Figure 5. Disrupting the conformational equilibrium inhibits substrate tail insertion but not later steps of degradation.

(A) Representative traces for the increase in acceptor fluorescence/FRET upon insertion of the ubiquitinated FAM-titin-I27^V15P-Cy5 substrate’s flexible initiation into the central pore of wild-type and Rpn5-VTENKIF mutant proteasomes with o-PA inhibited Rpn11, in the presence of ATP or ATPγS. The schematic below depicts the experimental setup, where FRET occurs when a substrate’s flexible initiation region labeled with an acceptor dye (blue star) enters and then stalls in the central pore of a proteasome containing inhibited Rpn11 (red cross) and a donor dye (red star) near the processing channel. The substrate’s ubiquitin modification is represented in pink, the Rpt ring is shown in light blue, the core particle in dark grey, and Rpn5 in orange. (B) Rate constants for the single-turnover, ubiquitin-dependent degradation of ubiquitinated G3P model substrate, either without stalling the proteasome (left) or after stalling translocation for 3 min with o-PA inhibited Rpn11 and restarting by the addition of Zn²⁺ (right). Rates were determined from single-exponential fits of the appearance of fluorescently tagged peptide products on SDS PAGE gels. Error bars represent SEM for the fit, N ≥ 3, technical replicates. (C) Ubiquitin-dependent degradation rates for wild-type, Rpn5-VTENKIF and Rpt6-EQ mutant proteasomes degrading the destabilized FAM-titin-I27^V13P/V15P-35mer tail or the non-destabilized FAM-titin-I27-35mer tail substrate under single-turnover conditions. Shown are the rate constants for the dominant fast phase derived from a double-exponential fit of the degradation kinetics (N = 3, technical replicates, error bars represent SD).

Figure 5—figure supplement 1. Tail insertion can be rate limiting for mutant proteasomes with compromised conformational equilibria.

(A) FRET-based assay monitoring the insertion of the ubiquitinated Cy5-labeled titin-I27^V15P substrate tail into the central pore of Cy3 labeled, o-PA-inhibited wild-type or Rpn5-VTENKIF mutant proteasomes. Representative traces (left) show the reciprocal change in Cy3 and Cy5 fluorescence, indicative of FRET, and the dependence of substrate-tail insertion on fully assembled proteasomes. Middle, traces for the substrate-tail insertion of wild-type and Rpn5-VTENKIF mutant proteasomes, monitored through Cy5 fluorescence and fit to Equation 2 (solid line). Residuals of the fit are shown below the traces, and derived kinetic parameters are shown on the right (N = 3, technical replicates, error represents SEM). (B) FRET-based tail-insertion assay, as described in (A), comparing wild-type, Rpn5-VTENKIF, and Rpt6-EQ mutant proteasomes (N ≥ 3, technical replicates, error represents S.E.M.).

Figure 5—figure supplement 2. Gel based degradation assay analysis.

(A) Representative SDS-PAGE gels for the end-point analyses of ubiquitinated TAMRA-G3P-degradation reactions by wild-type, Rpn5-VTENKIF, and Rpt6-EQ mutant proteasomes after restart from an o-PA-induced stall. The fluorescence signal of the substrate-attached TAMRA dye was use for in-gel detection and quantifications, which are plotted below the gels. Data were fitted to Equation 3 (N = 3, technical replicates, error bars represent SD). (B) Representative SDS-PAGE gels for the analyses of non-destabilized FAM-titin-I27 degradation by wild-type and mutant proteasomes under single-turnover conditions. Quantified FAM-fluorescence values for ubiquitinated substrate and peptide products are plotted below the gels (N = 3, technical replicates, error bars represent SD).