicles called autophagosomes
We show that LC3B–ATG4 removes LC3 from proteins enzyme (ATG3) conjugates are distinct from the LC3B–ATG3 thioester intermediate formed before lipidation, and we biochemically demonstrate that ATG4B can cleave LC3B–ATG3 conjugates
We found that the expression of LC3B G120 in ATG4B KO cells resulted in the accumulation of conjugates with a strikingly similar pattern to the Q116P G120 mutant, suggesting that LC3B conjugates accumulate as a result of impaired ATG4B-mediated deconjugation
Summary
Deconjugation-resistant LC3B reveals presence of high molecular weight LC3B conjugates in cells. We treated lysates from ATG4B KO cells expressing pre-primed LC3B G120 and deconjugation-resistant LC3B Q116P G120 with purified recombinant GST-tagged ATG4B or catalytic inactive mutant C74S as a negative control (Fig. 2B) This revealed that 3xFLAG–LC3B G120 conjugates were biochemical substrates of ATG4B, because their detection was lost upon treatment with active ATG4B WT but not inactive ATG4B C74S. We generated HeLa ATG3 KO cells using CRISPR-Cas (validated by Western blotting in Fig. S3B) to test in parallel the function of putative 3xFLAG–ATG3 modification site mutants in restoring endogenous LC3B lipidation This could help to rule out inactivity being the reason for loss of LC3B conjugation. Our study demonstrates that human ATG4 proteases function as deLC3ylation enzymes by counteracting the covalent attachment of LC3/GABARAP to other proteins in the cell such as residue Lys-243 of ATG3 (Fig. 3E)
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