Fig. 2: Ubp6 and USP14 mutants that are refractory to proteasome activation.
From: Allosteric control of Ubp6 and the proteasome via a bidirectional switch
a Activity of Ubp6 mutants in the presence or absence of proteasome, with Ubp6-AA highlighted. b Ub-AMC hydrolysis by Ubp6-AA (60 nM). c Positions of I329 and L330 (cyan) in the modeled Ubp6-Rpt1 interface. (PDB: 7QO3, 7QO4). d Concentration-dependence of Ubp6 activity on Ub-AMC (1 μM) in the presence of ubp6Δ hul5Δ proteasome (1 nM). The data were fit to a hyperbolic curve, yielding a Kd of 3.3 nM for wild-type Ubp6 and 4.0 nM for Ubp6-AA. See also Supplementary Fig. 7. e Activity of Ubp6-AA on HA-Ubn-NCB1 in the presence of proteasome (and ADP to prevent substrate degradation). Such deubiquitination is strictly proteasome dependent18. f Activity of the USP14-V343A L344A with or without proteasome. Wild-type values are independently set to 100% but differ by over 100-fold, reflecting the extent of activation. n = 2 independent samples per experiment were examined, normalized mean activity is plotted. This experiment was repeated independently with closely consistent results. g Sequence alignment between Ubp6 and USP14. Ubiquitin clash for free Ubp6 is shown in Supplementary Fig. 6a. Stippled lines, β-strand-forming residues in Ubp6 (see Supplementary Fig. 6c). Figure 5e describes BL2 contacts. Source data are provided as a Source Data file.
