ZFAND5 enhanced basal ATPase activity and degradation of Ub conjugates by pure 26S proteasomes. ZFAND5 enhanced basal ATPase activity and degradation of Ub conjugates by pure 26S proteasomes. (A) ZFAND5 increased ATP hydrolysis by 26S proteasomes, which (10 nM) were incubated with or without ZFAND5 (2.5 μM) at 37 °C. At each time point, production of inorganic phosphate (Pi) was measured using the malachite green assay. All values are the means of at least three independent experiments ± SD. **P < 0.01. (B) Point mutations in either or both zinc fingers of ZFAND5 activated ATP hydrolysis much less than WT ZFAND5. The WT or ZFAND5 mutants were incubated with proteasomes, and the production of Pi was measured. **P < 0.01, WT ZFAND5 vs. control. *P < 0.05, ZFAND5 mutants vs. control. (C) ZFAND5 accelerated the breakdown of ubiquitinated DHFR, but only if the A20 domain was intact. Degradation of 32P-labeled ubiquitinated DHFR (Ub5-DHFR) by 26S proteasomes (1 nM) was measured with or without the WT or the ZFAND5 mutants (0.25 μM) at 37 °C. The reactions were stopped at the indicated time points by addition of TCA, and the TCA-soluble radioactivity was measured (Left). The mean values of Ub5-DHFR degradation at 20 min were shown for comparison (Right). All values are the means of at least three independent experiments ± SD. Donghoon Lee et al. PNAS 2018;115:41:E9550-E9559 ©2018 by National Academy of Sciences