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. 2019 Nov 28;8:e49806. doi: 10.7554/eLife.49806

Figure 4. Proteasomes with impaired conformational switching display various degradation defects.

(A) Michaelis-Menten analysis based on initial rates for ubiquitin-dependent degradation of FAM-titin-I27V15P under multiple-turnover conditions. Km and kcat values are shown below with errors representing SEM from the fit. Rpt4-EQ had too low activity to be fit. (B) Representative anisotropy traces for the single-turnover degradation of ubiquitinated FAM-titin-I27V15P by wild-type and Rpt4-EQ mutant proteasomes (C) SDS-PAGE analysis of end-point samples from single-turnover degradation reactions, visualizing fluorescence of the FAM-titin-I27V15P model substrate in its ubiquitinated, de-ubiquitinated, and degraded form.

Figure 4.

Figure 4—figure supplement 1. Analysis of substrate processing by free regulatory particle during proteasome degradation.

Figure 4—figure supplement 1.

(A) Normalized fluorescence anisotropy measurements showing the processing of ubiquitinated TAMRA-G3P-substrate by reconstituted Rpt4-EQ regulatory particle (RP) alone, by proteasomes reconstituted with Rpt4-EQ regulatory particle at stoichiometric amounts (2RP:1CP = 1X RP + Core) or in two-fold access (4RP:1CP = 2X RP + Core), and by core particle alone, normalized to the degradation by reconstituted wild-type 26S proteasome (N = 3; error presented = SD). (B) SDS-PAGE analysis of end-point samples from single-turnover degradation reactions performed in (A), visualizing the fluorescence of TAMRA-labeled G3P-substrate (left) and total protein at (right). (C) Normalized fluorescence anisotropy measurements showing the processing of ubiquitinated TAMRA-G3P-substrate by proteasomes reconstituted with mutant regulatory particles, normalized to reconstituted wild-type 26S proteasome. (N = 3; error present = SD). (D) SDS-PAGE analysis of end-point samples from single-turnover degradation reactions performed in (C), visualizing the fluorescence of TAMRA-G3P-substrate (left) and total protein (right). (E) Normalized fluorescence anisotropy measurements showing the processing of ubiquitinated TAMRA-G3P-substrate by wild-type, Rpn5-VTENKIF-, and Rpt6-EQ-mutant proteasomes reconstituted with a stoichiometric amount (2RP:1CP = 1 XRP) or two-fold excess of RP (4RP:1CP = 2 XRP). (N = 3, error presented = SD). (F) SDS-PAGE analysis of end-point samples from single-turnover degradation reactions performed in (D), visualizing the fluorescence of TAMRA G3P-substrate (top) and total protein (bottom).
Figure 4—figure supplement 1—source data 1. Source data for Michaelis-Menten analyses of substrate degradation by wild-type, Rpn5-VTENKIF, Rpt4-EQ, and Rpt6-EQ mutant proteasomes, and source data for substrate processing by the corresponding regulatory particles.