S5b Participates in the Assembly Pathway of the 19S Regulatory Particle of the Proteasome

Molecular Cell

Article Hsm3/S5b Participates in the Assembly Pathway of the 19S Regulatory Particle of the Proteasome Benoıˆt Le Tallec,1 Marie-Be´ne´dicte Barrault,1 Raphae¨l Gue´rois,2,3 Thibault Carre´,1 and Anne Peyroche1,* 1Laboratoire

du Me´tabolisme de l’ADN et Re´ponses aux Ge´notoxiques, SBIGeM de Biologie Structurale et Radiobiologie, SB2SM CEA, iBiTecS, Gif-sur-Yvette, F-91191, France 3CNRS, URA 2096, Gif-sur-Yvette, F-91191, France *Correspondence: [email protected] DOI 10.1016/j.molcel.2009.01.010 2Laboratoire

SUMMARY

The 26S proteasome, the central enzyme of the ubiquitin-proteasome system, is comprised of the 20S catalytic core particle (CP) and the 19S regulatory particle (RP), itself composed of two subcomplexes, the base and the lid. 20S proteasome assembly is assisted by several chaperones. Integral subunits of the RP participate in its assembly, but no external factors have been identified so far. Here we characterize the yeast Hsm3 protein, which displays unique features regarding 19S assembly. Hsm3 associates with 19S subcomplexes via a carboxy-terminal domain of the Rpt1 base subunit but is missing in the final 26S proteasome. Moreover, Hsm3 is specifically required for the base subcomplex assembly. Finally, we identify the putative species-specific 19S subunit S5b as a functional homolog of the Hsm3 chaperone in mammals. These findings shed light on chaperone-assisted proteasome assembly in eukaryotes. INTRODUCTION The ubiquitin-proteasome system (UPS) is a major proteolytic system in the cytosol and nucleus of all eukaryotic cells (Voges et al., 1999). The 26S proteasome, the central enzyme of this pathway, regulates various essential cellular processes by degrading proteins in most cases conjugated to ubiquitin (Wolf and Hilt, 2004; Goldberg, 2007). The 26S proteasome comprises the catalytic core particle (CP or 20S particle) capped by one or two regulatory particles (RPs or 19S caps or PA700 in mammals), forming RP1CP and RP2CP complexes, respectively (Glickman et al., 1998b). CP proteasome has a barrel-shaped structure consisting of two inner heptameric rings of b type subunits and two outer heptameric rings of a type subunits (Groll et al., 1997). The proteolytic active sites, carried by three of the seven b subunits, are located in the central cavity of the proteasome barrel. These b subunits are synthesized as precursors, which upon assembly of the 20S particle undergo an autolytic process liberating their catalytic threonine residue (Wolf and Hilt, 2004). The entry of substrates into the

cavity of the 20S is restricted by a lattice-like structure formed by the N termini of the a subunits (Groll et al., 2000). The 19S RP activates the 20S by opening this gate (see Saeki and Tanaka, 2007, for review). In yeast, Blm10, a HEAT repeat protein, has been reported to attach directly to the a ring surface of the 20S and could act as an alternative activator, similarly to the homologous mammalian PA200 activator (Schmidt et al., 2005; Iwanczyk et al., 2006). The 19S RP, which functions in substrate recognition, deubiquitination, unfolding, and translocation through the channel of the 20S, provides the ATP and ubiquitin dependence on the CP (see Goldberg, 2007, for review). The 19S RP can be subdivided into two subcomplexes, namely the base and the lid (Glickman et al., 1998a). Rpn10 is thought to lie between the base and the lid and to stabilize this association (Glickman et al., 1998a). The crystal structure of the yeast 20S CP is known (Groll et al., 1997), but that of the 19S RP is not yet available. The base contains six homologous ATPases subunits of the AAA family (referred to as Rpt1-6 in yeast), plus two nonATPases, Rpn1 and Rpn2 (see Voges et al., 1999, for review). By analogy to proteasome-like ATPases complexes in prokaryotes, the 19S ATPases are presumed to assemble into a sixmembered ring that directly abuts the 20S particle (see Smith et al., 2006, for review). Each of the Rpt proteins is highly conserved evolutionarily, showing sequence identities around 70% between yeast and human orthologs, whereas Rpts are roughly 40% identical to each other within the same species (Glickman et al., 1998b). Each 19S ATPase appears to perform distinct functions in protein degradation (Rubin et al., 1998; Smith et al., 2007). Rpn1 and Rpn2, two non-ATPase subunits of the base, share about 20% of sequence identity. They contain Arm/HEAT repeats and are structurally related to each other, forming a-helical solenoids, but assume distinct functions (Glickman et al., 1998b; Kajava, 2002). Recent data have suggested that Rpn2, but not Rpn1, can attach directly to the a ring surface of the 20S CP (Rosenzweig et al., 2008). Finally, an additional 19S subunit, S5b, which interacts in vitro with a trimer containing Rpn1, Rpt2, and Rpt1, is supposed to be only present in mammals (Deveraux et al., 1995; Gorbea et al., 2000). The lid contains multiple non-ATPase subunits (Rpn3, Rpn5-9, Rpn11-12, Rpn15/Sem1), most of which are essential for viability in yeast, but their precise roles are still elusive except for Rpn11, which displays a deubiquitinating activity (Verma et al., 2002; Yao and Cohen, 2002).

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Molecular Cell Hsm3/S5b Controls 19S Base Assembly via Rpt1

Proteasome biogenesis is a highly orchestrated multistep event involving the biosynthesis of all subunits, their assembly, and maturation processes. Both specific intrinsic 20S subunits and proteasome-dedicated chaperones assist 20S-assembling steps (see Heinemeyer et al., 2004; Ramos and Dohmen, 2008, for reviews). Mutations in different base or lid subunits (Rpn2, Rpn5, Rpn6, Rpn9, Sem1) have been shown to induce defects in the assembly of the 19S subcomplexes (Takeuchi et al., 1999; Santamaria et al., 2003; Funakoshi et al., 2004; Isono et al., 2004, 2005, 2007). However, no external factors have been identified for the assembly of the 19S RP to date. In a previous study, we described a striking phenotype related to the DNA-damage response that allowed us to isolate two 20S proteasome-specialized chaperone pairs in budding yeast (Le Tallec et al., 2007). Here we characterize another suppressor isolated in the same screen, named HSM3. HSM3 is a poorly characterized gene, which might be involved in mutagenesis control (Fedorova et al., 2000; Merker et al., 2000). Our biochemical and genetic data indicate that Hsm3 is involved in 26S proteasome assembly. Hsm3 is mainly and strongly associated with the base subcomplex of the 19S RP and to a far lesser extent with the complete 19S RP. By contrast, Hsm3 is missing in the 26S proteasome. Moreover, in the absence of Hsm3, strong defects are specifically observed in the assembly of the base of the 19S. Hsm3 exerts its functions by directly binding a carboxy-terminal domain of the ATPase Rpt1. Finally, we provide evidence that the human putative species-specific 19S subunit S5b is conserved throughout evolution and is structurally and functionally related to the Hsm3 yeast chaperone. RESULTS HSM3 Is Genetically Linked to Proteasome Functions Searching for new regulatory elements of DNA damage response in yeast, we have previously uncovered two chaperone pairs (Poc1/2, alias Pba1/2 and Poc3/4) for 20S proteasome assembly (Le Tallec et al., 2007). Deletion of these genes suppresses the toxicity of a conditional dominant lethal allele of RAD53, encoding a hyperactive form of the DNA damage checkpoint Rad53 kinase, hereafter referred to as RAD53-DL (Le Tallec et al., 2007). Among RAD53-DL suppressors, we also identified SEM1, which encodes a component of the 19S RP lid subcomplex (Funakoshi et al., 2004; Krogan et al., 2004; Sone et al., 2004), and HSM3, a poorly characterized gene (Fedorova et al., 2000; Merker et al., 2000) (Figure 1A). In the presence of RAD53-DL, sem1D hsm3D strain did not grow better than the corresponding single mutants, indicating that these two genes belong to an epistatic group with respect to RAD53-DL suppression (Figure 1A). We have shown that several proteasome mutants displayed a hyperresistance toward the carcinogenic alkylating agent 4-nitroquinoline 1-oxide (4NQO) (Le Tallec et al., 2007) (Figure 1B). sem1D and hsm3D strains also exhibited this phenotype (Figure 1B). Since there was no additive resistance to 4NQO when hsm3D mutation was combined with poc1D (pba1D) (Figure 1C), HSM3 also belongs to the proteasome epistatic group for this phenotype. hsm3D cells grew normally at 30 C but exhibited a strong growth defect at 37 C in several genetic backgrounds

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 mg/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 rpn4D single mutants.

(Figure 1D and data not shown). HSM3 deletion caused synthetic lethality with a deletion of RPN4, which encodes a transcriptional regulator of proteasome genes (Xie and Varshavsky, 2001) (Figure 1E). These genetic data suggested a role for Hsm3 in proteasome functions even at 30 C. Hsm3 Associates with the Base Subcomplex of the 19S RP of the Proteasome We assayed for a physical interaction between Hsm3 and the proteasomal complexes. Tandem affinity purification (TAP) and

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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 mg of total proteins for Rpn1TAP and Rpn9-TAP and to 200 mg 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 b5 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.

Myc epitope-tagged Hsm3 strains displayed a wild-type phenotype in the presence of 4NQO and at 37 C, indicating that these proteins are functional (see Figure S1 available online). All five 19S ATPases subunits we tested, namely Rpt1, Rpt2, Rpt3, Rpt5, Rpt6, and the non-ATPase subunit Rpn2, efficiently coimmunoprecipitated with Hsm3-myc or Hsm3-TAP (Figures 2A and 2B), suggesting that Hsm3 can interact with some base subunits containing complexes. Moreover, Rpn1-TAP protein also effectively retained Hsm3-myc protein (Figures 2C and 2D), indicating that at least some of the Rpn1-containing complexes are associated with Hsm3. Note that immunoprecipitation experiments do not discriminate between direct or indirect interactions. On the other hand, neither Hsm3-myc nor Hsm3-TAP was able to retain any subunit of the CP (Figures 2A and 2B), and neither b5/Pre2 nor a1/Scl1 TAP-tagged 20S subunits coimmunoprecipitated Hsm3-myc (Figure 2C). We also analyzed the distribution of Hsm3 in relation to proteasome subunits using gel filtration chromatography. The majority of Hsm3-TAP protein was detected in elution peaks corresponding to high molecular size species (between 200 and 600 kDa)

(Figure 2E). The same results were obtained with the untagged version of Hsm3 (data not shown). Consistently with our immunoprecipitation experiments, Hsm3 was not significantly detected at positions of 26S proteasomes (fractions 3–7). Combined, these results strongly suggest that Hsm3 interacts with complexes containing some subunits of 19S RP but is excluded from the 26S proteasome formed upon the association of the 19S RP with the 20S CP. Whereas Hsm3-TAP was able to efficiently immunoprecipitate Rpt5 and Rpn2 base subunits, it barely retained myc-tagged Sem1, Rpn12, or Rpn11 lid subunits (Figure 2B and Figure S2). Consistently, lid TAP-tagged subunits Rpn5, Rpn6, Rpn9, and Rpn12 hardly retained Hsm3-myc (Figures 2C and 2D). Although faint, the signal corresponding to the interaction between lid subunits and Hsm3 is significant (Figure 2C and Figure S2). The preferential interaction of Hsm3 with base subunits compared to lid subunits was also observed with the nontagged version of Hsm3 (data not shown). Depending on experimental conditions, Rpn10 cofractionated with the base or the lid (Glickman et al., 1998a; Saeki et al., 2000). TAP-tagged Hsm3 barely

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Figure 3. The Absence of Hsm3 Impairs Proteasome Functions (A) Exponential cultures of wild-type (WT), hsm3D, or sem1D cells were shifted at 37 C for 6 hr. Resulting extracts were fractionated onto a Superose 6 column and analyzed by western blot with anti20S a/b subunits (upper panel) or tested for chymotrypsin-like activity (lower panel). (B) Wild-type (WT) or hsm3D strains were shifted for 6 hr at 37 C. Extracts equivalent to 100 mg 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 hsm3D (D) strains expressing base-tagged (Rpn1-TAP or Rpn10-myc) or lidtagged (Sem1-myc) subunits were cultured for 6 hr at 37 C. Extracts equivalent to 50 mg of protein were resolved on native PAGE followed by western blotting using anti-Myc antibodies (left panel) or anti-20S a/b subunits. Note that Rpn1TAP 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), sem1D, or hsm3D 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.

RP, which are devoid of Rpn10, and suggest that this association is weakened once the base associates with the lid.

immunoprecipitated Rpn10 subunit in comparison to Rpn1-TAP or Rpn9-TAP proteins (Figure 2B). This is reminiscent of what was observed between Hsm3 and lid subunits (Figure 2C and Figure S2). Increasing salt concentration leads to sequential dissociation of the different 26S proteasome subcomplexes (Leggett et al., 2002). At very high salt concentration (1 M NaCl), the lid and the base subcomplexes were almost completely dissociated (Figure 2D), but Hsm3-myc was still tightly associated with Rpn1 (Figure 2D). By contrast, Hsm3-myc interaction with Rpn6 could no longer be detected in these conditions (Figure 2D), suggesting that the weak or substoichiometric interaction observed between the lid and Hsm3 is mediated by the base subcomplex. In gel filtration assay, peak of Hsm3 elution is at around 500 kDa, and Hsm3 also eluted into lower complexes (Figure 2E). This is compatible with its association with the entire 19S base but also with early intermediates containing base subunits. Altogether, our data indicate that Hsm3 is primarily associated with base-like subcomplexes of the 19S

Hsm3 Participates in 26S Proteasome Assembly We asked whether the absence of Hsm3 had any functional impact on maturation and activity of the 26S proteasome. Since the deletion of HSM3 or SEM1 leads to growth impairment at 37 C (Figure 1D), we anticipated that proteasome defects in hsm3D or sem1D cells would be exacerbated at this temperature, possibly because these conditions correspond to an extra burden on proteasome demand. Like several mutants affected in either proteasome biogenesis or function, hsm3D strains accumulated large amounts of high molecular weight ubiquitin (Ub) conjugates at 37 C (Figure S3). We next investigated proteasome maturation defects in the absence of Hsm3. Size-fractionated extracts from wild-type and hsm3D cells shifted to 37 C for 6 hr were analyzed by immunoblotting with anti-core subunit antibodies (Figure 3A, upper panel). In hsm3D or sem1D extracts, a lesser amount of 20S subunits was detected in the heaviest fractions (one through six) that correspond to 26S proteasome species in wild-type background. Chymotrypsin-like activity in fractions one through seven was also significantly reduced in extracts from either hsm3D or sem1D cells compared to the wild-type (Figure 3A, lower panel). At the same time, more

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a/b-containing species accumulated in fractions seven through ten in the mutant cells compared to the wild-type (Figure 3A, upper panel), as already reported for sem1D cells (Funakoshi et al., 2004). These observations suggest that fewer 26S proteasomes are assembled and 20S species accumulate in the mutant cells. To confirm these observations, total cell extracts were subjected to nondenaturating gel analysis, which allowed us to resolve several proteasome-related species (Elsasser et al., 2005; Lehmann et al., 2008). Using native gel electrophoresis followed by in-gel peptidase activity staining, we observed that fewer RP-CP complexes were present in hsm3D mutant (Figure 3B). In the presence of low levels of SDS, we detected more free 20S proteasome in the absence of Hsm3 (Figure 3B). These results are consistent with the gel filtration analysis. The specific biochemical defects in hsm3D mutants were only detected at 37 C, but synthetic letality with rpn4D and hyperresistance toward 4NQO at 30 C point to a function of Hsm3 at normal growth temperature. In other experimental conditions (used for Figures 3C and 3D), 26S active species were detected in native gel electrophoresis as a single band that displayed hydrolytic activity after staining the gel in the presence of the LLVY-AMC fluorogenic substrate and that was revealed by both anti-20S subunits and RP subunits (Rpn1-TAP, Sem1-Myc, Rpt1/2/5) (Figures 3C and 3D). We also identified a RP-like complex, which was corevealed with anti-TAP-Rpn5/6/9/12, anti-Myc-Sem1, or Rpn10 antibodies and also with anti-Rpn1-TAP, Rpt1, Rpt2, and Rpt5 antibodies, but not with several anti-20S antibodies (Figures 3C and 3D and data not shown). Importantly, we never detected any catalytic activity in association with these species (Figure 3C). Strikingly, this RP-like complex almost completely disappeared in hsm3D cells shifted to 37 C (Figure 3C, lanes 2, 4, and 6), suggesting that in this context there is less free RP. This remarkable alteration was also observed in a rpt1 thermosensitive strain (referred to as cim5-1 [Ghislain et al., 1993]) and in a sem1D strain (Figure 3C), highlighting similarities between hsm3D cells and RP mutants. These observations suggest that the few 19S RPs assembled in such a context are trapped by the excess of 20S CP. The deletion of BLM10 partially suppressed the thermosensitivity of hsm3D cells at 37 C (Figure S4). Therefore, the presence of Blm10 is detrimental to hsm3D cells. Blm10, which associates with the a ring, might compete with the few competent RPs assembled in the absence of Hsm3. Hsm3 Is Specifically Required for 19S Base Subcomplex Assembly We next further examined the composition of 26S proteasome subcomplexes in hsm3D cells shifted at 37 C for 6 hr. The association between Rpn1-TAP and Rpn2, Rpt1, Rpt3, Rpt5, or Rpt6 was severely impaired in hsm3D cells, whereas its association with Rpt2 seemed less affected (Figures 4A and 4C). Remarkably, the impairment of these associations was not sensitive to high salt concentrations, indicating that assembly, rather than stability, is compromised in the absence of Hsm3 (Figure S5). We also observed that in the absence of Hsm3, some base subunits (Rpt2, Rpt6) were at least partially dissociated from Rpn2-myc, whereas association with Rpt1 appeared less impaired (Figure 4B). Moreover, Rpn1-TAP or Rpn2-myc could

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 hsm3D (D) 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 mg 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 hsm3D (D) strains expressing Rpn2-myc. (C) As in (A) except that sem1D strain was also used for comparison with hsm3D strain. (D) As in (A) except that Rpn12-myc protein was used to immunoprecipitate lid subunits containing complexes.

no longer efficiently immunoprecipitate the core subunits of 20S proteasome in hsm3D cells (Figures 4A–4C and Figure S5), and reciprocally, immunoprecipitation experiments using myc-tagged Pre9/a3 also showed that the association between 20S proteasome and base subunits was severely reduced (Figure S6). No significant impairment in the association between Rpn1 and other base subunits was detected in sem1D cells (Figure 4C). However, the absence of Sem1 affected the interactions within the lid subcomplex (Figure 4D and Figure S7A), as illustrated by a poor coimmunoprecipitation between Rpn12 and Rpn8 (Figure 4D), whereas the absence of Hsm3 did not (Figure 4D and Figures S7A and S7B). In summary, the absence of Hsm3 specifically induces severe defects in the structural integrity of the base of the 19S, but not of the lid, whereas the absence of Sem1 affects the association between lid subunits without major perturbations within the base. This is consistent with an independent assembly of the two subcomplexes as previously noticed (Isono et al., 2007). In addition, we observed that the growth defect of sem1D at 34 C was alleviated by the deletion of HSM3 (Figure S8). The absence of Hsm3

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might overcome a dead-end assembly pathway taking place in sem1D mutant, for example by reducing the formation of the potentially toxic base-20S intermediates. Such interactions were previously observed between chaperones of the 20S assembly (Li et al., 2007, and our unpublished data). Hsm3 Interacts with a C-Terminal Domain of Rpt1 We clearly established that Hsm3 is part of base-like subcomplexes; however, the previous experiments did not allow us to point out direct partners. To elucidate this point, we carried out an interactor hunt using a fragment library screening approach in the two-hybrid system to probe which part of the base binds Hsm3. We first verified the functionality of the Gal4DBD- Hsm3 fusion (Figure S9). Using this functional Gal4DBD-Hsm3 as a bait, we mainly isolated Rpt1-containing fragments from the genomic library. The shortest one corresponded to the 90 last amino acids of Rpt1 (Figures 5A and 5B). This interaction was also observed when bait and prey were inverted (Figure S10). Using Gal4-DBD-Rpt1[377-467] as a bait, we isolated different fragments of Hsm3 and established that a fusion protein lacking the 115 first amino acids of Hsm3 was competent for the interaction (Figure 5B). To confirm that this interaction was direct, we investigated an in vitro interaction using bacterially coexpressed full-length Hsm3 protein fused to His6-GST (rHsm3) and Rpt1[377-467] fused to MBP (rRpt1[377-467]). First, we observed that coexpression resulted in a dramatic increase in the levels of soluble rHsm3 and rRpt1[377-467] (Figure 5C, left panel). This is strongly indicative of a stable association between the two proteins. Indeed, recombinant rRpt1[377-467] protein copurified with rHsm3 protein on Nickel resin (data not shown), and reciprocally, rHsm3 protein coeluted with rRpt1[377-467] protein immobilized on amylose beads (Figure 5C, right panel). Moreover, both recombinant proteins appear to associate with a 1:1 ratio when purified successively on amylose resin and Ni2+ beads. Our results strongly support that Hsm3 primarily interacts with the base of the RP via the carboxy-terminal part of Rpt1. Rpt1 and Hsm3 Are Important In Vivo Partners To establish whether the interaction between Hsm3 and Rpt1 is important in vivo, we tested the effects of overexpressing each of these proteins in a context of deficiency for the other one. Overexpression of Rpt1 could compensate for the absence of Hsm3 as judged by the restoration of normal growth at 37 C and of wild-type sensitivity toward 4NQO (Figure 5D, middle panel). To evaluate the effect of Hsm3 overexpression on Rpt1-related functions in vivo, we used rpt1ts/cim5-1 mutant, which confers a thermosensitive growth defect, as previously described (Ghislain et al., 1993), but also a hyperresistance to 4NQO (Figure 5D, lower panel). This observation pointed out that hyperresistance toward 4NQO is a broad hallmark of the impairment of 26S proteasome function. Overexpressing Hsm3 could partially compensate for the growth defects observed at semirestrictive temperature (34 C) in the cim5-1(rpt1ts) mutant, even though no significant effect was seen at 37 C or in the presence of 4NQO (Figure 5D, lower panel). These observations are rather specific, since the overexpression of Rpt6 or Sem1 did not rescue the thermosensitivity of hsm3D cells, nor did the overexpression of HSM3 improve cim3-1 (rpt6ts) or sem1D growth at

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[377-467]). (B) Schematic representation of Rpt1 and Hsm3 proteins highlighting the domains of each protein sufficient for promoting the interaction in the twohybrid system. AAA indicates delimitations of AAA+ ATPase domain of Rpt1. (C) Interaction of recombinant Hsm3 and Rpt1[377-467]. (Left panel) Solubility of individually expressed or coexpressed recombinant His-GST-Hsm3 (rHsm3) or Rpt1[377-467]-MBP (rRpt1[377-467]) proteins produced in E. coli cells was analyzed by Coomassie blue staining after SDS-PAGE. Asterisks indicate rHsm3 and rRpt1[377-467]. (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 m) containing HSM3 or RPT1 or no gene () was introduced into congenic hsm3D, rpt1ts (cim5-1), or wild-type (WT) strains. Tenfold serial dilutions of the transformants were spotted onto selective plates. Plates were incubated for 3 days at indicated temperatures. Plates containing 4NQO (0.15 mg/mL) were incubated at 30 C.

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restrictive temperatures (data not shown). These results strongly suggest that the main function of Hsm3 is closely linked to Rpt1. Hsm3 Shows Structural and Functional Homologies with the Human S5b Arm/HEAT Repeat Protein Base ATPases subunits are strongly conserved throughout evolution (Glickman et al., 1998a). It is thus likely that homologs (at least functional homologs) of the potent base assembly factor Hsm3 exist in mammals. We thus applied an in-depth sequence search procedure to identify specific homologs for Hsm3 in higher eukaryotes (Becker et al., 2006; Le Tallec et al., 2007). The presence of Arm/HEAT repeats is predicted all along Hsm3 sequence. Although this fold superfamily is widespread, in-depth profile-profile analysis (Soding, 2005) identified the human S5b protein encoded by PSMD5 gene (Deveraux et al., 1995) as a remote homolog of Hsm3 (Figure S11 and Supplemental Experimental Procedures). Strikingly, S5b has been identified as a component of proteasomes prepared from red blood cells and found in association with Rpt1-Rpt2 or Rpt1-Rpt2Rpn1 when cotranslated in reticulocyte lysates along with these base subunits (Gorbea et al., 2000). However, specific homologs of S5b protein had not been clearly identified in other species, and S5b was not systematically recovered by others in mammalian 26S or 19S preparations. Thus it had been suggested that S5b could be a species-specific subunit of 19S proteasome. Our study offers the alternative explanation that S5b and Hsm3 are remote homologs. We observed that a myc-tagged S5b expressed from a pcDNA3 derivative plasmid in human HEK293T cells efficiently coimmunoprecipitated with several human RP subunits (Rpt1/2 and Rpn2/7), but not with 20S subunits (Figure 6A). Reciprocally, when a6 subunit was immunoprecipitated, several Rpt subunits were coimmunoprecipitated, whereas S5b-myc was not (Figure S12). This is consistent with S5b not being part of 26S proteasome but rather interacting specifically with the RP. Besides, S5b interacted with both yeast Rpt1[377-467] and the equivalent C-terminal fragment of human Rpt1 (hRpt1[344-434]) in the yeast two-hybrid system (Figure 6B). This observation is consistent with the inferred functional conservation of Hsm3 and S5b. From the global multiple sequence alignment (Figure S11), a structural model for S5b was built (Figure S13) showing a central patch that appeared specifically conserved with two invariant residues, R184 and D220 in S5b found in close proximity (corresponding to R195 and D230 in Hsm3, respectively). We thus hypothesized that Hsm3 R195 residue might be involved in the interaction with Rpt1. We mutagenized this residue from arginine to either alanine (R195A) or glutamate (R195E) in the yeast HSM3 gene and tested the ability of mutated proteins to interact with yRpt1[377-467] in the two-hybrid system. In both cases, mutation of R195 in Hsm3 protein impaired the association with Rpt1 for each reporter gene (Figure 6C). Substitution of arginine for glutamate almost completely abolished the two-hybrid interaction, whereas arginine-to-alanine substitution displayed more modest effects (Figure 6C). Western blot analysis revealed that the different hybrid proteins were expressed to similar levels under these experimental conditions (Figure 6C, bottom panel). High conservation and this result point to a critical role of this arginine residue in the interaction of Hsm3 with Rpt1.

Figure 6. Hsm3 and the S5b Mammalian Protein Are Related to Each Other (A) S5b coimmunoprecipitates with RP subunits. HEK293T cells were transfected with pcDNA3-33myc-S5b or with pcDNA3-33myc-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[377-467]) or the corresponding domain of human Rpt1 (hRpt1[344-433]) 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[377-467]. 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.

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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 (hsm3D), 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.

DISCUSSION Hsm3 Is a Chaperone of the 19S RP of the Proteasome Hsm3 harbors several general characteristics of proteasomerelated proteins. First, its absence is correlated with hyperresistance toward 4NQO, accumulation of poly-Ub conjugates, and synthetic growth defects when combined with rpn4D mutant. Second, Hsm3 is physically and tightly associated with the base subcomplex of the RP via its interaction with the carboxy-terminal part of the Rpt1 ATPase. Moreover, Hsm3 displays unique features related to proteasome functions: it is required for the integrity of the base subcomplex of the 19S particle but is absent from the 26S proteasome. Hence, Hsm3 should be considered as a bona fide 19S assembly chaperone as illustrated in Figure 7. Regulation of Base Assembly by Hsm3 One of the most striking effects of the absence of Hsm3 is its impact on the organization of the base subcomplex of the RP. We showed that in the absence of Hsm3, Rpn1 interacts almost no longer with Rpn2 and Rpt1, and far less efficiently with Rpt3/ 5/6, whereas its interaction with Rpt2 is less impaired. On the other hand, the interaction of Rpn2 with Rpt2, Rpt5, and Rpt6 is severely impaired, whereas its interaction with Rpt1 seems less affected. Hence, it appears that in the absence of Hsm3, aberrant base subcomplexes could be formed. These results indicate that Hsm3 is required for a well-matched association between Rpn1, Rpn2, and the whole ATPase ring and thus for assembly of the base. Since overexpression of Rpt1 almost suppresses all the growth defects due to the invalidation of HSM3, it is likely that the action of Hsm3 is in great part linked with the role of Rpt1 in RP assembly.

Our study shows that Hsm3 is mainly associated with a base subcomplex devoid of Rpn10. It suggests that Rpn10 joins the RP complex later than Rpn1, Rpn2, and the ATPases. This is consistent with a previous observation that described the formation of a base lacking Rpn10 in a rpn9D lid mutant (Takeuchi et al., 1999). Hence, Rpn10 can be biochemically related to the base but seems to correspond to a separate module for assembly. Hsm3 Is Conserved from Yeast to Mammals Since S5b was not identified in other species but was associated in a stoichiometric manner with several isolated RP base subunits (Gorbea et al., 2000), it has been proposed that S5b is a species-specific RP subunit. Our study demonstrates that a unique homologous protein can be found in a large spectrum of eukaryotic organisms, showing that S5b function is not limited to mammals. In this study, we provide evidence that S5b shares functional features with Hsm3. Our data indicate that S5b preferentially interacts with the RP not associated with the 20S complex. S5b has been shown to form a complex with Rpt1Rpt2 dimer and also with Rpn1-Rpt1-Rpt2 trimer (Gorbea et al., 2000). Indirect data have suggested that S5b could interact with the N-terminal domain of Rpt1. However, we show that S5b can interact with the carboxy-terminal domain of Rpt1 (both yRpt1 and hRpt1). Interactions between the C. elegans and the D. melanogaster Hsm3 homologs and the Rpt1 subunit have been detected in high-throughput interaction studies (Walhout et al., 2002; Giot et al., 2003; Li et al., 2004), strengthening the functional significance of the proposed sequence homology. Arm/HEAT Repeats in Protein Regulating Proteasome Structure and Function Hsm3/S5b are Arm/HEAT repeats containing proteins that form elongated a solenoid shape structures. Analysis of circular dichroism spectrum confirmed that most of the recombinant Hsm3 is in the a helix conformation (F. Ochsenbein and A.P.,

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unpublished data). Six lid subunits, Rpn1 and Rpn2 base subunits and several other proteasome-associated proteins such as Blm10/PA200, contain similar Arm/HEAT domains (Kajava, 2002; Kajava et al., 2004). Hence, this structural motif is common in proteasome-associated proteins. However, at least some of these proteins might use different modes and/or domains of interaction with their proteasomal targets. Rpn1 and Rpn2 have been shown to interact with several ATPases of the base. Rpn1 can associate with an Rpt1-Rpt2 dimer (Gorbea et al., 2000). It is unlikely that the chaperoning role of Hsm3 is through mimicking these interactions inside the base. First, we show that Hsm3 interacts with a complex containing both ATPase subunits and the non-ATPase subunits Rpn1/2. Second, S5b has been shown to form a tetramer along with the Rpn1Rpt1-Rpt2 trimer (Gorbea et al., 2000). Hsm3/S5b Interacts with the C-Terminal Domain of Rpt1 Individual 19S ATPase’s C termini have distinct functions, and some are involved in the complex formation with 20S and/or in gating (Smith et al., 2007). The loss of the penultimate tyrosine in the C-terminal tail of Rpt1 markedly interferes with 26S stability, since substitution into an alanine of this conserved tyrosine in Rpt1 prevents the purification of 26S particles by standard affinity purification methods (Smith et al., 2007). Hsm3 interacts directly with the carboxy-terminal domain of Rpt1. Truncation of the penultimate tyrosine of Rpt1 or its replacement by an alanine does not significantly perturb its interaction with Hsm3 in the two-hybrid system (our unpublished data). Hence, a lack of interaction between Hsm3 and Rpt1 is unlikely responsible for the assembly defects observed in the penultimate tyrosine mutant of Rpt1. Nevertheless, it does not preclude the attractive possibility that Hsm3 regulates the proteasome assembly by regulating the accessibility of the C-terminal tail of Rpt1. Mechanism of Hsm3 Dissociation from 19S Complexes Given the strong interaction of Hsm3 with the base compared to the substoichiometric interaction with the whole 19S complex, it is plausible that 19S assembly completion, i.e., lid association with the base, might be at least partially responsible for Hsm3 being released from the base. In lid assembly mutants, Hsm3 would be improperly released from the nascent RP. Our experimental lines fit with this hypothesis: we observed an accumulation of Hsm3 associated with the base in sem1D cells that are deficient for lid assembly (our unpublished data). Since a fraction of Hsm3 is still associated with the whole RP, it is possible that the dissociation of Hsm3 is under the control of an additional mechanism, perhaps coupled with the association with the 20S and/or the gate opening. EXPERIMENTAL PROCEDURES Standard Techniques Standard techniques, yeast strains, and plasmids used in this study are described in the Supplemental Experimental Procedures. Immunoprecipitation Experiments Cleared extracts were prepared from TAP-tagged strains in a mortar in the presence of liquid nitrogen as previously described (Le Tallec et al., 2007).

Routinely, 200 mg of total proteins (but 1 mg for Hsm3-TAP extracts) was incubated for 3 hr at 10 C in the presence of antiPan mouse Dynabeads (Dynal Invitrogen) first incubated in presence of 0.1% BSA and equilibrated in lysis buffer supplemented with 100 mM NaCl and 0.5% NP-40. After washing, the bound proteins were eluted with Laemmli sample buffer. Immunoblot analysis for the TAP tag was performed using PAP complex (Sigma-Aldrich). Other immunoprecipitation experiments were performed as described in Le Tallec et al. (2007). Gel Filtration Analyses Gel filtration analyses of yeast whole-cell extracts were performed as previously described (Le Tallec et al., 2007). In experiments designed to analyze mutant defects, wild-type and mutant cells were shifted at 37 C for 6 hr before being harvested. Native Polyacrylamide Gel Electrophoresis Fifty to one hundred and fifty micrograms of protein equivalent of total lysates (prepared as for gel filtration analyses) were loaded. For Figure 3B, lysates were subjected to electrophoresis onto native 3.5%–6.0% gradient polyacrylamide gels, and samples were run overnight at 45 mA at 4 C as described in Lehmann et al. (2008). For Figures 3C and 3D, native gel electrophoresis was carried out as described in Dohmen et al. (2005). Samples were run at 30 mA for 15 min and then at 12 mA for 16 hr at 4 C. Staining native gels for LLVYAMC hydrolysis activity was done in 50 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 1 mM ATP, 0.05 mM LLVY-AMC (Biomol) in the presence or absence of 0.02% SDS. Blotting was performed in the presence of 0.1% SDS as described in standard techniques (Supplemental Experimental Procedures). Assays for Proteolytic Activities Assays were carried out from CMY960, BLT117, and BLT118 cells shifted at 37 C for 6 hr. Chymotrypsin-like activity was measured as described in Le Tallec et al. (2007). Two-Hybrid Experiments The hunt procedure was carried out as described in Fromont-Racine et al. (1997). See the Supplemental Data for details. Purification of Recombinant Hsm3 and Rpt1 Proteins His-GST-HSM3 and/or Rpt1[377-467]-MBP (maltose-binding protein) were (co)expressed in E. coli using a derivative of pRSF-Duet-1 vector (Novagen). Sequences and details on constructions are available on demand. Wholecell extracts from transformed BL21 (DE3) cells were prepared as described in Le Tallec et al. (2007). For purification of MBP fusion proteins, soluble lysates were loaded onto amylose resin (New England Biolabs). After several washes with buffer C0 (Tris 50 mM [pH 7.5], 200 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.05% Tween-20), the bound proteins were eluted by addition of maltose (10 mM final). Ni-NTA magnetic agarose beads (QIAGEN) were added to fractions in buffer C (Tris 50 mM [pH 7.5], 300 mM NaCl, 0.05% Tween-20) in the presence of 10 mM imidazole. Beads were washed with buffer C plus 20 mM imidazole. Bound proteins were eluted in the same buffer adjusted to 250 mM imidazole. SUPPLEMENTAL DATA The Supplemental Data include 13 figures, 1 table, Supplemental Discussion, Supplemental Experimental Procedures, and Supplemental References and can be found with this article online at http://www.cell.com/molecular-cell/ supplemental/S1097-2765(09)00037-9. ACKNOWLEDGMENTS We thank Willy Aucher for providing us Y187 strain transformed with FRYL library; M. Rechsteiner and E. Rousseau for their gift of S5b and hRpt1 cDNAs, respectively; C. Mann for his gift of strains, anti-Cim3, and anti-Cim5 antibodies; J. Dohmen for his gift of proteasome tools; and F. Ochsenbein for circular dichroism spectroscopy experiments. We are grateful to

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M.-C. Marsolier-Kergoat for her continuous support, her critical reading of the manuscript, and helpful discussions. B.L.T. was supported by a grant from the Association pour la Recherche sur le Cancer. This work was financed by the CEA, by the Association pour la Recherche sur le Cancer, and by the Agence Nationale de la Recherche. Received: July 15, 2008 Revised: October 3, 2008 Accepted: January 9, 2009 Published: February 12, 2009 REFERENCES Becker, E., Meyer, V., Madaoui, H., and Guerois, R. (2006). Detection of a tandem BRCT in Nbs1 and Xrs2 with functional implications in the DNA damage response. Bioinformatics 22, 1289–1292. Deveraux, Q., Jensen, C., and Rechsteiner, M. (1995). Molecular cloning and expression of a 26 S protease subunit enriched in dileucine repeats. J. Biol. Chem. 270, 23726–23729. Dohmen, R.J., London, M.K., Glanemann, C., and Ramos, P.C. (2005). Assays for proteasome assembly and maturation. Methods Mol. Biol. 301, 243–254. Elsasser, S., Schmidt, M., and Finley, D. (2005). Characterization of the proteasome using native gel electrophoresis. Methods Enzymol. 398, 353–363.

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