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. 2019 Apr 4;177(2):286-298.e15.
doi: 10.1016/j.cell.2019.02.031. Epub 2019 Mar 28.

The 26S Proteasome Utilizes a Kinetic Gateway to Prioritize Substrate Degradation

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Free PMC article

The 26S Proteasome Utilizes a Kinetic Gateway to Prioritize Substrate Degradation

Jared A M Bard et al. Cell. .
Free PMC article

Abstract

The 26S proteasome is the principal macromolecular machine responsible for protein degradation in eukaryotes. However, little is known about the detailed kinetics and coordination of the underlying substrate-processing steps of the proteasome, and their correlation with observed conformational states. Here, we used reconstituted 26S proteasomes with unnatural amino-acid-attached fluorophores in a series of FRET- and anisotropy-based assays to probe substrate-proteasome interactions, the individual steps of the processing pathway, and the conformational state of the proteasome itself. We develop a complete kinetic picture of proteasomal degradation, which reveals that the engagement steps prior to substrate commitment are fast relative to subsequent deubiquitination, translocation, and unfolding. Furthermore, we find that non-ideal substrates are rapidly rejected by the proteasome, which thus employs a kinetic proofreading mechanism to ensure degradation fidelity and substrate prioritization.

Keywords: 26S proteasome; AAA(+) protease; ATP-dependent protein degradation; ubiquitin-proteasome system; unnatural amino-acid incorporation.

Conflict of interest statement

Declaration of Interests: The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. Site-specific labeling of the 26S proteasome and a steady-state assay for its conformations.
(A) SDS-PAGE analysis of base and lid with AzF incorporated into Rpt5 and Rpn9, and then labeled with Cy5 and Cy3, respectively. (B) Schematic for the in vitro reconstitution of the 26S proteasome. (C) Comparison of the substrate-free and substrate-engaged states. The distance between Rpt5-Q49 and Rpn9-S111 changes by 37 Å during the conformational transition. The AAA+ ATPases are blue, Rpn11 is green, and the rest of the regulatory particle as well as the 20S core are grey. Models were generated from surface representations of atomic models (PDB: 5mp9, 5mpd, 5mpb). The red and purple spheres indicate the labeled residues in Rpt5 and Rpn9, respectively. (D) Steady-state FRET between Cy3-labeled lid (RpnS111AzF-Cy3) and Cy5-labeled base (Rpt5Q49AzF-Cy5) under various conditions, normalized to the signal for proteasome with ATP alone. Ubp6C118A is catalytically inactive, Ub4 represents a linearly fused tetra-ubiquitin, and the ubiquitinated substrate is titin-I27V15P-23-K-35. Shown are the mean and individual measurements for N = 3. See also Figure S1.
Figure 2.
Figure 2.. Substrate engagement triggers the conformational switch of the proteasome.
(A) Schematic for the substrate processing pathway. Ubiquitin chains (pink) target a substrate (gold) to the 26S proteasome in the s1 state. The substrate’s unstructured tail is inserted into the pore of the AAA+ motor (blue), where it interacts with pore loops (red) that drive translocation. After substrate engagement, the regulatory particle changes its conformation to an s3-like state and Rpn11 (green) shifts to a central position that allows translocation-coupled deubiquitination. Ubiquitin-chain removal is followed by mechanical substrate unfolding and threading of the polypeptide into the 20S core for proteolytic cleavage. (B) Single-turnover degradation of ubiquitinated 5-FAM-titin-I27V15P-23-K-35 by reconstituted 26S proteasome is tracked by SDS-PAGE and visualized by 5-FAM fluorescence. (C) Single-turnover degradation of ubiquitinated 5-FAM-titin-I27V15P-23-K-35 is tracked by fluorescence anisotropy in the presence of ATP or ATPγS. The total time for degradation is derived from the sum of the time constant τ for the exponential decay of anisotropy and the time t0 for the initial anisotropy increase. (D) Substrate-tail insertion is tracked by FRET between the 26S proteasome reconstituted with Cy3-labeled base (Rpt1-I191AzF-Cy3) and ubiquitinated titin-I27V15P-23-K-35 modified with Cy5 on its unstructured tail. At t=0, an excess of substrate was added to proteasomes with oPA-inhibited Rpn11 in the presence of either ATP (purple) or ATPγS (grey). A control with non-ubiquitinated, Cy3-labeled titin-I27V15P-23-K-35 substrate is depicted in black. Shown is the signal of the Cy3 channel, normalized to initial fluorescence. (E) Conformational state of the proteasome over time, tracked by FRET between Cy5-labeled base (Rpt5Q49AzF-Cy5) and Cy3-labled lid (Rpn9S111AzF-Cy3). At t=0, an excess of ubiquitinated titin-I27V15P-23-K-35 or buffer was added to double-labeled proteasomes with oPA-inhibited Rpn11. Shown is the Cy5 channel, normalized to initial fluorescence. The total time for the conformational change is derived from the sum of the time constant τ for the exponential increase of FRET and the time t0 for the initial delay. (F) Overlay of fluorescence traces for tail insertion and conformational change from D and E reveal a delay of 400 ms. (G) Deubiquitination tracked by FRET between Cy3-labeled ubiquitin and a Cy5 label attached adjacent to the single ubiquitinated lysine in titin-I27V15P-23-K-35. At t=0 the substrate was mixed with excess proteasome in the presence of ATP or ATPγS. Shown is the Cy5 channel, normalized to initial fluorescence. (H) Time constants of the substrate-processing steps as calculated from each assay individually or after accounting for the preceding steps. All curves in CG are representative traces, and time constants are derived from averaging fits of independent experiments, shown with s.d. (N ≥ 3). See also Figure S1 and S2.
Figure 3.
Figure 3.. Increasing substrate stability slows degradation, but multiple ubiquitin chains can be rapidly removed.
(A) Single-turnover degradations of substrates with mutations in the titin-I27 folded domain tracked by anisotropy of N-terminally attached 5-FAM. Stabilities were determined by denaturant-induced equilibrium unfolding (see Fig. S3). (B) Singleturnover degradations of the same substrates as in A, but tracked by SDS-PAGE. (C) Single-turnover degradations of substrates with up to 3 lysine-attached ubiquitin chains in the unstructured tail tracked by anisotropy of 5-FAM. All curves are representative traces, and time constants are derived from averaging fits of independent experiments, shown with s.d. (N ≥ 3). See also Figure S3 and S4.
Figure 4.
Figure 4.. Substrates with poor initiation regions do not stably engage with the proteasome.
(A) Single-turnover degradations of substrates with varied unstructured initiation regions analyzed by SDS-PAGE. (B) Tail insertion kinetics of substrate variants tracked by FRET between the substrate and the proteasome as in Fig. 2D. The corresponding time constants are listed in Table 1. (C) Conformational change of the proteasome after substrate addition, tracked by FRET between the base and the lid as in Fig. 2E. (D) Competitive inhibition of titin-I27V15P-23-K-35 degradation by substrates with varied tails (same substrates as in C). The percent inhibition is derived from the initial degradation rates in the presence and absence of competitor. All curves are representative traces (N ≥ 3). See also Figure S5 and S6.
Figure 5.
Figure 5.. An extra ubiquitin chain promotes complete degradation of a substrate with a ubiquitin-obstructed initiation region.
(A) Single-turnover processing of substrates with varied unstructured initiation regions and ubiquitination states analyzed by SDS-PAGE. The substrate alone sample was generated by incubation of ubiquitinated substrate with Usp2. Coomassie-stained Rpn1 and Rpn2 are shown as a loading control. (B) Quantification of the fluorescence intensities shown in (A). The intensities for ubiquitinated substrate during proteasomal processing are normalized to the intensities for ubiquitinated substrate alone, while the intensities for deubiquitinated substrate and peptide product are normalized to the Usp2-treated samples (shown with means and s.d., N ≥ 3). The curves for the ubiquitinated substrate panel are taken from exponential fits (see Table S8).
Figure 6.
Figure 6.. Model for kinetic proofreading by the proteasome.
Ubiquitin binding and tail insertion constitute a dynamic gateway to proteasomal substrate processing, with both fast on and off rates. If the substrate has the necessary requirements for degradation, engagement with the AAA+ motor reduces koff for tail insertion and accelerates the conformational switch of the proteasome, thereby committing substrates to translocation-coupled deubiquitination, rate-limiting unfolding, and degradation.

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