Fig. 2
From: Insights into degradation mechanism of N-end rule substrates by p62/SQSTM1 autophagy adapter
Oligomerization of p62 affects the binding affinity and degradation of R-BiP. a Binding affinity measurements using FITC-labeled R-BiP peptide against increasing concentrations of the ZZ-domain at pH 8.0. The ZZ-domain fused with dimeric GST (red line) showed higher affinity than that with the flag-tag (blue line), which has extremely weak binding affinity as shown in the inset. The error bars represent standard error of the mean (S.E.M.) of more than three independent experiments. b The SEC-MALS results with MBP-PB1-ZZ WT (red line) and mutants K7A (green line) and D69A (sky blue line) at pH 8.0. The horizontal line represents the measured molar mass. Each species is indicated by an arrow with experimental (SEC-MALS) molar mass. WT showed a higher oligomeric state whereas the K7A and D69A mutants mainly adopted a monomeric state with minor dimeric species. c The SDS-PAGE results with MBP-PB1-ZZ WT and D69A mutant. The left blue gel is stained with Coomassie Brilliant Blue and the right shows the results of the Western blot. The D69A mutant adopted exclusively a monomeric state whereas WT showed oligomeric forms even under denaturing conditions. d Binding affinity measurements using FITC-labeled R-BiP peptide against increasing concentrations of MBP-PB1-ZZ WT (blue line) and mutants K7A (green line) and D69A (red line) at pH 8.0. The error bars represent standard error of the mean (S.E.M.) of more than three independent experiments. e Degradation assay of R-BiP generated from Ub–R-BiP using oligomerization defect mutants (K7A and D69A) in HeLa cells in the absence of MG132. Cells were treated with 50 μg/ml cycloheximide, and then subjected to immunoblotting of R-BiP. Oligomerization defect mutants are unable to degrade R-BiP protein in the cell (see also Supplementary Fig. 7 for p62 degradation). Uncropped images of Western blots are shown in Supplementary Figure 11
