FIGURE 3.

p97 hexamer disassembly in vitro is not affected by nucleotides or by TUG N-terminal domains and requires stoichiometric amounts of TUG. A, recombinant His₆-p97 was preincubated with ATP, ADP, or ATPγS at final concentrations of 1 mm for 10 min at 4 °C, and then GST or GST-TUG 238–550 was added, and the incubation was continued for an additional 30 min at 4 °C. The molar ratio of TUG to p97 was 3:1. p97 oligomeric status was analyzed by sedimentation on sucrose density gradients, followed by immunoblotting of the fractions as indicated. The positions of molecular weight markers are shown at the top. The experiment was repeated three times with similar results. B, recombinant His₆-p97 was incubated for 30 min at 4 °C with GST alone or with GST TUG 1–164 and/or GST TUG 165–550, as indicated. Molar ratios of 3:1 (TUG/p97) were used. p97 complexes were then separated by sedimentation on sucrose gradients, and the fractions were immunoblotted to detect p97. The experiment was performed twice with similar results. C, GST-TUG 238–550 was incubated with His₆-p97 at the indicated molar ratios of TUG/p97 monomer, and then complexes were sedimented on sucrose gradients. Fractions were immunoblotted to detect p97.