1. Ecm29 Fulfils Quality Control Functions in Proteasome Assembly
Andrea Lehmann, Agathe Niewienda, Katharina Jechow, Katharina Janek, Cordula Enenkel 
Molecular Cell 
Volume 38, Issue 6, Pages (June 2010)
DOI: /j.molcel
Copyright © 2010 Elsevier Inc. Terms and Conditions

2. Molecular Cell 2010 38, 879-888DOI: (10.1016/j.molcel.2010.06.016)
Copyright © 2010 Elsevier Inc. Terms and Conditions

3. Figure 1
Ecm29 Binds RP-CP-RP and Blm10-CP-RP with Incompletely Matured CP
(A) The protein level of Ecm29 is increased in cells with impaired CP function. HA-tagged Ecm29 is hardly detectable in wild-type cells (lane 1), unless proteasomal subunits are tagged with HA epitopes (β5-HA, lane 2; Ump1-HA, lane 3). Ecm29-HA levels are significantly elevated in ump1Δ cells (lane 4). Consistent with previous observations, the protein level of proteasomal subunits as shown for Rpn5 is also increased in ump1Δ cells, due to the stabilization of Rpn4 that augments the transcription of proteasomal genes (Lehmann et al., 2008). Kar2 was shown as loading control. Minor bands that are visible below Ecm29-HA most likely represent degradation bands. For pulse-chase analysis, see Figure S1.
(B) Ecm29 is present within RP-CP-RP and Blm10-CP-RP. Lysates of ump1Δ cells expressing GFP-labeled CP and HA-tagged Ecm29 were subjected to native PAGE. GFP-labeled CP were visualized by phosphofluoroimaging and assigned to RP-CP-RP, Blm10-CP-RP, RP-CP, Blm102-CP, Blm10-CP, and CP, as previously described (Lehmann et al., 2008). Ecm29-HA was detected by immunoblotting. No cross-reaction was observed in lysates without HA-tagged protein (data not shown).
(C) By using HA affinity chromatography under native conditions, Ecm29-associated RP-CP were isolated from ump1Δ cells expressing Ecm29-HA and GFP-labeled CP. Ecm29-HA-bound RP-CP-RP and Blm10-CP-RP were resolved by native PAGE and visualized by GFP imaging. The protein bands were excised, subjected to SDS PAGE, and silver stained. Differences in the protein pattern of CP subunits might be due to alternatively configured α rings within Blm10-CP-RP (Lehmann et al., 2008).
(D) Ecm29-associated RP-CP were affinity purified from ump1Δ cells expressing Ecm29-protein A and β5-HA by using IgG Sepharose under ATP-depleting conditions. Proteins were separated by SDS-PAGE and probed for unprocessed (pro) β5, incompletely (i) processed β5, and matured (m) β5. Control for unspecific CP binding, lane 1; coprecipitation of Ecm29-protein A and β5-HA, lane 2; lysate, lane 3.
(E) Lysates of ump1Δ (lane 1) and ecm29Δ ump1Δ (lane 2) cells expressing β5-HA were analyzed by immunoblotting. Rpn5 was used as loading control.
(F) By using IgG Sepharose chromatography, RP-CP assemblies were affinity purified from ump1Δ and ecm29Δ ump1Δ cells expressing Rpn1-protein A (RP base) and β5-HA. IgG Sepharose-bound proteins were eluted and separated by SDS-PAGE, followed by Coomassie blue staining and immunoblotting against β5-HA.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2010 Elsevier Inc. Terms and Conditions

4. Figure 2
The Equilibrium between RP-Associated CP and RP-Free CP Is Shifted toward RP-Associated CP in ecm29Δ Cells
(A) Lysates of wild-type and ecm29Δ cells expressing GFP-labeled CP and HA-tagged Ump1 were subjected to native PAGE. CP were visualized by GFP imaging (lanes 1 and 2). Bands were assigned to RP-CP-RP, Blm10-CP-RP, RP-CP, Blm102-CP, Blm10-CP, and CP, as described above. The gel was blotted and probed for Ump1-associated precursor complexes (lanes 3 and 4) and RP (anti-RP base Rpt1; lanes 5 and 6). Peptide cleavage activities using chromogenic substrate Suc-Leu-Leu-Val-Tyr-AMC (Y) were assayed in gel in the presence and absence of 0.02% SDS. RP- and Ump1-associated precursor complexes (RP-pro-CP) are marked.
(B) Lysates of wild-type (lane 1) and ecm29Δ (lane 2) cells were probed for Ecm29, β5-GFPS, and Ump1-HA by immunoblotting. Rpn5 and Kar2 were used as loading controls.
(C) Lysates of wild-type and ecm29Δ cells expressing GFP-labeled CP and Ump1-HA were probed for ubiquitin and polyubiquitylated proteins.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 3 ECM29 Genetically Interacts with UMP1
Wild-type (WT), ecm29Δ, ump1Δ, and ecm29Δ ump1Δ cells were grown on YPD plates at the indicated temperatures for 2 days.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4 RP-Associated Precursor Complexes Accumulate in ecm29Δ Cells
(A) Lysates of wild-type (lane 1) and ecm29Δ cells (lane 2) expressing Ump1-GFPS instead of endogenous Ump1 were resolved by native PAGE and analyzed by GFP imaging and in-gel activity assays using substrate Y.
(B) Ump1-GFPS-associated precursor complexes were affinity purified from ecm29Δ cells under native conditions, resolved by native PAGE, and visualized by GFP imaging. The fluorescent protein bands were assigned to pro-CP species (lanes 1–4) and RP-pro-CP (lane 5), excised from the gel, subjected to SDS-PAGE, and silver stained. Blm10, RP, and CP subunits were assigned. Affinity-purified RP-CP was loaded for comparison. See also RP-pro-CP in Figure S3.
(C) Ecm29-associated RP-CP was affinity purified from wild-type cells expressing Ecm29-protein A and either β5-HA or β3-HA. To induce proteasome biogenesis, cells were fed up during logarithmic phase. Lysates were prepared without ATP and loaded on IgG Sepharose beads (lane 1). Unspecific proteins were washed off the resin with increasing salt. Tightly bound proteins were eluted with acetic acid. In the upper panel, m-β5 and pro-β5 were mainly detected in the wash (TE, lane 2; TE 0.3 M NaCl, lane 3). Residuals of m-β5 and i-β5 were coeluted with Ecm29-protein A by acetic acid (lane 4). In the lower panel, the affinity resin was washed (TE 0.3 M NaCl, lane 2), and tightly bound proteins were eluted with acetic acid (lane 3). Protein samples were subjected to SDS-PAGE followed by immunoblotting against HA-tagged subunits, α4, and protein A-tagged Ecm29.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5
Remodeling of Ecm29-Bound RP-CP Assemblies Containing Incompletely Matured CP into Regular Enzymes by the Addition of Ecm29-HA
(A) Lysates of ump1Δ and ecm29Δ ump1Δ cells expressing GFP-labeled CP were subjected to native PAGE. CP configurations were visualized by GFP imaging and assigned as above. It is important to note that RP-pro-CP are not detected in ecm29Δ ump1Δ cells, since the Ump1-dependent checkpoint is overcome (Li et al., 2007).
(B) With the aid of Ecm29-HA, CP configurations of ecm29Δ ump1Δ cells are converted into CP configurations similar to those observed in ump1Δ cells. Lysates of ecm29Δ ump1Δ cells expressing GFP-labeled CP were incubated with buffer (lane 1), with purified Ecm29-HA (lane 2), with lysate of ecm29Δ ump1Δ cells lacking Ecm29-HA (lane 3), and with lysate of ump1Δ cells containing Ecm29-HA (lane 4; the additional latter two lysates contained no GFP-labeled CP). CP configurations were separated by native PAGE and visualized by phosphofluoroimaging. Ecm29-HA was purified from yeast, subjected to SDS-PAGE, and stained with Coomassie blue (lane 5). Due to induced proteasomal gene expression, CP subunits are not limiting in lysates of ump1Δ cells (Ramos et al., 1998; Xie and Varshavsky, 2001).
(C) The dissociation of RP-CP assemblies by means of Ecm29-HA is coupled with the release of matured CP. To follow CP maturation, lysate of ecm29Δ ump1Δ cells expressing β5-HA instead of endogenous β5 was incubated for half an hour with lysate of ecm29Δ ump1Δ cells lacking Ecm29-HA (upper panel) and with lysate of ump1Δ cells containing Ecm29-HA (lower panel; the additional lysates contained no HA-tagged version of β5). The mixtures were subjected to native PAGE. To assign CP configurations, proteasome activity was visualized by in-gel assays using the chromogenic substrate Suc-Leu-Leu-Val-Tyr-AMC (Y) in the presence of 0.02% SDS. The native PAGE was subjected in second dimension to SDS-PAGE, blotted, and probed for β5 processing. Unprocessed (pro) β5, incompletely (i) processed β5, and matured (m) β5 are marked. As additional control, see Figure S4.
(D) Overexpression of HA-tagged Ecm29 behind the inducible GAL1 promoter. Cell extracts were probed for HA-tagged Ecm29 and Kar2 by immunoblotting (upper panel). Growth of cells expressing HA-tagged Ecm29 behind the inducible GAL1 promoter was monitored on plates containing complete medium with either glucose or galactose (lower panel). If Ecm29 dissociates regular RP-CP, the overexpression would have been lethal.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2010 Elsevier Inc. Terms and Conditions

8. Figure 6
Reconstitution of CP Maturation with Purified Ecm29-RP-CP-RP/Ecm29-RP-CP-Blm10 and Recombinant β3-HIS6
(A) Recombinant β3-HIS6 was expressed in E. coli, purified, subjected to SDS-PAGE, and stained with Coomassie blue, as shown in the separate lane below. Ecm29-RP-CP-RP and Ecm29-RP-CP-Blm10 were purified from ump1Δ cells expressing Ecm29-HA and GFP-labeled CP, as shown before (Figure 1C). The mixture of Ecm29-RP-CP-RP and Ecm29-RP-CP-Blm10 was incubated with recombinant β3-HIS6 (+ β3-HIS6) or mock treated (− β3-HIS6). The reactions were subjected to native PAGE. CP configurations were visualized by GFP imaging, and the major bands were assigned with numbers (upper left panel): Ecm29-RP-CP-RP (band 1), Ecm29-RP-CP-Blm10 (band 2), and CP (band 3). The native PAGE was blotted and probed for β3 using anti-β3 antibodies (upper right panel). Peptide hydrolyzing activities were visualized by in-gel activity assays using substrate Y without SDS (lower left panel) and with SDS (lower right panel).
(B) To follow CP maturation, the bands corresponding to Ecm29-RP-CP-RP (lane 1), Ecm29-RP-CP-Blm10 (lane 2), and CP (lane 3) were excised and subjected to SDS-PAGE, followed by immunoblotting against β5-GFPS, α4, and β3-HIS6 by using antibodies raised against GFP, α4, and HIS6, respectively. The detection of α4 served as loading control.
Molecular Cell  , DOI: ( /j.molcel )
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9. Figure 7 Model of Ecm29 Function
(A) Ecm29 is primarily nuclear, suggesting that quality control of RP-CP assembly mainly takes place in the nucleus of proliferating yeast cells. Proliferating ump1Δ cells expressing GFP-labeled Ecm29 were monitored by direct fluorescence microscopy using the GFP filter (left panel) and Nomarski optics (right panel).
(B) Ecm29 is a scaffold protein that controls the integrity of RP-CP assemblies, as shown here for RP-CP-RP lacking β3. The RP is colored gray, the CP α ring purple, and the CP β ring blue. Ecm29 as marked by a red solenoid recognizes RP-CP-RP in which CP maturation is stalled due to the lack of β3 subunits. Remnants of β5 propeptides are determinants of stalled CP maturation and marked by dotted ellipses in red. As soon as β3 subunits are incorporated, remnants of β5 propeptides are cleaved off, and the active sites are liberated. Ecm29 is degraded by the nascent RP-CP-RP. This results in the destabilization of RP-CP interaction. The idle catalytic chamber of the CP is closed, as marked by filled ellipses in purple, and the CP is released as latent enzyme.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2010 Elsevier Inc. Terms and Conditions