Figure 6.

SHW1 enhances the ability of COP1 to degrade HY5. Recombinant COP1-His, GST-SHW1, and GST-HY5 fusion proteins were purified from Escherichia coli (BL21DE3), and in vitro ubiquitination assays were performed. A, Ubiquitination assays were performed using UBE1 (E1), UbcH5b (E2), and His-tagged ubiquitin (His-6-Ub). Recombinant COP1-His was preincubated with 20 mm zinc chloride. Ubiquitinated HY5 was detected using antiubiquitin antibody. *, Nonspecific band. B, Immunoblot against HY5 antibody shows that increasing concentration of SHW1 results in more ubiquitinated HY5 in vitro. GST was used as a negative control. C, Quantification of the ubiquitinated HY5 level in the presence of COP1 with increasing concentrations SHW1. For experimental detail, see Figure 5B. D, A working model illustrates the regulatory role of SHW1 on HY5 stability. SHW1 interacts with COP1 and promotes COP1-mediated degradation of HY5.