FIGURE 13.

The Cdc34 acidic tail forms cross-links to a residue located in the Cul2 basic canyon. A, time course for a diubiquitin synthesis assay for WT Cdc34 alone or in the presence of either WT Cul1-Rbx1, WT Cul2-Rbx1, or R648C Cul2-Rbx1. Notice that both WT Cul1-Rbx1 and WT Cul2-Rbx1 stimulate the Cdc34-dependent formation of diubiquitin by ∼20-fold, and the reduction in the activity of R648C Cul2-Rbx1 as compared with WT Cul2-Rbx1 is only ∼3-fold. B, Cul2 Western blot of reactions between either Cdc34 1C or Cdc34 2C proteins that had been activated with BMOE and WT Cul2-Rbx1. C, graph showing the fraction of Cul2 protein that cross-linked to either Cdc34 1C or the Cdc34 2C proteins. Error bars, S.E. of measurement from duplicate data points. D and E, same as in B and C, except cross-linking reactions contained R648C Cul2-Rbx1. Quantitation of the levels for the fastest migrating Cdc34-Cul2 product species is shown as gray bars; product levels for the next fastest species are shown as black bars.