Figure 4

Arg68 of LC3B is essential for protein-protein interaction with ATG4B and for C-terminal cleavage efficiency of human Atg8 family proteins. A. Post-translation modification pattern of wild type (WT) and mutants of LC3B. Myc-LC3B-G120A mutant shows the position of pro-form protein. Long exposure (lower panel) shows the PE-conjugated forms. p: pro-form; I: the -I form; II: the -II form. B. The function of R68 of LC3B in post-translational modification is conserved among human Atg8 family members. Compared with wild type protein, R68A mutant of LC3A, R74A mutant of LC3C, R65A mutant of GABARAP or GABARAPL1 shows accumulated pro-form of each protein. Long exposure shows the -II form in GABARAP and GABARAPL1. C. Disruption of the salt bridge between R68 of LC3B and D171 of ATG4B weakens their interaction. GST pull-down assay was performed between GST-ATG4B with LC3B or LC3B -R68A protein in the presence of 1mM PMSF. D. GST pulldown assay was performed between LC3B with GST-ATG4B or GST-ATG4B-D171A mutant proteins in the presence of 1mM PMSF. E. Arg68-Asp171 salt bridge is crucial for the cleavage efficiency of LC3B by ATG4B. Purified GST-ATG4B, GST-ATG4B-D171A, LC3B and LC3B-a mutant proteins were subjected to in vitro cleavage assay in a time dependent manner. The cleavage pattern at indicated time point is shown by SDS-PAGE followed by coomassie bright blue staining. Molecular weights (kDa) were shown. IB: immunoblotting.