FIGURE 4.

Ubiquitin is conjugated preferentially to the ubiquitin domain of the RFP-Ub. A, illustration of the three possible scenarios for RFP-Ub ubiquitination. In the left panel, ubiquitin conjugates only to a lysine residue on the ubiquitin portion of the chimera. In the middle panel, ubiquitin is linked to both the RFP and the ubiquitin domains of the RFP-Ub fusion. The right panel represents a mode of action in which ubiquitin is linked to the RFP and the ubiquitin domains of the RFP-Ub. B, polyubiquitination reactions of RFP-Ub beads were conducted as described in the legend to Fig. 3. Each reaction was divided, and one-half was subjected to TEV protease digestion. The TEV-treated and -untreated portions were separated side by side on SDS-PAGE and analyzed by Western blot analysis with anti-RFP antibody. Left, representative reactions of E2s that did not directly ubiquitinate the RFP portion (E2A and E2E2); right, E2s that directly add ubiquitin to the RFP portion (E2C and Ubc5C). Similar analysis of all other E2s is depicted in supplemental Fig. S9. IB, immunoblot.