The insufficiency of ATG4A in macroautophagy

J Biol Chem. 2020 Sep 25;295(39):13584-13600. doi: 10.1074/jbc.RA120.013897. Epub 2020 Jul 30.

Abstract

During autophagy, LC3 and GABARAP proteins become covalently attached to phosphatidylethanolamine on the growing autophagosome. This attachment is also reversible. Deconjugation (or delipidation) involves the proteolytic cleavage of an isopeptide bond between LC3 or GABARAP and the phosphatidylethanolamine headgroup. This cleavage is carried about by the ATG4 family of proteases (ATG4A, B, C, and D). Many studies have established that ATG4B is the most active of these proteases and is sufficient for autophagy progression in simple cells. Here we examined the second most active protease, ATG4A, to map out key regulatory motifs on the protein and to establish its activity in cells. We utilized fully in vitro reconstitution systems in which we controlled the attachment of LC3/GABARAP members and discovered a role for a C-terminal LC3-interacting region on ATG4A in regulating its access to LC3/GABARAP. We then used a gene-edited cell line in which all four ATG4 proteases have been knocked out to establish that ATG4A is insufficient to support autophagy and is unable to support GABARAP proteins removal from the membrane. As a result, GABARAP proteins accumulate on membranes other than mature autophagosomes. These results suggest that to support efficient production and consumption of autophagosomes, additional factors are essential including possibly ATG4B itself or one of its proteolytic products in the LC3 family.

Keywords: GABARAP like-1; LC3-interacting region (LIR); MST4; autophagy; autophagy-related protein 4A (ATG4); cell biology; delipidation; liposome; membrane reconstitution; phosphorylation.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Apoptosis Regulatory Proteins / metabolism
  • Autophagy-Related Proteins / genetics
  • Autophagy-Related Proteins / metabolism*
  • Cysteine Endopeptidases / genetics
  • Cysteine Endopeptidases / metabolism*
  • HEK293 Cells
  • Humans
  • Macroautophagy*
  • Microtubule-Associated Proteins / metabolism

Substances

  • Apoptosis Regulatory Proteins
  • Autophagy-Related Proteins
  • GABARAP protein, human
  • MAP1LC3A protein, human
  • Microtubule-Associated Proteins
  • ATG4A protein, human
  • Cysteine Endopeptidases