Figure 2. Structure-function relationship of LC3-deconjugation by RavZ.
(A) Schematic diagram of proteo-membrane containing LC3-PE. The fatty acid chains of PE are buried in the lipid bilayer, serving as the membrane anchor. (B) LC3 proteins containing different fragments derived from the PE. Ethanolamine, EA; phosphoethanolamine, pEA; glycerophosphoethanolamine, GpEA and diacetyl glycerophosphoenthanolamine, DAGpEA. (C) In vitro cleavage of semisynthetic LC3 proteins by Atg4B and RavZ.
Figure 2—figure supplement 1. In vitro cleavage of LC3 proteins with various C-terminal modifications.
(A) In vitro LC3 cleavage assay by SDS-PAGE. RavZ only cleaves LC3-PE but not pro-LC3, while Atg4B processes both of them. (B) RavZ and Atg4 deconjugate LC3-PE at different sites. ESI-MS spectra of MBP-LC3-PE before and after Atg4B and RavZ treatments were shown. MBP-LC3-PE, Mw calculated 60078, found 60075; MBP-LC31–120, Mw calculated 59391, found 59402; MBP-LC31–119, Mw calculated 59344, found 59344. (C) The C-terminal lipidated peptide of LC3-PE is insufficient for RavZ proteolysis. The C-terminal lipidated peptide of LC3-PE and chimeric PE-modified Rab7 were subjected to RavZ treatment. The lipidated peptides were solved in the HEPES buffer containing detergent (30 mM HEPES 7.4, 50 mM NaCl, 2 mM DTT, 0.1% Triton X-100). RavZ (2 µM) was added to the peptide solution (2 µM) and chimeric PE-modified Rab7 (7 µM). After overnight incubation at 37°C, the reactions were subjected to LC-MS. No cleavage products were observed in these conditions. Right panel shows ESI-MS spectra of EGFP-Rab7 thioester, EGFP-Rab7-PE chimeric protein and RavZ-treated EGFP-Rab7-PE. Chimeric EGFP-Rab7-PE protein, Mw calculated 50500, found 50485.
Figure 2—figure supplement 2. Structure-function relationship study of LC3-deconjugation by RavZ.
(A) ESI-MS spectra of modified LC3 (S1–S4) before (black line) and after Atg4B (red line) or RavZ treatment (blue line). 0.7 µM LC3 proteins were incubated with 0.7 µM Atg4B or RavZ for 8 hr at 37°C. (B) Kinetics of RavZ-medicated cleavage reactions with LC3-PE (16:0) and LC3-PE (6:0) (S5). 7 µM LC3-PE (16:0) or LC3-PE (6:0) was incubated with RavZ (0.7 µM) at 37°C. The reaction was quenched at different time points and subjected to SDS-PAGE. The graphs in the lower panel show reaction progress. The red solid line shows the fitting to a single-exponential equation to obtain t1/2. (C) Enzyme kinetics of RavZ reaction. Varying concentrations of LC3-PE (16:0) or LC3-PE (6:0) were incubated with RavZ (0.7 µM) for 8 min or for 140 min at 37°C, respectively. The reaction was quenched and subjected to SDS-PAGE. In the lower panel, the reaction rates were plotted against substrate concentrations. The red solid line shows the fitting to Michaelis-Menten equation. (D) Summary of Michaelis-Menten kinetic parameters. n = 3 independent experiments. Mean and SD are presented. (E) In vitro cleavage assay of LC3-C16 (S6). LC3-C16 was treated with Atg4B or RavZ (0.7 µM) at 37°C for 2 hr and subjected to SDS-PAGE. (F) Kinetics of RavZ20-331-medicated cleavage reaction with LC3-PE (16:0). The same conditions were used as in (B). (G) The exponential decay curve showing the half-time (t1/2) of LC3-PE cleavage activity of RavZ20-331. n = 3 independent experiments. Mean and SD are presented.