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• W2126391194 abstract "Article24 July 2008free access Dissecting β-ring assembly pathway of the mammalian 20S proteasome Yuko Hirano Yuko Hirano Laboratory of Frontier Science, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan Search for more papers by this author Takeumi Kaneko Takeumi Kaneko Laboratory of Protein Metabolism, Department of Integrated Biology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Kenta Okamoto Kenta Okamoto Department of Structural Biology and Biomolecular Engineering, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan Search for more papers by this author Minghui Bai Minghui Bai Laboratory of Protein Metabolism, Department of Integrated Biology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Hideki Yashiroda Hideki Yashiroda Laboratory of Protein Metabolism, Department of Integrated Biology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Kaori Furuyama Kaori Furuyama Laboratory of Protein Metabolism, Department of Integrated Biology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Koichi Kato Koichi Kato Department of Structural Biology and Biomolecular Engineering, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan Division of Biomolecular Functions, Department of Life and Coordination-Complex Molecular Science, Institute for Molecular Science, National Institutes of Natural Sciences, Okazaki, Japan Search for more papers by this author Keiji Tanaka Keiji Tanaka Laboratory of Frontier Science, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan Search for more papers by this author Shigeo Murata Corresponding Author Shigeo Murata Laboratory of Protein Metabolism, Department of Integrated Biology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Yuko Hirano Yuko Hirano Laboratory of Frontier Science, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan Search for more papers by this author Takeumi Kaneko Takeumi Kaneko Laboratory of Protein Metabolism, Department of Integrated Biology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Kenta Okamoto Kenta Okamoto Department of Structural Biology and Biomolecular Engineering, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan Search for more papers by this author Minghui Bai Minghui Bai Laboratory of Protein Metabolism, Department of Integrated Biology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Hideki Yashiroda Hideki Yashiroda Laboratory of Protein Metabolism, Department of Integrated Biology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Kaori Furuyama Kaori Furuyama Laboratory of Protein Metabolism, Department of Integrated Biology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Koichi Kato Koichi Kato Department of Structural Biology and Biomolecular Engineering, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan Division of Biomolecular Functions, Department of Life and Coordination-Complex Molecular Science, Institute for Molecular Science, National Institutes of Natural Sciences, Okazaki, Japan Search for more papers by this author Keiji Tanaka Keiji Tanaka Laboratory of Frontier Science, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan Search for more papers by this author Shigeo Murata Corresponding Author Shigeo Murata Laboratory of Protein Metabolism, Department of Integrated Biology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Author Information Yuko Hirano1, Takeumi Kaneko2, Kenta Okamoto3, Minghui Bai2, Hideki Yashiroda2, Kaori Furuyama2, Koichi Kato3,4, Keiji Tanaka1 and Shigeo Murata 2 1Laboratory of Frontier Science, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan 2Laboratory of Protein Metabolism, Department of Integrated Biology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan 3Department of Structural Biology and Biomolecular Engineering, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan 4Division of Biomolecular Functions, Department of Life and Coordination-Complex Molecular Science, Institute for Molecular Science, National Institutes of Natural Sciences, Okazaki, Japan *Corresponding author. Laboratory of Protein Metabolism, Department of Integrated Biology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Tel.: +81 3 5841 4803; Fax: +81 3 5841 4805; E-mail: [email protected] The EMBO Journal (2008)27:2204-2213https://doi.org/10.1038/emboj.2008.148 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The 20S proteasome is the catalytic core of the 26S proteasome. It comprises four stacked rings of seven subunits each, α1–7β1–7β1–7α1–7. Recent studies indicated that proteasome-specific chaperones and β-subunit appendages assist in the formation of α-rings and dimerization of half-proteasomes, but the process involved in the assembly of β-rings is poorly understood. Here, we clarify the mechanism of β-ring formation on α-rings by characterizing assembly intermediates accumulated in cells depleted of each β-subunit. Starting from β2, incorporation of β-subunits occurs in an orderly manner dependent on the propeptides of β2 and β5, and the C-terminal tail of β2. Unexpectedly, hUmp1, a chaperone functioning at the final assembly step, is incorporated as early as β2 and is required for the structural integrity of early assembly intermediates. We propose a model in which β-ring formation is assisted by both intramolecular and extrinsic chaperones, whose roles are partially different between yeast and mammals. Introduction The ubiquitin–proteasome system is the main pathway for ATP-dependent non-lysosomal degradation of intracellular proteins in eukaryotes (Coux et al, 1996; Baumeister et al, 1998). Protein degradation achieved by this system is involved in various cellular processes, including cell cycle regulation, stress response, intracellular signalling, transcription regulation, and acquired immunity (Glickman and Ciechanover, 2002). Proteins involved in such regulatory processes as well as damaged proteins are recognized by the ubiquitin system and are marked for degradation by covalent attachment of polyubiquitin chains. Polyubiquitinated proteins are then recognized and degraded by the 26S proteasome, a 2.5-MDa multisubunit protease. The 26S proteasome is composed of a catalytic core particle, called the 20S proteasome, bound at one or both ends by a 19S regulatory particle. The 20S proteasome is a cylindrical structure comprised of 28 subunits arranged in four stacked seven-membered rings. Each ring contains seven different subunits, whereby the two outer rings are formed by non-catalytic α-type subunits, named α1–α7, and the two inner rings are formed by the β-type subunits, β1–β7, three of which are catalytic (β1, β2, and β5) (Baumeister et al, 1998). Each of the 14 different proteins occupies a defined position within the 20S proteasome (Groll et al, 1997; Unno et al, 2002). Vertebrates encode four additional catalytic β-subunits; three interferon-γ-inducible β1i, β2i, and β5i and one thymus-specific β5t, which are incorporated in place of their most closely related β-subunits, thus forming distinct subtypes of proteasomes with altered catalytic activities called immunoproteasome and thymoproteasome (Tanaka and Kasahara, 1998; Murata et al, 2007). The integrity of the 20S proteasome is assured by correct assembly of the 14 α-subunits and 14 β-subunits. All of the active β-subunits as well as non-catalytic β6 and β7 are synthesized with N-terminal propeptides, which are removed autocatalytically at the final step of the assembly to expose the catalytic threonine residues of β1, β2, and β5. The N-terminal active sites of β-subunits are on the inner surface of the β-rings, whereas the C termini of β-subunits are on the outer surface of the 20S proteasome (Groll et al, 1997). It has been shown that efficient assembly of the 20S proteasome is orchestrated by proteasome-specific chaperones such as PAC1 (Pba1 or POC1 in yeast), PAC2 (Pba2 or POC2 in yeast), PAC3 (Pba3, Dmp2, or POC3 in yeast), PAC4 (Pba4, Dmp1, or POC4 in yeast), hUmp1 (also known as POMP, proteassemblin in mammals and as Ump1 in yeast), the N-terminal propeptides of β-subunits, and C-terminal tails of β-subunits, which provide specific subunit interactions with cis- and trans-β-rings (Heinemeyer et al, 2004; Ramos et al, 2004; Hirano et al, 2005, 2006; Murata, 2006; Le Tallec et al, 2007; Li et al, 2007; Kusmierczyk et al, 2008; Yashiroda et al, 2008). Proteasome assembly proceeds through distinct assembly intermediates. The earliest intermediate observed in mammalian cells is an α-ring that is comprised of all seven α-subunits, a PAC1–PAC2 heterodimer, and a PAC3–PAC4 complex (Hirano et al, 2005, 2006; Le Tallec et al, 2007). Both PAC1–PAC2 and PAC3–PAC4 are involved in the formation of α-rings. Recently, Pba3–Pba4 or Dmp1–Dmp2, yeast orthologues of PAC3–PAC4, has shown to catalyse correct subunit orientation of an α-ring (Kusmierczyk et al, 2008; Yashiroda et al, 2008). PAC1–PAC2 prevents non-productive dimerization of α-rings. The α-ring serves as a scaffold for the assembly of β-subunits. Another intermediate is the 13S complex composed of one α-ring, unprocessed β2, β3, β4, and (h)Ump1, both in yeast and mammals (Frentzel et al, 1994; Nandi et al, 1997; Li et al, 2007). Recent studies in yeast showed that the addition of the other β-subunits except β7 form the subsequent intermediate referred to as a ‘half-mer’ precursor complex (Li et al, 2007; Marques et al, 2007). The 16S complex containing all the subunits and hUmp1 has been described also in mammalian cells (Schmidtke et al, 1997; Witt et al, 2000). A ‘half-proteasome’ is often used as a general term for assembly intermediates containing unprocessed β-subunits and (h)Ump1. Studies in yeast have shown that dimerization of the half-mer is driven by the propeptide of β5 and the C-terminal tail of β7, whose incorporation into the half-proteasome is coupled with the dimerization, where the role of Ump1 is proposed to be an assembly checkpoint factor that inhibits the dimerization until a full set of β-subunits are recruited on the α-ring (Ramos et al, 2004; Li et al, 2007). Removal of β-propeptides and degradation of Ump1 coincide with completion of proteasome maturation, followed by degradation of PAC1–PAC2 (Ramos et al, 1998; Hirano et al, 2005). PAC3 is released from the intermediates during the maturation process (Hirano et al, 2006). Several studies in yeast reported that the propeptides and the C-terminal tails of certain β-subunits have important roles in proteasome biogenesis. The propeptide of β5 is crucial for the incorporation of β5 during proteasome formation and is thus essential for life (Chen and Hochstrasser, 1996). The propeptides of β1 and β2 are dispensable for cell viability but are known to protect the N-terminal catalytic threonine residue against Nα-acetylation. In addition, mutants lacking these two propeptides displayed modest defects in proteasome biogenesis (Arendt and Hochstrasser, 1999). The C-terminal tail of β2, which wraps around β3 within the same β-ring, is also essential for proteasome biogenesis in yeast (Groll et al, 1997; Ramos et al, 2004). The C-terminal tail of β7, which is inserted into a groove between β1 and β2 in the opposite ring, also has an important function in dimerization of half-proteasomes as well as stabilization of active conformation of β1 (Groll et al, 1997; Ramos et al, 2004). In mammals, analysis of propeptides has been mainly conducted in the context of immunoproteasome formation, but there is little or no information on the C-terminal tails of β2 and β7, whose location in the mammalian proteasome closely resembles those of yeast β2 and β7 in the yeast proteasome (Unno et al, 2002). Here, we describe a series of biochemical experiments employing RNA interference of each β-subunit, which resulted in the accumulation of distinct assembly intermediates. By characterizing these intermediates, we clarified the order of β-subunit incorporation on the α-ring. We also assessed the roles of propeptides and C-terminal tails of β-subunits in mammalian proteasome biogenesis, which revealed that these appendages mostly function in a manner similar to yeast counterparts but also displayed some phenotypes not observed in yeast. Furthermore, we identified a novel function of hUmp1 in stabilizing assembly intermediate of proteasomes that has not been appreciated in yeast. Results Ordered assembly of β-subunits on α-ring During the assembly pathway from the α-ring through the half-proteasome, each β-subunit assembles on the α-ring. To clarify the order of incorporation of β-subunits, we used the strategy of small interfering RNA (siRNA)-mediated knockdown of each β-subunit, which was expected to result in arrest of the assembly process before the incorporation of the targeted subunit and accumulation of a specific intermediate. The total level of the different subunits as well as proteasome activity assessed by the peptide-hydrolysing activity of HEK293T cells transfected with siRNA targeting each β-subunit or hUmp1 was markedly reduced compared with those of control cells, suggesting that the biogenesis of proteasomes is severely impaired in each knockdown cell (Supplementary Figure S1). Each cell extract was resolved by native PAGE, followed by active staining or immunoblot analyses for α6- and all β-subunits (Figure 1). Immunoblot for α6 revealed accumulation of different complexes (molecular weight, 232–669 kDa) in each knockdown cell, as well as the normal α-ring in control cells, which have been shown to be a distinct assembly intermediate comprising all the seven α-subunits, PAC1–PAC2, and PAC3 (Hirano et al, 2006) (Figure 1A). Besides the major, fast-migrating band, a more slowly migrating minor band was observed for each knockdown cell except β2, β3, and hUmp1 knockdown (Figure 1A). Both the major and minor species did not show any peptide-hydrolysing activity, which was observed only in the complex of approximately 700-kDa, that is, 20S proteasomes (Figure 1B), indicating that they are assembly intermediates of 20S proteasomes. Figure 1.Accumulation of distinct assembly intermediates in each β-subunit knockdown cells. The cell extracts (40 μg) used in Supplementary Figure S1 were separated by native PAGE. Assembly intermediates were detected by immunoblotting using the indicated antibodies (A, C–I) The bands corresponding to α-ring and the 20S proteasome as well as the locations of molecular size markers are depicted by arrowheads. (B) The peptide-hydrolysing activity was assayed by active staining of the gel using Suc-LLVY-MCA in the presence of SDS. Note that the 26S proteasome did not move inside the native PAGE gel. Download figure Download PowerPoint Among the seven β-subunits, β2 was the first assembled on the α-ring based on the finding that β2 was detected in all the intermediates except for that in its own knockdown (Figure 1D) and the intermediate that accumulated in β2-knockdown cells did not contain any β-subunit (Figure 1C–I, lanes for β2 RNAi). The assembly of β3 followed that of β2; β3 was detected in the intermediates observed in β1-, β4-, β5-, β6-, and β7-knockdown cells, and thus the incorporation of β3 should precede these subunits (Figure 1E). This view was further confirmed by the fact that the intermediate in β3-knockdown cells contained only β2 among the β-subunits (Figure 1C–I, lanes for β3 RNAi). β3 assembly was followed by β4 incorporation, which would result in the formation of the 13S complex, comprising the α-ring plus β2, β3, and β4, as suggested by the presence of β4 in the intermediate in β1, β5, β6, and β7 knockdown (Figure 1F), consistent with the previous reports that identified the 13S complex as a distinct entity of proteasome precursors (Frentzel et al, 1994; Nandi et al, 1997; Li et al, 2007). β5 was the next β-subunit incorporated into the 13S complex because β5 was detected only in the intermediates in β1-, β6-, and β7-knockdown cells (Figure 1G). The assembly of β6 followed that of β5, as evidenced by the presence of β6 in the intermediates of β1 and β7 knockdown (Figure 1H). β7 was likely the last β-subunit incorporated in the precursor proteasomes because β7 was not found in any of the intermediate complexes (Figure 1I) and because the intermediate observed in β7-knockdown cells contained all the β-subunits with the exception of β7 (Figure 1C–I, lanes for β7 RNAi). The behaviour of β1 was rather elusive. The intermediate in β1-knockdown cells contained β2, β3, β4, β5, and β6 (Figure 1C–I, lanes for β1 RNAi), whereas β1 was already included in the intermediates of β4, β5, β6, and β7 knockdown (Figure 1C). The former observation suggests that β1 was incorporated following β2, β3, β4, β5, and β6, and that β1 is required for β7 incorporation. The latter observation suggests that the presence of β2 and β3 is sufficient for the incorporation of β1 and that β1 can be incorporated anytime during the maturation pathway from the complex containing both β2 and β3 through the half-mer. Association of PA28, Hsp90, and Hsc70 with 20S proteasome precursors When the same panel was probed for PAC1, the major assembly intermediate bands were associated with PAC1 (Figure 2A), which has been shown to be retained in the proteasome precursor until the completion of the assembly (Hirano et al, 2005). However, the slowly migrating minor bands above the major bands did not contain PAC1, whereas the composition of each major and minor bands in terms of α- and β-subunits is identical (Figures 1 and 2A). It is also curious that the intermediate in β2-knockdown cells was apparently larger than the α-ring (Figure 1A), although the subunit composition is supposed to be identical to that of the α-ring. To address the identity of these bands, we tested whether PA28, PA200, Hsp90α, and Hsc70, which have been reported to be involved in proteasome biogenesis (Schmidtke et al, 1997; Preckel et al, 1999; Fehlker et al, 2003; Imai et al, 2003; Marques et al, 2007), associate with the intermediates. Figure 2.Release of PAC3 is coupled with β3 incorporation. The same panels in Figure 1 were probed with anti-PAC1 (A) and -PAC3 (B) antibodies. The arrow indicates PAC3 species dissociated from proteasome precursors (B). Download figure Download PowerPoint PA28 was associated with the slow-migrating minor bands but not with the primary bands, different from PAC1 and Hsp90α, which were detected only in the major bands (Supplementary Figure S2A and B). Hsc70 was observed in both the major and the minor bands (Supplementary Figure 2C). Neither Hsp90α nor Hsc70 was detected in the α-ring. By contrast, PA200, whose yeast orthologue Blm10 was shown to associate with nascent proteasomes (Fehlker et al, 2003; Marques et al, 2007), was not observed in the intermediates, whereas its association with 20S proteasomes was detected (Supplementary Figure S2D, arrowhead). However, we cannot conclude that PA200 is not bound to assembly intermediates as free forms of PA200, which probably dissociated from 20S or nascent proteasomes during native PAGE analysis, were found (Supplementary Figure S2D, arrow). The association of Hsp90 and Hsc70 with the assembly intermediates accounts for the increased size of the intermediate in β2-knockdown cells and suggests that recruitment of these chaperones precedes β2 and hUmp1 incorporation. The minor bands are characterized by the association of PA28, a 200-kDa heterohexameric complex. At present, we do not know whether these molecules really have some functions in the proteasome biogenesis or are associated with the intermediates as an experimental artefact. Further studies are needed to answer this question. Release of PAC3 upon incorporation of β3 We previously showed that precursor proteasomes purified with tagged hUmp1 did not contain PAC3 and demonstrated that PAC3 is released during the maturation pathway of the mammalian proteasome (Hirano et al, 2006). To elucidate the step where PAC3 was released, we took advantage of the knockdown experiments in which distinct assembly intermediates accumulated depending on which β-subunit was targeted (Figure 1). The same panel in Figure 1 was probed with anti-PAC1 and -PAC3 antibody. All the assembly intermediates as well as the α-ring were associated with PAC1 (Figure 2A) (Hirano et al, 2005). PAC3 is also associated with the α-ring in control cells as reported previously (Hirano et al, 2006). However, PAC3 was associated with intermediates of β2-, β3-, and hUmp1-knockdown cells but not with those of others, where PAC3 was found as fast migrating species, presumably as a free complex (Figure 2B, arrow). Considering the order of incorporation of β-subunits shown in Figure 1, the release of PAC3 is apparently coupled with the incorporation of β3. A new role of hUmp1 in the assembly pathway One intriguing difference in the phenotypes of loss of Ump1 orthologues between yeast and mammals is that knockdown of hUmp1 in mammalian cells did not result in the accumulation of intermediates containing unprocessed β-subunits (Figure 1, lanes for hUmp1 RNAi), whereas deletion of Ump1 in yeast caused apparent accumulation of such intermediates (Ramos et al, 1998). This observation in hUmp1-knockdown cells has also been shown in previous studies (Hirano et al, 2005, 2006). This finding raises the possibility that the role of Ump1 orthologues is different between yeast and mammals. To determine the step at which hUmp1 is incorporated, the same panel in Figure 1 was probed with anti-hUmp1 antibody. hUmp1 was included in a complex other than that in β2-knockdown cells (Figure 3A), indicating that the incorporation of hUmp1 precedes that of β3. On the other hand, the intermediate in the hUmp1 knockdown complex did not contain any of the β-subunits, including β2, a finding closely resembling that in β2-knockdown cells with regard to size and composition (Figures 1 and 2; compare lanes for β2 RNAi to lanes for hUmp1 RNAi). These results suggest that incorporations of β2 and hUmp1 are coupled with each other and that loss of either result in dissociation of the other. Figure 3.Role of hUmp1 in the structural integrity of early assembly intermediates. (A) The same panel in Figure 1 was probed with anti-hUmp1 antibody. (B–E) Extracts of HEK293T cells transfected with the indicated combinations of siRNAs were separated by native PAGE. Intermediate complexes were detected by immunoblotting using the indicated antibodies. The left lane representing β7 RNAi serves as a positive control for immunoblotting (C, D). Asterisks indicate nonspecific bands (A). (F) Flag–hUmp1 and each 20S subunit were co-translated and radio-labelled in reticulocyte lysates, immunoprecipitated with M2 agarose, and analysed by SDS–PAGE and autoradiography. ‘ALL' represents co-translation of all β-subunits together with hUmp1. Download figure Download PowerPoint To confirm this concept, hUmp1 was knocked down concurrently with β2, β3, or β4, and the resultant assembly intermediates were compared with those in β2, β3, or β4 single-knockdown cells. Intriguingly, the size of intermediates observed in the simultaneous knockdown of hUmp1 with β3 or β4 was similar to that of the complex in β2 single-knockdown cells (Figure 3B). Notably, the intermediate found in β3–hUmp1 double-knockdown cells lacked β2 (Figure 3C), which was found in the complex of β3 single-knockdown cells (Figure 1D). The β4 and hUmp1 double knockdown was associated with loss of both β2 and β3 in the intermediate (Figure 3C and D), which were clearly detected in the β4 single knockdown complex (Figure 1D and E). Furthermore, the complexes manifested in the double knockdown cells were associated with PAC3, consistent with the absence of β3, whose incorporation would detach PAC3 from the precursor proteasome (Figures 2B and 3E). To gain mechanistic insight into the early function of hUmp1, we tested the interactions between hUmp1 and each 20S proteasome subunit. hUmp1 could directly bind to β2 and β3 as well as some of the α-subunits (α2, α3, α5, and α7) (Figure 3F). This observation raises the possibility that hUmp1, either alone or as a complex with β2 and β3, is recruited on the α-ring through direct interaction between hUmp1 and certain α-subunits. Taken together, these results demonstrate that β2 is unable to associate with the α-ring without hUmp1 and suggest the important role of hUmp1 in promoting the maturation process beyond the α-ring, either by stabilizing the complex or by recruiting β2. Propeptides of β1, β6, and β7 are dispensable for proteasome maturation It has been reported that the propeptides and C-terminal tails of β-subunits have important roles in proteasome biogenesis in yeast (Chen and Hochstrasser, 1996; Ramos et al, 2004; Li et al, 2007; Marques et al, 2007), but little is known about their roles in the maturation of proteasomes in mammals. To elucidate the role of propeptides of β1, β2, β5, β6, and β7, and the C-terminal tails of β2 and β7 in mammals, we first established cell lines stably transfected with constructs encoding wild-type subunits (β1*, β2*, β5*, β6*, and β7*), mature subunits whose propeptides were replaced with ubiquitin (β1*Δpro, β2*Δpro, β5*Δpro, β6*Δpro, and β7*Δpro), and β2 and β7 lacking their C-terminal tails (β2*Δtail and β7*Δtail) (Supplementary Figure S3A). Synonymous mutations were introduced into these constructs so that they were not sensitive to siRNAs against each β-subunit used in Figure 1. These constructs were attached with C-terminal Flag tag to distinguish the expressed proteins from endogenous proteins. We first confirmed the expression of both precursor and mature forms of β-subunits in HEK293T cells transfected with constructs encoding wild-type and Δtail β-subunits (Supplementary Figure S3B). On the other hand, as expected, only mature forms of β-subunits were detected in cells transfected with ubiquitin-fused Δpro constructs (Supplementary Figure S3B). These ubiquitin-fused proteins are known to be cleaved rapidly by cellular deubiquitinating enzymes to generate free ubiquitin and the mature moiety of the proteasome subunit, so that the exposure and integrity of the N-terminal residue are ensured (Chen and Hochstrasser, 1996; Arendt and Hochstrasser, 1999; Jager et al, 1999). siRNA-mediated knockdown of endogenous subunits in these cells allowed us to determine the precise roles of propeptides and C-terminal tails. Expression of constructs encoding each wild-type subunit (β1*, β2*, β5*, β6*, and β7*) restored production of 20S proteasomes and rescued cells from death caused by siRNA treatment (data not shown), verifying that exogenously expressed constructs worked appropriately (Figures 4A, 5A and 6A; Supplementary Figures S4 and S5). Among the Δpro constructs, cells expressing β1*Δpro, β6*Δpro, and β7*Δpro grew apparently normal and produced 20S proteasomes at an amount comparable to wild-type expressing cells (Supplementary Figures S4 and S5; Figure 6A). These findings indicate that the propeptides of β1, β6, and β7 are not prerequisite for proteasome maturation in mammals, similar to the results in yeast. On the other hand, cells expressing β2*Δpro, β5*Δpro, β2*Δtail, and β7*Δtail were non-viable in the absence of their endogenous counterparts (data not shown), suggesting the indispensable roles of these propeptides and C-terminal tails in proteasome biogenesis in mammals. Figure 4.Both the propeptide and C-terminal tail of β2 are indispensable for β3 incorporation. Stable cell lines expressing the indicated mutant β2-subunits were treated with the siRNA targeting endogenous β2. Intermediate complexes were detected by immunoblotting using the indicated antibodies following native PAGE (A–D). Intermediates observed in β2*ΔTail cells can be divided into two species; faster migrating ones (arrowheads) and slower migrating ones (vertical bars). The free complex of PAC3 is depicted by an arrow (D). Download figure Download PowerPoint Figure 5.β5 propeptide is required for β6 incorporation. Stable cell lines expressing the indicated mutant β5-subunits were treated with the siRNA(s) for endogenous β5 or β6 (A–D), or for the indicated combinations (E, F). Cell extracts were resolved by native PAGE, followed by immunoblot analysis for the indicated antibodies. Download figure Download PowerPoint Figure 6.C-terminal tail of β7 is essential for the incorporation of β7 and dimerization of half-mers. Stable cell lines expressing the indicated mutant β7-subunits were treated with siRNA targeting endogenous β7. Cell extracts were resolved by native PAGE, followed by immunoblot analysis using the indicated antibodies. The free form of β7*Δtail is" @default.
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• W2126391194 title "Dissecting β-ring assembly pathway of the mammalian 20S proteasome" @default.
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• W2126391194 doi "https://doi.org/10.1038/emboj.2008.148" @default.
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