Assembly of the 26 S complex that degrades proteins ligated to ubiquitin is accompanied by the formation of ATPase activity.

In the ubiquitin pathway for intracellular protein breakdown, proteins ligated to ubiquitin are degraded by a large (26 S) ATP-dependent protease complex. It was found previously that the 26 S complex is assembled from three different enzyme components by a process that requires MgATP. In addition, MgATP is also required for the continued action of the 26 S complex in the breakdown of ubiquitin-protein conjugates. In the present study we have tried to gain some insight into the mode of action of ATP by following ATP hydrolysis by the 26 S complex and its three components. It was found that none of the three unassembled components had significant ATPase activity, but such activity appeared following their entry into the 26 S complex. The presence of all three components and of MgATP was required for the formation of complex-associated ATPase activity. GTP and UTP cannot replace ATP for complex assembly, but these nucleotides can substitute for ATP in the stimulation of the conjugate-degrading activity of the 26 S complex. Unlabeled GTP and UTP inhibit the hydrolysis of [gamma-32P] ATP by complex-associated ATPase, indicating that this activity is related to the latter site of ATP action in this system.