Delipidation of mammalian Atg8-family proteins by each of the four ATG4 proteases

Abstract

During macroautophagy/autophagy, mammalian Atg8-family proteins undergo 2 proteolytic processing events. The first exposes a COOH-terminal glycine used in the conjugation of these proteins to lipids on the phagophore, the precursor to the autophagosome, whereas the second releases the lipid. The ATG4 family of proteases drives both cleavages, but how ATG4 proteins distinguish between soluble and lipid-anchored Atg8 proteins is not well understood. In a fully reconstituted delipidation assay, we establish that the physical anchoring of mammalian Atg8-family proteins in the membrane dramatically shifts the way ATG4 proteases recognize these substrates. Thus, while ATG4B is orders of magnitude faster at processing a soluble unprimed protein, all 4 ATG4 proteases can be activated to similar enzymatic activities on lipid-attached substrates. The recognition of lipidated but not soluble substrates is sensitive to a COOH-terminal LIR motif both in vitro and in cells. We suggest a model whereby ATG4B drives very fast priming of mammalian Atg8 proteins, whereas delipidation is inherently slow and regulated by all ATG4 homologs.

Non-canonical autophagy drives alternative ATG8 conjugation to phosphatidylserine

“…These findings confirm that differential LC3 lipidation influences ATG4B-mediated deconjugation, revealing an important functional outcome for this alternative modification. Interestingly, these findings are consistent with a previous in vitro study of GABARAPL1 conjugated liposomes, delipidated by ATG4A, B or C 36 , indicating that a reduced rate of ATG8-PS cleavage is likely shared among multiple isoforms of both ATG8 and ATG4.…”

“…We note that full length ATG4C and ATG4D are active under these conditions, without caspase cleavage, unlike the bacterially purified proteins 36,37 , and ATG4A was omitted, as it appears GABARAP specific 36,38 ; RavZ, a bacterial effector protein known to cleave both ATG8-PE and PS 39,40 , was included as a control. As expected, all 4 enzymes can delipidate LC3B from PE liposomes, consistent with previous work 36 . Strikingly however, only ATG4D (and the RavZ control) can deconjugate LC3B from PS liposomes.…”

GABARAP Like-1 enrichment on membranes: Direct observation of trans-homo-oligomerization between membranes and curvature-dependent partitioning into membrane tubules

“…Thus, some property of membrane association changes the protein reactivity of these autophagosome markers. It appears that the increased surface density of these proteins may be sufficient to specifically capture cytosolic proteins harboring many low affinity LC3/Atg8 interaction sites 40 , and possibly to alter the activity of membrane targeted enzymes 41 , and thus local concentrations of these proteins are clearly important. Whether actual oligomers are forming however has been uncertain.…”

“…Reactions were run at 37°C for 90 minutes in SNH buffer (20 mM Tris at pH 8, 100 mM NaCl, and 5 mM MgCl2). Floation assay: Nycodenz liposome flotation was performed as described previously 41 . Lipidation samples were mixed with 150 uL 80% Nycodenz (100% (w/v) Nycodenz in SNH buffer).…”

On ATG4B as Drug Target for Treatment of Solid Tumours—The Knowns and the Unknowns

“…In mammals, there are four ATG4 isoforms that contribute to processing of the seven LC3/GABARAP family members. Each family member has a different preference for LC3/GABARAP substrates and a difference in the recognition and cleavage of the pro-peptide substrate as opposed to the lipidated LC3/GABARAP-II version has been proposed [38]. The in vitro activity of ATG4 family members has been established using tagged LC3/GABARAP constructs or cellular overexpression and using semi-quantitative methods such as Western blotting for determining the kinetic parameters [39].…”