Fig. 3
From: Insights into degradation mechanism of N-end rule substrates by p62/SQSTM1 autophagy adapter
Mutational effects of key determinants on the recognition of N-degrons. a Binding affinity measurements using FITC-labeled R-BiP peptide against increasing concentrations of p62 mutants (MBP-PB1-ZZ WT—blue line, D129N—red line, N132L—green line, R139D—violet line, D147R—orange line, and D149R—black line) at pH 8.0. The error bars represent standard error of the mean (S.E.M.) of more than three independent experiments. b Degradation assay of R-BiP generated from Ub–R-BiP using key determinant mutants (D129N, N132L, R139D, D147R, and D149R) in HeLa cells in the absence of MG132. Cells were treated with 50 μg/ml cycloheximide, and then subjected to immunoblotting of R-BiP. Recognition defect mutants are unable to degrade R-BiP protein in the cell. Uncropped images of Western blots are shown in Supplementary Figure 11. c Superposition of structures of R-BiP-bound ZZ-domain (green ribbon) and Scc1-bound UBR box (beige ribbon). Key residues in the ZZ-domain are marked with black dotted circles (center) with a close-up view of each region for details. The labeled residues for the ZZ-domain and UBR box are colored black and dark green (underlined), respectively, for clarity
