Fig. 6

pH-dependent assembly and disassembly of p62. a Monitoring of high-order oligomerization of p62 by particle turbidity with decreasing pH. Depending on the concentration of MBP-PB1-ZZ, the pH values showing maximal particle size differ slightly. At least three experiments were performed using various protein and HCl concentrations. b Representative negative-stain TEM images of MBP-PB1-ZZ at various pH conditions (8.0, 7.5, 7.0, 6.5, 6.0, 5.5, 5.0, 4.5, and 4.0). Filamentous p62 proteins of various lengths are formed at pH 6.5, 6.0 and 5.5. Relatively globular small oligomers are observed at pH 8.0, 7.5 and 7.0. Particle sizes are too small to observe at pH values below 5.0. The indicated scale bar represents 100 nm. c Monitoring of aggregation of R-BiP in absence/presence of p62 by particle turbidity with decreasing pH. Although the R-BiP protein is ordinarily denatured at acidic pH, denaturation is limited via protection by the p62 protein. At least three experiments were performed using various protein and HCl concentrations. The error bars represent standard error of the mean (S.E.M.). d Binding affinity measurements using FITC-labeled R-BiP peptide against increasing concentrations of p62 at various pH ranging from 4.5 to 9.0. Strong nano-molar scale binding was observed at pH 5.5 and 6.0, while no binding was observed under extremely acidic or basic conditions. The error bars represent standard error of the mean (S.E.M.) of more than three independent experiments