Human ATG4 autophagy proteases counteract attachment of ubiquitin-like LC3/GABARAP proteins to other cellular proteins

Jul 19, 2019The Journal of biological chemistry

Human ATG4 enzymes prevent attachment of LC3/GABARAP proteins to other cell proteins

AI simplified

Longevity & Aging on OpenScience ↗PubMed ↗DOI ↗

Abstract

A pre-processed LC3B mutant (Q116P) accumulated in multiple forms and at much higher molecular weights than free LC3B.

LC3/GABARAP proteins, previously known for lipid modification, may also modify larger cellular molecules.
The accumulation of deconjugation-resistant LC3B was observed in ATG4-deficient cell lines.
ATG3 was identified as a target for conjugation with multiple copies of LC3/GABARAP.
LC3B-ATG3 conjugates differ from the thioester intermediate formed during lipidation.
ATG4B can cleave LC3B-ATG3 conjugates, indicating a role in reversing this modification.
Lys-243 on ATG3 has been determined as a site for LC3B modification.

AI simplified

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").

Full Text

We can’t show the full text here under this license. Use the link below to read it at the source.

View full text (XML) ↗View full text ↗

Human ATG4 autophagy proteases counteract attachment of ubiquitin-like LC3/GABARAP proteins to other cellular proteins

Abstract

Full Text

You found one interesting study. We’ll send the next 7.

what lands in your inbox each week:

Recent issues from the longevity & aging brief

Abstract

Full Text

Related papers

You found one interesting study. We’ll send the next 7.

what lands in your inbox each week:

Recent issues from the longevity & aging brief