Figure 2. Proteasomes reconstituted with endogenous or heterologously expressed base exhibit similar degradation activities for a polyubiquitinated substrate.

(a) Degradation of a polyubiquitinated GFP fusion substrate by endogenous yeast holoenzyme or 26S proteasomes reconstituted with saturating recombinant base (ebase) or endogenous yeast base (ybase). Substrate degradation was monitored by the loss of GFP fluorescence and strictly required the addition of Rpn10, despite the presence of Rpn13 (see Fig. 1A). (b) Michaelis-Menten analyses of substrate degradation by proteasomes reconstituted with endogenous or recombinant base. Degradation reactions were performed using limiting base and excess core particle, lid and Rpn10 to ensure that reconstituted proteasome particles were singly capped. K_M and V_max values were 0.63 μM and 0.21 enz⁻¹ min⁻¹ for holoenzyme with endogenous base, and 0.35 μM and 0.18 enz⁻¹ min⁻¹ for holoenzyme with recombinant base.