.2016 Feb 19;351(6275):10.1126/science.aad9421 aad9421.
doi: 10.1126/science.aad9421.Rpn1 provides adjacent receptor sites for substrate binding and deubiquitination by the proteasome
Affiliations
- PMID:26912900
- PMCID: PMC4980823
- DOI: 10.1126/science.aad9421
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Rpn1 provides adjacent receptor sites for substrate binding and deubiquitination by the proteasome
Yuan Shi et al. Science..
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Abstract
Hundreds of pathways for degradation converge at ubiquitin recognition by a proteasome. Here, we found that the five known proteasomal ubiquitin receptors in yeast are collectively nonessential for ubiquitin recognition and identified a sixth receptor, Rpn1. A site ( T1: ) in the Rpn1 toroid recognized ubiquitin and ubiquitin-like ( UBL: ) domains of substrate shuttling factors. T1 structures with monoubiquitin or lysine 48 diubiquitin show three neighboring outer helices engaging two ubiquitins. T1 contributes a distinct substrate-binding pathway with preference for lysine 48-linked chains. Proximal to T1 within the Rpn1 toroid is a second UBL-binding site ( T2: ) that assists in ubiquitin chain disassembly, by binding the UBL of deubiquitinating enzyme Ubp6. Thus, a two-site recognition domain intrinsic to the proteasome uses distinct ubiquitin-fold ligands to assemble substrates, shuttling factors, and a deubiquitinating enzyme.
Copyright © 2016, American Association for the Advancement of Science.
Figures
Schematic (top) and model structure (bottom,left) mapping the UBL-binding Rpn1 T1 (indigo) and T2 (orange) sites. (Bottom,right) Enlarged region of the proteasome to illustrate the Rpn1 T1 and T2 sites bound to a ubiquitin chain (yellow) and deubiquitinating enzyme Ubp6 (green), respectively. PDB 4CR2 and 2B9R were used for this figure.
(A) Yeast strains with the indicated genotypes were serially diluted, transferred to agar plates, and incubated at 30°C. Media were YPD, YPD with 1M NaCl (top row), synthetic medium lacking arginine (Arg−), or Arg− with 6 mg/ml canavanine (bottom row).ΔUU designates therad23 dsk2 ddi1 background.(B) Proteasome association with ubiquitin conjugates evaluated by a mobility shift assay. RP complexes were purified from anrpn10-uim rpn13-pru strain and incubated with core particle. Reconstituted proteasomes were incubated with autoubiquitinated Cdc34, resolved via native PAGE, and visualized with a fluorogenic activity stain.(C) Known and candidate ubiquitin receptor proteins were expressed in bacteria as GST fusion proteins and immobilized on glutathione resin. Ubiquitinated T7-PY-Sic1 conjugates were used as ligand. Resin-bound proteins were resolved by SDS-PAGE, blotted, and probed with antibodies as indicated. Direct loading of ubiquitinated T7-PY-Sic1 is also included (input).(D)1H,15N HSQC spectra of15N Rpn1412–625 (black) and with equimolar ubiquitin (orange). A titration series is displayed to the right for D541 with increasing ubiquitin, as indicated.(E) Model ribbon diagram of the Rpn1 toroidal domain (PDB 4CR2 (47)) showing residues that shift following ubiquitin addition (D) in orange.(F) Ubiquitin amino acids with amide signals that shift by one standard deviation above average following Rpn1 T1 addition, as shown in fig S8B and S8D, are labeled and highlighted in orange on a ribbon diagram (PDB 2K39 (48)).
(A) Backbone heavy atoms for the ten lowest energy structures of the Rpn1 T1 site with H26–H31 superimposed. The N- and C-terminal residues and the individual helices are labeled.(B) Ribbon diagram of the lowest energy structure of the Rpn1 T1 site (blue) bound to two monoubiquitin molecules (orange and yellow). Heavy atoms for G76, the M1 backbone, and lysine sidechains are displayed with their oxygen and nitrogen atoms in red and blue respectively. Displayed sidechains for the ubiquitin at H28/H30 are labeled in orange whereas those from the ubiquitin centered at H26 are labeled in black. Dashed orange lines are drawn between G76 from the ubiquitin at H28/H30 and the closest lysine sidechains (K6 and K48) from the ubiquitin at H26; the distance between these two pairs of amino acids is <10Å and the flexibility of the lysine sidechain and C-terminal tail of monoubiquitin allows this distance to be readily shortened.(C) Zoomed-in view of (B) highlighting the Rpn1:ubiquitin contact surface with key amino acids displayed and labeled. Electrostatic interactions are indicated with a red dashed line.(D) Pull-down assay with His-scRpn1 full length protein and M1-, K6-, K11-, K27-, K29-, K33-, K48-, and K63-diubiquitin, as indicated (top and middle panels, left). Immunoblotting was done with anti-ubiquitin (top, left) or anti-His (middle, left) antibodies. Intrinsically disordered protein SocB-His (49) was used as a negative control with K48 diubiquitin, as indicated. Direct loading for 15% of the diubiquitin input for each chain type with immunoblotting by anti-ubiquitin antibody is included (bottom, left). The pull-down assay was repeated four times and the diubiquitin signal intensities were separately normalized to the strongest signal by using ImageJ. The average value and standard deviation is plotted (right).(E) Shifting for indicated Rpn1 T1 site residues plotted with increasing K48 diubiquitin and fitting to the listed Kd values.(F) ITC analysis of Rpn1412–625 binding to K48 diubiquitin. 1.91mM K48 diubiquitin was injected into a calorimeter cell containing 0.18mM Rpn1412–625 and the data were fit to a 2-site sequential binding mode to yield the indicated thermodynamic values.(G) As in (D) but with GST-hRpn1404–617 or GST (as a control). In this case, quantification on the right was done with two independent pull-down assays.
(A) Selected regions from1H,15N HSQC spectra of15N wild-type Rpn1412–625 (left) and15N Rpn1412–625-ARR (D541A/D548R/E552R, right) alone (black) and with ubiquitin (orange) at 4- and 5-fold molar excess, respectively.(B) GST pull-down assay with GST, GST-Rpn1412–625, GST-Rpn1412–625-ARR, or GST-Rpn1412–625 A514G/D517A/D541A/D548R/E552R (GAARR) and the indicated ubiquitin species. Immunoblotting was done with anti-ubiquitin (top) or anti-GST (bottom) antibodies.(C) Defective ubiquitin binding by the Rpn1-ARR mutant protein. Full length GST-Rpn1 fusion protein was expressed, purified, and tested for binding to ubiquitinated T7-PY-Sic1. GST-Rpn10 and GST-Rpn10-uim were included as positive and negative controls, respectively.(D) Rad23 variants were tested for binding to Rpn1-WT and Rpn1-ARR. A mixture of recombinant Rad23-Flag and Rad23 UBL-Flag (serving as an internal negative control) were used as ligands. UBA domains were absent from both constructs.(E) Rpn1 amino acids at the Rad23 UBL domain contact surface as determined by the data in fig S20 are highlighted in orange on the ribbon diagram of the Rpn1 T1 site.(F) ITC analysis of the Rpn1 T1 site binding to Rad23 UBL. 0.407mM Rad23 UBL was injected into a calorimeter cell containing 0.036mM Rpn1412–625 and the data were fit to a 1-site binding mode with the indicated thermodynamic parameters.
(A) Proteasome association with ubiquitin conjugates evaluated by mobility shift assay. Regulatory particle (RP) complexes were purified from the indicated yeast strains, incubated with purified core particle (CP) complex to form holoenzyme, and tested for association with ubiquitin conjugates as described for Figure 1B.(B) Proteasomes were prepared as described for (A), and assayed for degradation of a ubiquitinated fragment of cyclin B1 (Ubn-HA-NCyclinB). Rpn5 was probed as a loading control. (C) Synthetic canavanine sensitivity ofrpn1-ARR with other intrinsic ubiquitin receptor mutants. All strains were prepared in therad23 dsk2 ddi1 background, and carry additional mutations as indicated. Yeast cultures were serially diluted, transferred to synthetic media agar plates lacking arginine (Arg−), or Arg− with 2 mg/ml canavanine, followed by incubation at 30°C.(D) Proteasome association with Rad23 evaluated by mobility shift assay. Proteasomes were purified fromrad23 dsk2 ddi1 yeast strains bearing the indicated intrinsic ubiquitin receptor mutations, and incubated with ligand at 100-fold molar excess. Complexes including GST-Rad23-UBL were resolved by native PAGE, and assayed as described.(E) Proteasomes were purified in the absence of salt from yeast strains bearing mutations in intrinsic ubiquitin receptors, as indicated. Proteasomes and their associated proteins were resolved by SDS-PAGE, blotted, and probed with antibodies to proteasome-associated UBL protein Rad23, and to proteasome subunit Rpn5 as a loading control.(F)In vivo degradation of proteasome substrate Gic2.rpn10-uim rpn13-pru yeast strains bearing TAP-tagged Gic2 integrated at the native genomic locus, and other alleles as indicated, were treated with cycloheximide for the indicated times. Lysates were prepared and resolved by SDS-PAGE. Proteins were blotted and probed for Gic2-TAP, with Pgk1 as a loading control.(G) Therpn1-ARR allele confers sensitivity to 4-NQO. Cultures of strains carrying the indicated mutations were serially diluted, transferred to YPD plates either lacking or containing 0.1 mg/ml 4-NQO, and incubated at 30°C for two days.
(A, C) Lowest energy structures of Rpn1482–612:K48 diubiquitin in the extended (A) or contracted (C) binding mode. These structures were solved experimentally by using a suite of NMR experiments, as described in Materials and Methods by using the data listed in Table S2. An enlarged view is included in (C) to illustrate restricted accessibility of proximal ubiquitin G76. Displayed amino acid sidechains from distal ubiquitin (orange) are labeled in orange whereas those from proximal ubiquitin (yellow) are labeled in black. Blue coloring is used for Rpn1 with helices labeled in grey. (B) Model of Rpn1 T1:K48 tetraubiquitin by adding a ubiquitin (yellow) to each end of the K48 diubiquitin chain for the extended (left) and contracted (right) experimentally determined Rpn1 T1:K48 diubiquitin structure. The Rpn1 T1:K48 tetraubiquitin structures were energy minimized by using Schrödinger (www.schrodinger.com ).(D) Expanded view of the extended (top) or contracted (bottom) binding mode for Rpn1 T1:K48 diubiquitin to illustrate hydrophobic and electrostatic interactions at the contact surface.(E) Model of proteasome engaging a ubiquitinated substrate, generated with Rpn1 T1:K48 diubiquitin in the extended binding mode, Rpn13 Pru:ubiquitin (PDB 2Z59), hRpn10:K48 diubiquitin (PDB 2KDF), and human cyclin B1 (PDB 2B9R) placed into a proteasome cryoEM-based model (PDB 4CR2). Rpn1, blue and indigo; ubiquitin, yellow; Rpn13 Pru, navy; Rpn10, light blue; substrate, beige; ATPase ring, burgundy; CP, grey; remaining RP, white.
(A) Deuteration of recombinant Rpn1 (Rpn1free) compared with deuteration of Rpn1 in the context of the base (Rpn1base). Peptide residue numbers are shown at left, as well as the Rpn1 domain organization as described in the Supplement. Differences at each time point were calculated using Eq. 6 (see Supplement) and color-coded according to the scale at the bottom.(B) Deuteration differences between Rpn1base when bound to Ubp6 minus Rpn1base without Ubp6. Peptide residue numbers are identical to those in (A), and deuteration differences are indicated by the scale at the bottom.(C) Proteasomes were purified from strains bearing wild type or mutant Rpn1 alleles as indicated, employing mild washes. The YY mutant isD431Y Q434Y; AAAA isL430A D431A Q434A Q435A; and AKAA isL430A D431K Q434A Q435A. Aliquots of extracts and purified proteasomes were resolved by 10% SDS-PAGE, blotted, and probed with antibodies against Rpn1, Ubp6, and Rpn12.(D) Activation of Ubp6 by wild type and mutant regulatory particle. Ubp6 was incubated with RP purified from wild type andrpn1-AKAA strains and assayed for Ub-AMC hydrolysis activity. Concentration of RP was constant at 1 nM, and the concentration of Ubp6 was graded. Curve-fitting as shown yields a Kd value of 4.7 nM for wild type. For the AKAA mutant, a 29-fold higher concentration of Ubp6 would be required to achieve a hydrolytic rate corresponding to half-maximal for wild-type.
(A) Reconstituted proteasomes, prepared with regulatory particles isolated from wild type,rpn1-AKAA, orrpn1-ARR strains, were incubated with GST-Ubp6UBL or GST-Rad23UBL in molar excess, resolved by native PAGE, and assayed as described.(B) Ubp6 in RP complexes as indicated was evaluated for activation by the proteasome. Proteins were incubated with 1 μM Ub-AMC, and hydrolytic activity was monitored by the fluorescence of released AMC.(C) Toroidal domain of Rpn1 highlighting the T1 and T2 sites. Residues required for interaction with ubiquitin chains and Rad23 are shown in purple, and exposed residues implicated in Ubp6 binding are highlighted in orange. This image is generated by using PDB 4CR2.(D) Rpn1 T1 and T2 sites in the context of the proteasome, with sites colored as indicated as in (C). This image was generated by docking Ubp6 onto PDB 4CR2 based on the experimental data of Fig. 6B and 6C. The coloring scheme follows that of Fig. 5E and with Ubp6 in green. T2 amino acids L430, D431, Q434, and Q435 are highlighted in orange.
References
- Deveraux Q, Ustrell V, Pickart C, Rechsteiner M. A 26 S protease subunit that binds ubiquitin conjugates. J Biol Chem. 1994;269:7059–7061. - PubMed
- Fu H, et al. Multiubiquitin chain binding and protein degradation are mediated by distinct domains within the 26 S proteasome subunit Mcb1. J Biol Chem. 1998;273:1970–1981. - PubMed
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