Figure 4

Mutagenesis of CHIP:E2 interaction surfaces. A. In vitro ubiquitination measurements of CHIP with UbcH5 or Ubc13-Uev1a carried out using an ELISA-based assay. Wild-type or mutant His-CHIP bound to Nickel-NTA coated microplates was autoubiquitinated with FLAG-Ubiquitin in the presence of UbcH5b (dark grey bars). Mixed His- and Flag-tagged polyubiquitin chains were formed in Nickel-NTA coated microplates in the presence of untagged CHIP and Ubc13-Uev1a (light grey bars). The white bar ("*Ubc13-Uev1a") represents (minimal) autoubiquitination of His-CHIP by FLAG-Ubiquitin in the presence of Ubc13-Uev1a. "-CHIP" designates negative controls carried out with no CHIP in the reactions. Data are in arbitrary units and were normalized by the values measured for the respective E2 enzymes with wild-type CHIP. B. Autoubiquitination of CHIP in the presence of UbcH5 but not Ubc13-Uev1a, probed by western blotting. Anti-ubiquitin staining confirms the formation of polyubiquitin chains by Ubc13-Uev1a in a CHIP-dependent manner. Anti-CHIP staining shows that a fraction of CHIP is ubiquitinated (higher-molecular weight bands) in the presence of UbcH5 but not Ubc13-Uev1a. C. Ubiquitination measurements of wild-type CHIP with wild-type and mutant UbcH5 or Ubc13-Uev1a, as described for panel A.