Fig. 4.

Proteasome conformational mutants decrease the unfolding ability of the proteasome. (A) Unfolding ability assay, in which a substate is engaged by the proteasome (k_engagement), barnase is unfolded and degraded (k_deg^full-length), and then DHFR is either degraded (k_deg^frag) or irreversibly released (k_rel^frag); the ratio of these two rate constants gives the unfolding ability (U). (B–D) Degradation of 20 nM ubiquitinated Neh2Dual-Barnase∆K-Cy5-DHFRδK∆C by 100 nM WT (B), Rpn5-s3mut (C), or Rpn2-s3mut (D) proteasome. Black box shows the region of the gel containing full-length protein with or without ubiquitination. Red box shows the region of the gel containing the DHFR fragment. The amounts of full-length protein (open squares) and DHFR fragment (red circles) are shown as a percentage of the ubiquitinated full-length substrate presented to the proteasome at the beginning of the reaction; the full-length protein is quantified as the sum of ubiquitinated and nonubiquitinated full-length species so any deubiquitination is not misinterpreted as degradation. Dots are results from individual experiments, and error bars represent the SEM of 15 (A), 7 (B), or 6 (C) experiments. Curves are global fits to single exponentials. Sample gels (scanned for Cy5 fluorescence) are shown on the right. * marks a contaminant in the substrate that is not ubiquitinated or degraded, and ** marks dye-labeled peptides that are degradation products when DHFR is completely degraded. (E) Unfolding abilities (U) calculated from the curve fits shown in B–D, according to Eq. 1. Error bars are the SEM propagated from curve fitting the collected datasets. Two-tailed t tests were used to determine significant differences from WT (*).