Figure 5.

The formation of multiubiquitin chains depends on the integrity of the APC11 RING-H2 finger. (A) Schematic representation of APC11. Cysteines (C) and two histidine residues (H) are indicated. Boxed residues represent the predicted Zn coordinating amino acids. The cysteine residues that were changed to alanine (A) in this study are indicated by arrows. (B) Bacterial lysates containing GST-APC11 wild type and the indicated GST-APC11 mutants were immobilized on glutathione-beads, were washed, and were eluted with SDS/sample buffer. Shown is a 12% polyacrylamide gel stained with Coomassie blue. (C) Identical amounts of bacterial lysates as in B were bound to glutathione beads, were washed, and were incubated with a reaction mixture as in Fig. 2B for 30 min at 37°C. Proteins were visualized by immunodecoration with anti-GST antibodies followed by enhanced chemiluminescence.