FIGURE 4.

Ubp15 and USP7 flanking domains are not required for substrate selectivity, and Ubp15 shows complementary activity to the 26 S proteasome. A, time course of ¹²⁵I-labeled Pds1-Lys³³ deubiquitination as described in Fig. 3E by 160 nm full-length Ubp15 or truncated isoforms: ΔN (residues 199–1230) or ΔC (residues 1–562). B and C, quantitation of A. Levels of mono- (B) or tetraubiquitinated (C) Pds1-Lys³³ are normalized to their initial values (in arbitrary units) at time zero. D, time course of 0.5 μm Ub-AMC hydrolysis by 50 pm full-length Ubp15 or truncated isoforms: ΔN, ΔC, or isolated catalytic domain (ΔNΔC, residues 199–562). E, time course of ¹²⁵I-labeled CycB-Lys⁶⁰ deubiquitination by 6 nm full-length USP7 or 20 nm Ubp15 as compared with the isolated catalytic domain of USP7 (ΔNΔC; residues 213–549) at 20 μm, as indicated. F, Ubp15 preferentially targets ubiquitin conjugates that are not degraded by the 26 S proteasome. Time course of ¹²⁵I-labeled Pds1-Lys³³ deubiquitination by 160 nm recombinant Ubp15 as shown in A or 1 μl of immunoprecipitated yeast 26 S proteasome. G and H, quantitation of F. Levels of each ubiquitin conjugate are normalized to their initial values (in arbitrary units) at time zero for Ubp15 (G) and proteasome (H). See supplemental Table S1 for initial rates. All results are representative of two to three experiments.