FIGURE 3.

Two-step proteasome incorporation of Ubp6. A and B, SPR sensograms of Ubp6^UBL and Ubp6^ΔUBL binding kinetics to Rpn1. The response data for binding and dissociation (dotted lines) are shown for a series of concentrations (arrows). Values of best fit parameters to a single bimolecular interaction model (solid lines) are summarized in Table 3. RU, response units. C, immobilized Ubp6 and Rad23 lacking their UBL domains (Ubp6^ΔUBL and Rad23^ΔUBL) were tested for binding to 26 S proteasome-purified free of all UDPs or other transient proteasome-interacting proteins. Association of full-length Ubp6 and Rad23 is included as positive control. Eluate from each column was tested for proteasome activity (20 S CP) as well as immunoblotted (IB) for presence of 19 S subunits from the Base and Lid subcomplexes. D, truncated Ubp6-expressing strains were molecular weight-fractionated, and proteins copurifying with peak of proteasome activity were identified by immunoblotting. E, potency of Ubp6 to remove ubiquitin from Ub-GFP was monitored over 60 min, and % reaction progression was plotted over time (diamonds). The reaction was repeated in presence of equimolar Rpn1 (squares) or purified base-CP (lidless) proteasome lacking both endogenous Ubp6 and Rpn11 (circles). Neither Rpn1 (star) nor base-CP alone (×) exhibit deubiquitination activity in the absence of added Ubp6 (dotted lines). F, experiment similar to E but for truncated Ubp6^ΔUBL. Ubp6 activation is not dependent on UBL.