Mammalian 26S Proteasomes Remain Intact during Protein Degradation Franziska Kriegenburg, Michael Seeger, Yasushi Saeki, Keiji Tanaka, Anne-Marie B. Lauridsen, Rasmus Hartmann-Petersen, Klavs B. Hendil  Cell  Volume 135, Issue 2, Pages 355-365 (October 2008) DOI: 10.1016/j.cell.2008.08.032 Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 1 The Degradation System and Its Components (A) Polyubiquitylation of Sic1. 35S-labeled Sic1 was ubiquitylated for the indicated times and analyzed by SDS-PAGE and autoradiography. UbnSic1 denotes polyubiquitylated Sic1, which is too large to enter the stacking gel. (B and C) Proteasomes or proteasome components, purified from bovine blood cells, were analyzed by nondenaturing PAGE (B) and SDS-PAGE (C). Panel C, lane 4 shows human, protein A-tagged 26S proteasomes, precipitated from HeLa cell extracts with immunoglobulin-Sepharose. (D) Degradation of UbnSic1. Polyubiquitylated 35S-Sic1 (2.2 μM) was incubated for 60 min at 37°C with proteasomes as indicated (2.6 nM human 26S proteasomes or 20S proteasomes in 30 μl of buffer C) before degradation was determined as the increase in TCA-soluble radioactivity. Degradation was measured in the presence of 2 mM ATP (columns 1 and 2), or ATP was removed by addition of apyrase (0.6 units bound to 10 μl of Sepharose beads, columns 3 and 4). In column 4, AMP-PNP, 100 μM, was added along with the apyrase. Degradation depended on 26S proteasomes and ATP, and the noncleavable ATP analog could not substitute. Cell 2008 135, 355-365DOI: (10.1016/j.cell.2008.08.032) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 2 Polyubiquitylated Substrates Do Not Cause Release of Proteasome Subunits (A) Protein A-tagged, 35S-labeled 26S proteasomes from HeLa cells were bound to immunoglobulin-Sepharose via their protein A tag, diluted to around 2.7 nM, and incubated at 37°C in 30 μl of buffer C in the presence or absence of 2.2 μM UbnSic1 . The reaction mix was separated into supernatant (S) and pellet of beads (P) at 0 and 60 min before SDS-PAGE and autoradiography. There was a slow release of 26S proteasomes from beads into supernatant (compare lanes 6 and 2). This slow release was not increased by addition of substrate and no particular subunits, for instance from the RC, seem to have been preferentially released (compare lane 8 and lane 6). (B) This experiment is similar to that shown in (A) except that soluble, unlabeled human 26S proteasomes were added as a chasing agent (20-fold excess over the immobilized, labeled 26S proteasomes, lanes 5–8) together with the polyubiquitylated Sic1 substrate. (C) No subunit release, even at low substrate concentrations. The experiment was made as in (B) with unlabeled 26S proteasomes present but with molar ratios of UbnSic1:proteasomes as indicated. (D) No release of subunit S5a from proteasomes. Wild-type HeLa cells were metabolically labeled with 35S-methionine and 26S proteasomes were precipitated with an immobilized monoclonal antibody to subunit S5a/Rpn10. The beads with labeled 26S proteasomes were then incubated for 1 hr with UbnSic1 as in (A) and either with or without 10 nM of unlabeled recombinant S5a/Rpn10, as indicated. Release of radioactive subunits into the supernatant was followed as before. Addition of substrate and S5a caused no increase in release of proteasome subunits. (E) Immobilized 20S proteasomes are accessible to RCs. Human 20S proteasomes (≈0.1 μg) either in solution or bound to Sepharose beads with immobilized anti-proteasome antibody (MCP20) were incubated at 37°C for the indicated time in a final volume of 30 μl of buffer B with 2 μg of purified RC. Activation of proteasomes by RCs was determined as the increase in hydrolytic activity, measured with Suc-LLVY-AMC. Fluorescence was normalized to the activities at 45 min, which was 1595 units for soluble and 633 units for immobilized proteasomes. The rate of association of RC to 20S proteasomes was not decreased in immobilized proteasomes, which were therefore as accessible as soluble ones. Cell 2008 135, 355-365DOI: (10.1016/j.cell.2008.08.032) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 3 Degradation of Polyubiquitylated Sic1 Does Not Depend on Proteasome Concentration (A) Bovine 26S proteasomes at concentrations varying between 27 nM and 2.7 nM in 30 μl of buffer C were incubated with 2.2 μM 35S-UbnSic1. Samples were taken at intervals for determination of Sic1 degradation, measured as the increase in TCA-soluble radioactivity. Degradation is expressed as moles of Sic1 degraded per mole of 26S proteasome. (B) Surface plasmon resonance measurement of binding of regulatory complex to core 20S proteasomes. Bovine 20S proteasomes were coupled to a sensor chip, and RCs, at concentrations as indicated, were injected at 25°C in order to measure association. After 1 min and 45 s, the chip was washed with buffer in order to follow the dissociation. Data were fitted to a Langmuir binding model giving a rate constant for association of (6.1 ± 0.2) × 106 M−1 min−1 and a rate constant for dissociation of (0.0124 ± 0.003) min−1. (C) Rate of assembly of 26S proteasomes from 20S proteasomes and RCs in solution. Bovine 20S proteasomes at concentrations of either 90 nM or 9 nM were incubated at 37°C with a 2-fold molar amount of bovine RCs in a final volume of 30 μl of buffer B. Samples were taken at intervals, as indicated, diluted to equal protein concentration, and analyzed by nondenaturing PAGE. Blots were probed with an antibody (TBP1-19) to the RC (left panel). In the right panel, a sample taken at 30 min was similarly analyzed, but with an antibody to 20S proteasomes (MCP231). The two upper bands in the gels contain both RC and 20S proteasomes (compare right and left panels) and must therefore be double-capped and single-capped 26S proteasomes. (D) Assembly of 26S proteasomes in solution. Staining of the left blot from (C) was quantified and the timecourse of assembly of 26S proteasomes (sum of intensities of the two upper bands in each lane) is shown. The curves are not fitted to the data from (C) but show the theoretical timecourses expected from 2nd order kinetics: Y=a2kt1+akt, where Y is the amount of bound RCs in single-capped plus double-capped 26S proteasomes, k is the association rate constant, found by surface plasmon resonance spectrometry, a is the initial concentration of binding sites for RC on 20S proteasomes (18 × 10−8 M and 1.8 × 10−8 M, respectively), and t is the time in minutes. Filled and empty symbols refer to different ordinate axes, as indicated. (E) Analysis of released subunits by nondenaturing PAGE. 35S-labeled 26S proteasomes from HeLa cells, about 16 nM, were incubated at 37°C in 50 μl of buffer C. To check for proteasome disassembly, UbnSic1 was added to a concentration of 1.4 μM to one sample. After 1 hr the preparations were analyzed by nondenaturing PAGE. Autoradiography showed that the substrate formed so large complexes with the 26S proteasomes, that they did not even enter the stacking gel. However, no subunits of 26S proteasomes were seen in the gel. Cell 2008 135, 355-365DOI: (10.1016/j.cell.2008.08.032) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 4 Inactivated 20S Proteasomes Do Not Inhibit Protein Degradation by 26S Proteasomes (A) Outline of the experiment. Active 26S proteasomes were mixed with epoxomicin-inhibited 20S proteasomes (with red crosses). If the 26S proteasomes disassemble into RC and 20S proteasomes, as suggested in the cartoon, the released, active 20S proteasome would have to compete with the surplus of inactivated 20S proteasomes for the RC so that inactive 26S proteasomes are preferentially formed. (B) Active and inactivated 20S proteasomes react equally well with RCs. Active or epoxomicin-inactivated proteasomes, 0.1 μg, were incubated for 1 hr 30 min in 30 μl of buffer B with 2 mM ATP and with increasing amounts of bovine RCs, as shown. Assembly was assessed by nondenaturing PAGE and blotting with an antibody (MCP231) to the 20S proteasome. (C) Inhibited 20S proteasomes can interrupt formation of active 26S proteasomes from 20S proteasomes and RCs. Bovine 20S proteasomes, 0.1 μg, were incubated with 2 μg of bovine RCs in 30 μl of buffer B. The formation of active 26S proteasomes was measured as the increase in hydrolytic activity with Suc-LLVY-AMC. A 20-fold excess of inactivated 20S proteasomes was added to one series after 5 min (arrow). This caused cessation of activation. As a control, 20S proteasomes were incubated without addition of RCs. In this case, addition of inactivated 20S proteasomes had no influence on the hydrolytic activity. (D) Inactive 20S proteasomes have no influence on degradation of polyubiquitylated Sic1. 26S proteasomes, 4.8 nM, were incubated with 2.2 μM 35S-UbnSic1 in 30 μl of buffer C. After 5 min (arrow) a 20-fold molar excess of epoxomicin-inhibited 20S proteasomes was added to some of the samples. The degradation rate of UbnSic1, measured as release of TCA-soluble radioactivity, was not affected. (E) Inactive 20S proteasomes have no influence on degradation of polyubiquitylated UbcH10. The experiment was done as that shown in (D), but with polyubiquitylated UbcH10 (4.8 μM) as the substrate. Epoxomicin-inhibited 20S proteasomes were added after 15 min. (F) Degradation of endogenous protein in HeLa cell extracts. HeLa cells were metabolically labeled for 30 min in medium with 35S-methionine, 0.15 MBq/ml, 44 TBq/mmol. The cells were then harvested and homogenized in 10 volumes of buffer C. The lysate was centrifuged at 13000 rpm for 10 min and the protein concentration of the supernatant was determined. The supernatant was incubated at 37°C without additions or with either 250 μM MG132 or 0.38 mg/ml of epoxomicin-inhibited 20S proteasomes. Degradation was followed by TCA precipitation. Degradation was not influenced by the epoxomicin-inhibited proteasomes. Cell 2008 135, 355-365DOI: (10.1016/j.cell.2008.08.032) Copyright © 2008 Elsevier Inc. Terms and Conditions