1. Volume 33, Issue 3, Pages 389-399 (February 2009)
Hsm3/S5b Participates in the Assembly Pathway of the 19S Regulatory Particle of the Proteasome 
Benoît Le Tallec, Marie-Bénédicte Barrault, Raphaël Guérois, Thibault Carré, Anne Peyroche 
Molecular Cell 
Volume 33, Issue 3, Pages (February 2009)
DOI: /j.molcel
Copyright © 2009 Elsevier Inc. Terms and Conditions

2. Figure 1 HSM3 Is Genetically Linked to Proteasome Functions
(A) 10-fold serial dilutions of the indicated mutants in the RAD53-DL background (i.e., RAD53-GFP under the control of the tetracycline operator, which is repressed by doxycyclin) were spotted onto YPD plates under permissive conditions (with doxycyclin [+DOX]) or restrictive conditions (without doxycyclin [−DOX]). Plates were incubated at 30°C for 2 days.
(B) 10-fold serial dilutions of the indicated mutants were spotted onto YPD plates (no drug) or YPD containing 4NQO (0.3 μg/mL) and incubated for 2 days at 30°C.
(C) 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 strains are indicated on the right.
(D) Serial dilutions (10-fold) of the indicated strains were spotted onto YPD plates and incubated at the indicated temperatures for 2 days.
(E) The tetrads resulting from the sporulation of heterozygous double mutants were incubated at 30°C onto YPD plates. Circled are the colonies germinated from double mutant spores; squares indicate rpn4Δ single mutants.
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3. Figure 2 Hsm3 Mainly Interacts with a 19S Base-like Subcomplex
(A) Lysates from strains expressing or not expressing (−) Hsm3-myc or Rpn10-myc were immunoprecipitated with anti-Myc antibodies. Lysates (INPUT) and immunoprecipitates (IP myc) were analyzed by western blotting with antibodies indicated on the left. Arrowheads indicate 20S-core subunits. Asterisks correspond to nonspecific products including immunoglobulins.
(B) Lysates from strains expressing (or not [−]) Hsm3-TAP or Rpn1-TAP (base) or Rpn9-TAP (lid) were pulled down with IgG antibodies. Ten micrograms of lysates (INPUT) and immunoprecipitates (IP TAP) were analyzed by western blotting. IP equivalent to 25 μg of total proteins for Rpn1-TAP and Rpn9-TAP and to 200 μg of total proteins for Hsm3-TAP and control strain were analyzed.
(C) Lysates from strains expressing Hsm3-Myc combined with various TAP-tagged proteasome subunits were pulled down with IgG antibodies. The eluates were then analyzed by immunoblotting using PAP reagent to detect TAP proteins and anti-Myc antibodies. In the conditions we used, CP-RP association is mostly preserved.
(D) Lysates from strains expressing Hsm3-myc and Rpn1-TAP or Rpn6-TAP were immunoprecipitated with IgG antibodies in the presence of increased salt concentrations (100, 300, and 500 mM and 1M of NaCl). Immunoprecipitates were analyzed by western blotting.
(E) Crude extracts from strains expressing the indicated epitope-tagged proteins were fractionated by gel filtration on Superose 6. Relevant fractions were analyzed by western blot. Precursors (p) and mature (m) forms of β5 subunit are indicated. Arrowheads indicate the positions of the peaks of marker proteins. Each experiment was carried out at least three times, and representative results are shown.
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4. Figure 3 The Absence of Hsm3 Impairs Proteasome Functions
(A) Exponential cultures of wild-type (WT), hsm3Δ, or sem1Δ cells were shifted at 37°C for 6 hr. Resulting extracts were fractionated onto a Superose 6 column and analyzed by western blot with anti-20S α/β subunits (upper panel) or tested for chymotrypsin-like activity (lower panel).
(B) Wild-type (WT) or hsm3Δ strains were shifted for 6 hr at 37°C. Extracts equivalent to 100 μg were resolved on a native 3.5%–6.0% PAGE gel followed by an incubation in the presence of LLVY-AMC to measure peptidase activity of the different proteasome subcomplexes in the presence (+SDS) or absence (−SDS) of 0.02% SDS.
(C) Wild-type (WT) or hsm3Δ (Δ) strains expressing base-tagged (Rpn1-TAP or Rpn10-myc) or lid-tagged (Sem1-myc) subunits were cultured for 6 hr at 37°C. Extracts equivalent to 50 μg of protein were resolved on native PAGE followed by western blotting using anti-Myc antibodies (left panel) or anti-20S α/β subunits. Note that Rpn1-TAP proteins were also revealed by the anti-Myc western blot procedure. Arrows indicate various proteasome species. Gel was also stained for LLVY-AMC hydrolytic activity (peptidase assay; right panel).
(D) rpt1ts (cim5-1), sem1Δ, or hsm3Δ strains and wild-type strain (referred to as RPT1, SEM1, and HSM3) were cultured for 6 hr at 37°C. Extracts were analyzed as in (B). Arrows indicate various proteasome species.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
The Architecture of the Base of the 19S RP Is Severely Impaired in the Absence of Hsm3
(A) Extracts of wild-type (WT) or hsm3Δ (Δ) strains expressing Rpn1-TAP subunit cultured for 6 hr at 37°C were subjected to IgG pull-down assay. Ten micrograms of lysates (INPUT) and 25 μg of immunoprecipitates (IP TAP) were analyzed by western blotting with different antibodies as indicated on the right.
(B) As in (A) except that anti-myc immunoprecipitations were performed using extracts from wild-type (WT) or hsm3Δ (Δ) strains expressing Rpn2-myc.
(C) As in (A) except that sem1Δ strain was also used for comparison with hsm3Δ strain.
(D) As in (A) except that Rpn12-myc protein was used to immunoprecipitate lid subunits containing complexes.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5 Hsm3 Binds a C-Terminal Fragment of the Rpt1 AAA+ ATPase
(A) Diploids containing various combinations of Gal4DNA-binding domain (GAL4DBD) and Gal4-activating domain (Gal4AD) fusions as indicated were first tested for transcriptional activation of the HIS3 reporter gene onto plates containing 3-amino-triazol (3AT). Blue color formation in the presence of X-Gal indicating transcriptional activation of the second reporter gene LacZ was then monitored. The shortest Rpt1 fragment isolated corresponded to the last 90 amino acids of Rpt1 (Rpt1[ ]).
(B) Schematic representation of Rpt1 and Hsm3 proteins highlighting the domains of each protein sufficient for promoting the interaction in the two-hybrid system. AAA indicates delimitations of AAA+ ATPase domain of Rpt1.
(C) Interaction of recombinant Hsm3 and Rpt1[ ]. (Left panel) Solubility of individually expressed or coexpressed recombinant His-GST-Hsm3 (rHsm3) or Rpt1[ ]-MBP (rRpt1[ ]) proteins produced in E. coli cells was analyzed by Coomassie blue staining after SDS-PAGE. Asterisks indicate rHsm3 and rRpt1[ ]. (Right panel) Soluble fractions were loaded onto amylose resin. Bound proteins were eluted by adding maltose and analyzed by Coomassie blue staining.
(D) Multicopy vector (2 μ) containing HSM3 or RPT1 or no gene (−) was introduced into congenic hsm3Δ, rpt1ts (cim5-1), or wild-type (WT) strains. Ten-fold serial dilutions of the transformants were spotted onto selective plates. Plates were incubated for 3 days at indicated temperatures. Plates containing 4NQO (0.15 μg/mL) were incubated at 30°C.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6 Hsm3 and the S5b Mammalian Protein Are Related to Each Other
(A) S5b coimmunoprecipitates with RP subunits. HEK293T cells were transfected with pcDNA3-3×myc-S5b or with pcDNA3-3×myc-Gmh1 (encoding a nonproteasomal protein) as a control. Extracts (INPUT) were immunoprecipitated with anti-Myc antibodies (IP Myc) and analyzed by western blot using anti-Myc or antibodies raised against the proteins indicated on the left. Ig, immunoglobulins. Asterisk indicates a nonspecific crossreacting band.
(B) The last 90 aminoacids of yeast Rpt1 (yRpt1[ ]) or the corresponding domain of human Rpt1 (hRpt1[ ]) was fused to GAL4DBD, and S5b was fused to Gal4AD (Gal4AD-S5b+). Empty vectors were used as negative controls (GAL4DBD− and GAL4AD-S5b−). Diploids containing the various combinations of Gal4 fusions were plated to evaluate transcriptional activation of the HIS3 (3AT 30 mM) and of the LacZ (X-Gal) reporter genes.
(C) Wild-type (WT) or mutant versions (R195A or R195E) of Hsm3 fused to Gal4DBD or the empty vector (−) were transformed into Y187 strain. The transformants were mated with Y190 strain containing Gal4AD-Rpt1[ ]. Growth of diploids cells was tested in the presence of various concentrations of 3AT (0, 30, and 50 mM) to evaluate the HIS3 reporter activation. Blue color formation in the presence of X-Gal indicates a transcriptional activation of the LACZ reporter gene. (Lower panel) Total extracts obtained from equal amounts of the different diploid cultures were subjected to western blotting using anti-Gal4DBD antibodies. Asterisks indicate nonrelevant crossreacting proteins that serve as loading controls.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7 Model Illustrating the Role of Hsm3 in 19S RP Assembly
Hsm3 binds the carboxy-terminal part of Rpt1 ATPase and promotes base assembly. The Rpn1-Rpt2-Rpt1 trimer that has been shown to interact with S5b (the human homolog of Hsm3) is highlighted with black lines. In the absence of Hsm3 (hsm3Δ), base assembly is compromised: Rpn1 interaction with Rpn2 and Rpt1 appears to be severely altered as Rpn2 interaction with Rpt2 does (dashed blue lines), whereas Rpn1-Rpt2 and Rpn2-Rpt1 interactions are mostly maintained (continuous orange lines). Interactions between Rpt subunits have not been directly tested. Upon lid binding, most of Hsm3 would be released from the nascent RP. See text for details.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions