FIGURE 6.

The Cdc34 acidic tail forms cross-links to multiple positions in the Cul1 basic canyon. Cross-linking was performed in the presence of BMOE-activated Cdc34 proteins for 60 s prior to quenching. A, Cul1 Western blot of reactions between either Cdc34 1C or Cdc34 2C proteins and K435C Cul1-Rbx1 in the presence of BMOE. Note that very little product is formed between Cdc34 1C and Cul1, whereas almost all Cul1 is cross-linked to the Cdc34 2C proteins. B, color overlay of reactions between WT (red) or K435C (yellow) Cul1-Rbx1 and Cdc34. Note the difference in electrophoretic mobilities for WT Cul1-Cdc34 and K435C Cul1-Cdc34 products, indicating that these cross-links probably involve different cysteine residues on Cul1. C and D, same as in A and B, except with K472C Cul1-Rbx1. E and F, same as in A and B, except with K515C Cul1-Rbx1. G and H, same as in A and B, except with K645C Cul1-Rbx1. Notice that the electrophoretic mobilities of the Cul1-Cdc34 products are identical in the WT and K645C Cul1 color overlay. However, there is an increase in signal for the reactions containing K645C Cul1-Rbx1 compared with reactions containing WT Cul1-Rbx1 (see Fig. 4D), suggesting that at least some cross-linking is occurring between Cys-645 and the Cdc34 2C proteins. I, heat map for the values of the fraction of Cul1 converted to Cdc34-Cul1 cross-links for WT and all five cysteine mutant Cul1-Rbx1 proteins. In cases where more than one Cdc34-Cul1 product is observed (e.g. K472C Cul1 and K515C Cul1), the values for the fraction converted have been combined. Values represent the average from duplicate measurements.