Fig. 5. In vitro characterization of the UmuD9-4/D′ heterodimer. (A) Heterodimerization of UmuD9-4 with UmuD′. UmuD and UmuD9-4 were each individually incubated with UmuD′ at 25°C for 30 min and electrophoresed through a 12% native polyacrylamide gel. UmuD, UmuD9-4 and UmuD′ were also electrophoresed to determine mobility of the homodimer species. Visualization of the reactions was accomplished by staining with Coomassie Brilliant Blue. The UmuD/D′ sample was purified from E.coli as a heterodimer complex (Woodgate et al., 1989). Migration of the homodimer species and UmuD/D′ is indicated. Concentrations of the various components are described in Materials and methods. (B) In vitro stability of UmuD9-4/D′ in the presence of ClpXP. Purified UmuD or UmuD9-4 was incubated in the presence of UmuD′ at 25°C for 30 min, followed by incubation with ClpP ± ClpX at 37°C for the designated times. Reactions were stopped with the addition of 4× SDS sample buffer, followed by freezing in dry ice. Samples were electrophoresed, visualized, and quantitated as described in Figure 1. Kinetics of degradation are for UmuD′ in each respective heterodimer. UmuD is unaffected by ClpXP. Open squares, UmuD/D′ + ClpX; filled squares, UmuD/D′ – ClpX; open triangles, UmuD9-4/D′ + ClpX; filled triangles, UmuD9-4/D′ – ClpX.