Volume 27, Issue 4, Pages 660-674 (August 2007) 20S Proteasome Assembly Is Orchestrated by Two Distinct Pairs of Chaperones in Yeast and in Mammals  Benoît Le Tallec, Marie-Bénédicte Barrault, Régis Courbeyrette, Raphaël Guérois, Marie-Claude Marsolier-Kergoat, Anne Peyroche  Molecular Cell  Volume 27, Issue 4, Pages 660-674 (August 2007) DOI: 10.1016/j.molcel.2007.06.025 Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 1 UMP1, PRE9, POC1, POC2, POC3, and POC4 Belong to a Common Group Regarding the DNA Damage Response (A and B) Ten-fold serial dilutions of the indicated mutants in the RAD53-DL background were spotted onto YPD plates under permissive conditions (with doxycyclin [+ Dox]) or restrictive conditions (without doxycyclin [−Dox]). The wild-type strain (WT) was spotted as a reference. Plates were incubated at 30°C for 2 days unless indicated otherwise. (C) Ten-fold serial dilutions of the indicated mutants were spotted onto YPD plates (no drug), YPD containing either 4NQO (0.4 μg/mL) or MMS (0.025% [v/v]), and incubated for 3 days at 30°C unless indicated otherwise. (D) Early exponential phase cells were plated onto YPD plates with the indicated concentrations of 4NQO. After incubation for 3 days at 30°C, viable colonies were counted. Relevant genotypes of the different congenic strains are indicated on the right. Molecular Cell 2007 27, 660-674DOI: (10.1016/j.molcel.2007.06.025) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 2 POC Genes Are Genetically Linked to Proteasome Functions (A and C) Heterozygous double mutants were sporulated. The resulting tetrads were dissected and incubated at 30°C (A) or at the indicated temperatures (C) onto YPD plates. Circled are the colonies germinated from double mutant spores. (B and D) Ten-fold serial dilutions of the indicated strains were spotted onto YPD plates and incubated at the indicated temperatures for 2 days. Molecular Cell 2007 27, 660-674DOI: (10.1016/j.molcel.2007.06.025) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 3 Poc Proteins Are Components of 20S Proteasome Precursor Complexes (A) Lysates from strains expressing (+) or not (−) Poc3-HA and/or Pre9-myc were immunoprecipitated with either anti-HA or anti-Myc antibodies. Lysates (INPUT) and immunoprecipitates (IP) were analyzed by western blot as indicated on the left. (B) Anti-Myc immunoprecipitations were performed from strains expressing Poc-myc proteins (+) or not (−). Pellets (IP) were analyzed with anti-“core” subunits and anti-Myc antibodies. (C) Coimmunoprecipitation of precursor (p) or mature (m) forms of epitope-tagged β2/Pup1 was examined by western blot analysis with the appropriate antibodies. (Upper panel) Lysates (INPUT) from strains expressing Poc3-HA, Ump1-HA, or α3/Pre9-HA in combination with β2/Pup1-myc were subjected to anti-HA immunoprecipitation (IP). (Lower panel) Lysates (INPUT) from strains expressing Poc2-Myc or Poc3-Myc in combination with β2/Pup1-HA were subjected to anti-Myc immunoprecipitation (IP). (D) Crude extracts from strains expressing the indicated epitope-tagged versions of the proteins were fractionated by gel filtration on Superose 6. Relevant fractions were analyzed by western blot. Arrowheads indicate the positions of the peaks of marker proteins. Each experiment was carried out at least three times, and representative results are shown. Molecular Cell 2007 27, 660-674DOI: (10.1016/j.molcel.2007.06.025) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 4 Poc1/Poc2 and Poc3/Poc4 Define Two Levels in the CP Maturation Pathway and Form Two Pairs (A) Congenic deletion strains with the relevant genotypes indicated were tested for survival in the presence of various concentrations of 4NQO. See Figure 1C. (B) Lysates from strains expressing either Poc1-HA, Poc2-myc, or both (left panel) and Poc3-HA, Poc4-myc, or both (right panel) were immunoprecipitated with either anti-HA or anti-Myc antibodies. Lysates (INPUT) and immunoprecipitates (IP) were analyzed by western blot as indicated on the right. (C) Interaction of recombinant Poc3 and Poc4. (Left panel) Coexpression increases the solubility of Poc3 and Poc4 in E. coli cells. Solubility of individually expressed or coexpressed recombinant Poc3-Stag (rPoc3-Stag) or 6xHis-Poc4 (rHis-Poc4) was analyzed. After SDS-PAGE, whole-cell extracts (WCE) and soluble (S) proteins were stained with Coomassie blue. (Right panel) Copurification of rPoc3/rPoc4. Soluble fractions (S) were loaded on Ni-NTA beads. Bound proteins were eluted (E) by adding imidazole, and the different fractions were analyzed by western blot using anti-His or anti-Stag antibodies. Coexpression led to increased solubilization of each protein. To get this bias round, soluble extracts from coexpressing cells (lane 6, left panel) were diluted with wild-type E. coli soluble extracts. The resulting diluted extracts (lane 5) were used for loading on Ni-NTA beads. (D) Purified recombinant Poc3/Poc4 from (C) were loaded on a Superdex 75 column, and fractions were analyzed by western blot. Arrowheads indicate size markers. Molecular Cell 2007 27, 660-674DOI: (10.1016/j.molcel.2007.06.025) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 5 Poc3 and Poc4 Are Required for Efficient CP Proteasome Maturation (A) Levels of ubiquitin conjugates in crude extracts were analyzed by anti-polyubiquitin (Poly-Ub) western blot in congenic strains, as indicated. Immunoblotting with anti-60 kDa vacuolar ATPase (Vac. ATPase) was used as a loading control. (B) POC3, POC4, PRE9, or UMP1 was deleted in strains expressing β2/Pup1-myc (upper panel) or β5/Pre2-myc (lower panel). Whole-cell extracts of congenic strains with the relevant genotype indicated above each lane were immunoblotted with anti-Myc. Unprocessed precursors (p) and processed mature (m) forms of β subunits are indicated. The asterisk indicates processing intermediates of β5/Pre2-myc previously observed in ump1Δ cells (Ramos et al., 1998). (C) SDS-PAGE and anti-Myc immunoblot analyses of Superose 6 column fractions from extracts of wild-type and congenic poc3Δ or poc4Δ cells expressing β5/Pre2-myc. Asterisks indicate degradation products. Arrowheads indicate processing intermediates of β5/Pre2. IN, input. (D) Equal amounts of protein prepared from wild-type (WT), poc3Δ, and poc4Δ cells were loaded on Superose 6. Chymotrypsin-like activity was measured in resulting fractions. Molecular Cell 2007 27, 660-674DOI: (10.1016/j.molcel.2007.06.025) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 6 Steady-State Localization of Poc3 and Poc4 Is Ump1 Dependent (A) Size-fractionated extracts were analyzed by western blot using anti-HA antibodies. Poc3-HA profile was analyzed in wild-type and ump1Δ cells (upper panels). Ump1-HA profile was analyzed in wild-type and poc3Δ strains (lower panels). (B) Wild-type (UMP1) or mutant (ump1) strains expressing Poc3-myc (left panels) or Poc4-myc (right panels) were subjected to indirect immunofluorescence method using anti-Myc antibody. Nuclei were stained with DAPI. Molecular Cell 2007 27, 660-674DOI: (10.1016/j.molcel.2007.06.025) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 7 Mammalian Orthologs of Poc3 and Poc4 Interact with the 20S Proteasome and with Each Other (A) Extracts from HEK293T cells transfected with pcDNA3 (lane 1), pcDNA3-myc-mPAC3 (lane 2), or pcDNA3-myc-mPAC4 (lane 3) were immunoprecipitated with anti-Myc antibodies and analyzed by western blot using anti-Myc and anti-α proteasome subunits (α-sub) antibodies. Ig, immunoglobulin chains. (B) HEK293T cells were transfected with pcDNA3-3xFLAG-mPAC4 along with either pcDNA3 (lane 1) or pcDNA3-myc-mPAC3 (lane 2). Extracts (INPUT) were immunoprecipitated with anti-Myc antibodies. The pellet fractions were analyzed by western blot using anti-Myc or anti-FLAG M2 antibodies. (C) In mammalian cells, PAC4 stabilizes PAC3 and vice versa. (Left panel) HEK293T cells were transfected with pcDNA3-3xFLAG-mPAC3 and with a control siRNA or mPAC3 siRNAs (mPAC3-a and mPAC3-b) in the presence (+) or the absence (−) of pcDNA3-3xmyc-mPAC4. (Right panel) HEK293T cells were transfected with pcDNA3-3xFLAG-mPAC4 and with a control siRNA or PAC4 siRNAs (PAC4-a and PAC4-b) in the presence (+) or the absence (−) of pcDNA3-3xmyc-mPAC3. Blots were also probed with anti-α subunits antibodies (α-sub). (D) Functional impact of PAC3 and PAC4 RNAi-based gene silencing in HEK293T cells. (Left panel) PAC3 and PAC4 silencing impairs cell proliferation. Cells transfected twice with siRNA (20 mM final) at intervals of 48 hr were counted 96 hr after siRNA transfection. The bars showing the relative viability represent the mean calculated from duplicates of three independent experiments. Standard deviations are depicted above the bars. (Right panel) Relative amounts of α6 subunit in total extracts were estimated by western blot using anti-α6 antibody. GAPDH signal was used as a loading control. Molecular Cell 2007 27, 660-674DOI: (10.1016/j.molcel.2007.06.025) Copyright © 2007 Elsevier Inc. Terms and Conditions