Activation of the 26S ATPases, unlike peptidases, absolutely requires Ubl domains. (A) Full-length Ddi2 (WT), as well as Ddi2 lacking its Ubl domain (∆Ubl), stimulated peptidase activity, as performed in Fig. 3A. 6Ub denotes linear hexa-ubiquitin chains. Error bars are SEM (n = 3). (B) Full-length Rad23A (WT), as well as Rad23A lacking its Ubl domain (∆Ubl), stimulated peptidase activity as in A. Error bars represent the range from two independent experiments. (C) Rates of ATP hydrolysis as the percent of the activity of 26S proteasomes with casein present. Rad23A (black bar) but not Rad23A ∆Ubl (checkered bar) stimulated ATP hydrolysis. Casein without Rad23A (gray bar) did not stimulate ATP hydrolysis. (D) Rates of ATP hydrolysis as percent of 26S proteasomes activity alone. Ddi2 with casein (black bar) but not Ddi2 ∆Ubl with casein (checkered bar) stimulated ATP hydrolysis. Casein without Ddi2 (gray bar) did not stimulate ATP hydrolysis. n = 3. *P < 0.05, **P < 0.01.