Advances in Biological Regulation xxx (2013) 1–8
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The mechanism for molecular assembly of the proteasome Kazutaka Sahara, Larissa Kogleck, Hideki Yashiroda, Shigeo Murata* Laboratory of Protein Metabolism, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan
a b s t r a c t In eukaryotic cells, the ubiquitin proteasome system plays important roles in diverse cellular processes. The 26S proteasome is a large enzyme complex that degrades ubiquitinated proteins. It consists of 33 different subunits that form two subcomplexes, the 20S core particle and the 19S regulatory particle. Recently, several chaperones dedicated to the accurate assembly of this protease complex have been identified, but the complete mechanism of the 26S proteasome assembly is still unclear. In this review, we summarize what is known about the assembly of proteasome to date and present our group’s recent findings on the role of the GET pathway in the assembly of the 26S proteasome, in addition to its role in mediating the insertion of tail-anchored (TA) proteins into the ER membrane. Ó 2013 Elsevier Ltd. All rights reserved.
Introduction The 26S proteasome is responsible for the degradation of the majority of regulatory proteins and aberrant proteins of the cytosol, nucleus, and organelles inside cells, thus playing a central role in various cellular processes such as cell cycle progression, DNA repair, apoptosis, signal transduction, immune response, development, and protein quality control (Tanaka, 2009). Degradation of proteins by the proteasome is usually mediated by covalent attachment of ubiquitin chains to lysine residues within a target protein.
* Corresponding author. Tel.: þ81 3 5841 4803; fax: þ81 3 5841 4805. E-mail address: [email protected] (S. Murata). 2212-4926/$ – see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jbior.2013.09.010
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The 26S proteasome is a large ATP-dependent protease complex that recognizes and degrades polyubiquitinated proteins selectively (Hershko and Ciechanover, 1998). Its highly specialized function requires a complex structure which consists of 33 different subunits. In this review, we focus on the mechanism of proteasome assembly, especially on the recent advances in this area of study. Topics Architecture of the proteasome The 26S proteasome is composed of two particles; the 20S core particle (CP) and the 19S regulatory particle (RP) (Murata et al., 2009) (Fig. 1). The RP is attached to one or both ends of the CP and is the first step in the degradation process of target proteins by recognizing and removing ubiquitin chains, unfolding substrate proteins, and feeding them into the interior cavity of the CP, where they are degraded. Both the CP and the RP are complexes made up of 14 and 19 different subunits, respectively. The CP is a barrel shaped enzyme complex formed by an axial stack of four heteroheptameric rings; two outer a-rings consisting of seven homologous a-subunits (a1-a7) and two inner b-rings consisting of seven homologous b-subunits (b1-b7). The two abutting b-rings form a catalytic cavity, in which the proteolytic active sites of the CP are exposed. Catalytic activity is conveyed by b1, b2, and b5, which have caspase-like, trypsin-like, and chymotrypsin-like activity, respectively. These three subunits cleave peptide bonds after particular amino acid residues in a substrate protein (Borissenko and Groll, 2007). The RP can be further divided into the base and the lid subcomplexes. The base is composed of six homologous AAA-ATPase subunits Rpt1–6 and three non-ATPase subunits, Rpn1, 2, and 13. The six ATPase subunits form a hexametric ring and serve to unfold and translocate the substrate proteins into the CP in an ATP dependent manner. Another role of the base is to open the center of the a-ring in the CP, which is usually in a closed state. This is exerted by the carboxyl terminal HbYX (hydrophobic, tyrosine, any) motifs in Rpt2 and Rpt5, each of which extends into a pocket formed by two adjacent asubunits (Gillette, et al., 2008; Smith et al., 2007; Rabl, et al., 2008). Rpn13 and another RP subunit Rpn10 directly bind ubiquitinated proteins and function as ubiquitin receptors. The lid is composed of
Fig. 1. The assembly pathway of the 26S proteasome. The 33 different subunits are incorporated into the 26S proteasome in a precise and highly specialized manner. The assembly of the CP and the base is regulated by proteasome-dedicated chaperones. In contrast, the specific chaperones for the lid assembly have not been identified. See main text for details.
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nine non-ATPase subunits, Rpn3, 5–9, 11,12, and 15/Sem1. The major function of the lid is to deubiquitinate the captured substrates, which is catalyzed by the metalloisopeptidase Rpn11 (Verma et al., 2002; Yao and Cohen, 2002). Some prokaryotic species also have proteasomes, but the subunit organization is much less complex. The a- and b-rings of prokaryotic CPs are a homoheptamer of a single type of a-subunit and bsubunit, respectively. The RP is a homohexameric ring of a single ATPase subunit. The assembly of the prokaryotic proteasome is therefore not a difficult task and can be reconstituted in Escherichia coli (Zühl et al., 1997; Zwickl et al., 1999). In contrast, the assembly of eukaryotic proteasomes requires high precision and accuracy, ensuring that every subunit occupies a particular, predetermined place within the newly synthesized proteasome. Recent studies have identified many chaperones dedicated to this highly complex proteasome assembly. Here, we summarize the current understanding of the assembly mechanism of the proteasome. 20S core particle assembly by dedicated assembly chaperones CP assembly is mediated by at least five conserved extrinsic chaperones PAC1–4 and UMP1/POMP in mammals and Pba1–4 and Ump1 in yeast (Hirano et al., 2005, 2006; Hoyt et al., 2008; Kusmierczyk et al., 2008; Le Tallec et al., 2007; Murata et al., 2009; Ramos et al., 1998; Yashiroda et al., 2008) (Fig. 1). The assembly of the CP starts with the a-ring formation supported by two heterodimeric chaperone complexes PAC1-PAC2 and PAC3-PAC4 in mammals and Pba1-Pba2 and Pba3-Pba4 in yeast. The precise mechanism of a-ring formation is unknown, but since knockdown of PAC3 and PAC4 causes marked reduction in completed a-ring structures in mammalian cells, it is suggested that PAC3-PAC4 is required for a-ring formation (Hirano et al., 2006). At the very least, the role of PAC1-PAC2 is to prevent dimerization of a-rings, which would cause stalling of CP biogenesis, since knockdown of PAC1 and PAC2 causes accumulation of a-ring dimers (Hirano et al., 2005). A structural analysis of the Pba1-Pba2CP complex revealed that Pba1 and Pba2 have HbYX motifs at their carboxyl termini and bind to the outer surface of the a-ring, which should prevent attachment of the RP to the a-ring prior to finishing CP assembly (Hirano et al., 2005; Stadtmueller et al., 2012). After the assembly of the a-ring is completed, b-subunits are incorporated onto the a-ring. This process is assisted both by propeptides and C-terminal tails of b-subunits as well as by the assembly chaperones. Each b-subunit is recruited onto the a-ring in a defined order of b2, b3, b4, b5, b6, b1, and b7, as revealed in knockdown experiments of each b-subunit in mammalian cells (Hirano et al., 2008). b2 is the first subunit to be assembled onto the a-ring. This b2 recruitment is dependent on another dedicated chaperone, Ump1. The dislocation of the PAC3-PAC4 complex from the a-ring occurs when b3 is incorporated. A structural analysis of the yeast Pba3-Pba4-a5 complex revealed steric hindrance between Pba3-Pba4 and b4, further confirming the release of PAC3-4/Pba3-4 early on during b-subunit assembly (Yashiroda et al., 2008). Half-CPs dimerize upon incorporation of b7 as the last subunit and form mature CPs, followed by the cleavage of b-subunit propeptides and the degradation of UMP1 and PAC1-PAC2 (Hirano et al., 2008; Li et al., 2007; Marques et al., 2007). Zygote-specific CP assembly chaperone During the maternal-to-zygotic transition (MZT) after fertilization, massive maternal proteins are degraded by the UPS. This process is important for the replacement of oogenic proteins by proteins newly synthesized from the zygotic genome. We recently identified a molecule named ZPAC for zygotespecific proteasome assembly chaperone, which is specifically expressed in mouse gonads (Shin et al., 2013). In zygotes, the expression of ZPAC as well as the proteasome activity is transiently increased at the MZT. ZPAC directly binds to Ump1 and is specifically associated with precursor forms of the CP. Knockdown of ZPAC in zygotes reduces the CP amount and causes accumulation of precursor CPs, resulting in early developmental arrest of embryos. The exact mechanism in which ZPAC helps CP assembly is unknown, but deletion of ZPAC results in a decrease of Ump1, and vice versa, suggesting a role of ZPAC in stabilizing Ump1 proteins. This finding demonstrates that a cell-type specific assembly strategy could occur and raises the possibility that other assembly chaperones working in a defined cell-type or only under particular conditions may exist. Please cite this article in press as: Sahara K, et al., The mechanism for molecular assembly of the proteasome, Advances in Biological Regulation (2013), http://dx.doi.org/10.1016/j.jbior.2013.09.010
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Base assembly is also mediated by dedicated chaperones The RP consists of the base and the lid subcomplexes. The base and the lid are assembled independently, and subsequently associate with each other to form the RP. The assembly of the base is facilitated by another set of dedicated chaperones called p28/gankyrin, p27, S5b, and Rpn14 in mammals and Nas6, Nas2, Hsm3, and Rpn14 in yeast (Funakoshi et al., 2009; Kaneko et al., 2009; La Tallec et al., 2009; Park et al., 2009; Roelofs et al., 2009; Saeki et al., 2009) (Fig. 1). In mammalian and yeast cells, the base subunits and these chaperones are assembled into three modules, the p28(Nas6)-Rpt3-Rpt6-Rpn14 module, the p27(Nas2)-Rpt4-Rpt5 module and the S5b(Hsm3)-Rpt1Rpt2-Rpn1 module. These three modules come together to form the heterohexameric ATPase ring plus Rpn1 complex, which then assembles with the Rpn2-Rpn13 complex. The base chaperones then dislocate from the subunits during the assembly. These base assembly chaperones seem to define the order of assembly of the three modules, at least in mammalian cells. They also play an important role in preventing premature association of the base with the CP by binding to the carboxyl termini of the Rpt subunits. In addition, two other molecules have been shown to be involved in the base assembly. Ubp6, a proteasome-associated deubiquitinating enzyme in yeast, is included in the Hsm3-Rpt1-Rpt2-Rpn1 module and removes polyubiquitinated proteins from the module by deubiquinating them, thus promoting assembly of the base complex (Sakata et al., 2011). Another molecule is the ADPribosyltransferase tankyrase in drosophila. Tankyrase mediates ADP-ribosylation of PI31, which has been shown to regulate proteasome activity by an unknown mechanism. ADP-ribosylation of PI31 increases PI31 affinity for p27 and S5b, and thereby sequesters p27 and S5b away from the base subunits, thus promoting RP assembly (Cho-Park and Steller, 2013). This finding might provide a link between proteasome activity and cellular metabolic status. Stepwise assembly of the lid occurs without dedicated chaperones Recent studies in yeast suggest that the lid assembly is also a stepwise process (Fig. 1). The lid assembly starts with the assembly of two modules: the Rpn5-6-8-9-11 module and the Rpn3-7-15 module. These two modules associate with each other, forming a lid subcomplex without Rpn12. Cryo-EM analyses revealed that the C-terminal helices of Rpn3, 5–9, and 11 form a helical bundle that directs the ordered self-assembly of the lid subcomplex (Estrin et al., 2013). Although a genetic analysis suggests that Hsp90 has a role in the lid assembly, any dedicated chaperones for the lid have not been identified so far. Together with the observation that the lid subcomplex is reconstituted by mixing purified recombinant lid subunits and Hsp90 (Lander et al., 2012), it is suggested that the lid assembly occurs without any dedicated chaperones (Fukunaga et al., 2010). Rpn12 is subsequently recruited to the newly formed, incomplete lid subcomplex, which then enables the completed lid subcomplex to join together with the base (Tomko and Hochstrasser, 2011). The TRC/GET pathway participates in CP assembly Recently we identified the components of the guided entry of tail-anchored proteins (GET) pathway as factors involved in CP assembly by genetic analysis using budding yeast (Akahane et al., 2013). The GET pathway is responsible for the insertion of most of tail-anchored (TA) proteins into the endoplasmic reticulum (ER) (Schuldiner et al, 2008; Stefanovic and Hedge, 2007). Its mammalian counterpart is called the transmembrane recognition complex (TRC) pathway. The TRC/GET pathway comprises ribosome-bound Bag6-TRC35-Ubl4A/Sgt2-Get4-Get5, cytosolic TRC40/Get3 homodimer, and an ER-localized receptor complex WRB-CAML/Get1-Get2. This pathway starts with the capture of a C-terminal hydrophobic stretch of a TA protein by the BAG6/Sgt2 complex at the ribosome. Subsequently, the TA proteins are transferred to TRC40/GET3 and inserted into the ER with the help of the WRB-CAML/Get1-Get2 complex (Hedge and Keenan, 2011; Vilardi et al., 2011; Yamamoto and Sakisaka, 2012) (Fig. 2). Deletion of either GET1, GET2, or GET3 results in synthetic growth defects when crossed with deletion of genes involved in proteasome assembly such as UMP1, NAS6, and HSM3 in yeast. In Please cite this article in press as: Sahara K, et al., The mechanism for molecular assembly of the proteasome, Advances in Biological Regulation (2013), http://dx.doi.org/10.1016/j.jbior.2013.09.010
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Fig. 2. The TRC pathway. The TRC pathway delivers TA proteins into the ER membrane in 3 steps. Firstly, translated TA proteins at the ribosomes are captured by pre-targeting factor (Bag6-TRC35-Ubl4A in mammals, and Sgt2-Get4-Get5 in yeast). Secondly, TA proteins are then transferred to the TRC40/Get3 homodimer and recruited onto the ER membrane. Lastly, the delivered TA proteins are inserted into the ER via the receptor complex (WRB-CAML in mammals, and Get1-Get2 in yeast).
mammalian cells, knockdown of either TRC40 (Get3 homolog) or Bag6 (the most upstream component of the TRC pathway) causes the accumulation of CP assembly intermediates representing inefficient bsubunit assembly on the a-ring. The notable finding is that the association of CP intermediates with the ER membrane was reduced compared to control cells. These observations are consistent with the previous report showing an association of Ump1 and CP intermediates with the ER (Fricke et al., 2007) and strongly suggest that CP assembly occurs more efficiently on the ER than in the cytosol. Considering that all the components of the TRC/GET pathway are involved in CP assembly and that there is no physical interaction between the components of the TRC/GET pathway and CP subunits/chaperones, it is likely that a particular TA protein that is delivered to the ER by the TRC/GET pathway recruits CP intermediates and supports the accurate assembly of the CP. The finding suggests a possible link between CP biogenesis and ER membrane integrity (Fig. 3). It is suggested that nearly 400 TA proteins are encoded in the human genome (Kalbfleisch et al, 2007), and further studies should be focused on the identification of the responsible TA protein that plays a role in CP assembly. Bag6 is required for base assembly Interestingly, Bag6-knockdown causes not only a defect in CP assembly but also in base assembly. Bag6 directly interacts with Rpt5 and promotes the incorporation of Rpt4 into the p27-Rpt5 complex, thus facilitating the formation of the p27-Rpt4-Rpt5 module, which is required for efficient base assembly (Fig. 3). Bag6 is a multi-functional molecule. One of its important roles is as a ribosome-associating chaperone that recognizes aggregation-prone substrates in mammalian cells and regulates protein triage, in which proteins undergo either productive folding or degradation by the UPS (Kawahara et al., 2013; Lee & Ye, 2013). Therefore, it could be possible that an excess expression of misfolded proteins in cells affects proteasome assembly by engaging Bag6 to a function as protein quality control and thus Please cite this article in press as: Sahara K, et al., The mechanism for molecular assembly of the proteasome, Advances in Biological Regulation (2013), http://dx.doi.org/10.1016/j.jbior.2013.09.010
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Fig. 3. Involvement of the TRC pathway and Bag6 in proteasome assembly. The TRC pathway may be involved in the formation of the 20S proteasome. TRC40 and Bag6 play an important role in this function. TA proteins, the substrate of this pathway, may be directly involved in this function. Bag6 also plays a role in the formation of the base complex through assisting the formation of the p27Rpt4-Rpt5 module.
reducing the amount of Bag6 available for proteasome assembly. The finding may provide a link between protein homeostasis and proteasome biogenesis and raise the possibility that age-dependent accumulation of misfolded proteins in cells affects proteasome assembly and might be one of the causes of age-dependent decline in proteasome activity (Gregersen et al., 2006; Tonoki et al., 2009). Concluding remarks A growing line of evidence shows the involvement of the proteasome in various human diseases such as cancers, neurodegeneration, autoinflammation, aging, and autoimmunity. Increased proteasome activity is found in cancers, and treatment using proteasome inhibitors is now accepted as one of the most effective antineoplastic strategies (Dick and Fleming, 2010; Ruschak et al, 2011). On the other hand, decline in proteasome activity with age is linked to longevity in Drosophila melanogaster and Caenorhabditis elegans (Tonoki et al., 2009; Vilchez et al., 2012). These observations suggest the importance of maintaining the right balance of proteasome amount and activity in human cells. Proteasome assembly is an important process that determines the amount of active proteasomes. Recent progress in the study of proteasome assembly should provide suggestions on how we can manipulate proteasome activity and will help in understanding and treating human diseases. Please cite this article in press as: Sahara K, et al., The mechanism for molecular assembly of the proteasome, Advances in Biological Regulation (2013), http://dx.doi.org/10.1016/j.jbior.2013.09.010
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The mechanism for molecular assembly of the proteasome Kazutaka Sahara, Larissa Kogleck, Hideki Yashiroda, Shigeo Murata* Laboratory of Protein Metabolism, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan
a b s t r a c t In eukaryotic cells, the ubiquitin proteasome system plays important roles in diverse cellular processes. The 26S proteasome is a large enzyme complex that degrades ubiquitinated proteins. It consists of 33 different subunits that form two subcomplexes, the 20S core particle and the 19S regulatory particle. Recently, several chaperones dedicated to the accurate assembly of this protease complex have been identified, but the complete mechanism of the 26S proteasome assembly is still unclear. In this review, we summarize what is known about the assembly of proteasome to date and present our group’s recent findings on the role of the GET pathway in the assembly of the 26S proteasome, in addition to its role in mediating the insertion of tail-anchored (TA) proteins into the ER membrane. Ó 2013 Elsevier Ltd. All rights reserved.
Introduction The 26S proteasome is responsible for the degradation of the majority of regulatory proteins and aberrant proteins of the cytosol, nucleus, and organelles inside cells, thus playing a central role in various cellular processes such as cell cycle progression, DNA repair, apoptosis, signal transduction, immune response, development, and protein quality control (Tanaka, 2009). Degradation of proteins by the proteasome is usually mediated by covalent attachment of ubiquitin chains to lysine residues within a target protein.
* Corresponding author. Tel.: þ81 3 5841 4803; fax: þ81 3 5841 4805. E-mail address: [email protected] (S. Murata). 2212-4926/$ – see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jbior.2013.09.010
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The 26S proteasome is a large ATP-dependent protease complex that recognizes and degrades polyubiquitinated proteins selectively (Hershko and Ciechanover, 1998). Its highly specialized function requires a complex structure which consists of 33 different subunits. In this review, we focus on the mechanism of proteasome assembly, especially on the recent advances in this area of study. Topics Architecture of the proteasome The 26S proteasome is composed of two particles; the 20S core particle (CP) and the 19S regulatory particle (RP) (Murata et al., 2009) (Fig. 1). The RP is attached to one or both ends of the CP and is the first step in the degradation process of target proteins by recognizing and removing ubiquitin chains, unfolding substrate proteins, and feeding them into the interior cavity of the CP, where they are degraded. Both the CP and the RP are complexes made up of 14 and 19 different subunits, respectively. The CP is a barrel shaped enzyme complex formed by an axial stack of four heteroheptameric rings; two outer a-rings consisting of seven homologous a-subunits (a1-a7) and two inner b-rings consisting of seven homologous b-subunits (b1-b7). The two abutting b-rings form a catalytic cavity, in which the proteolytic active sites of the CP are exposed. Catalytic activity is conveyed by b1, b2, and b5, which have caspase-like, trypsin-like, and chymotrypsin-like activity, respectively. These three subunits cleave peptide bonds after particular amino acid residues in a substrate protein (Borissenko and Groll, 2007). The RP can be further divided into the base and the lid subcomplexes. The base is composed of six homologous AAA-ATPase subunits Rpt1–6 and three non-ATPase subunits, Rpn1, 2, and 13. The six ATPase subunits form a hexametric ring and serve to unfold and translocate the substrate proteins into the CP in an ATP dependent manner. Another role of the base is to open the center of the a-ring in the CP, which is usually in a closed state. This is exerted by the carboxyl terminal HbYX (hydrophobic, tyrosine, any) motifs in Rpt2 and Rpt5, each of which extends into a pocket formed by two adjacent asubunits (Gillette, et al., 2008; Smith et al., 2007; Rabl, et al., 2008). Rpn13 and another RP subunit Rpn10 directly bind ubiquitinated proteins and function as ubiquitin receptors. The lid is composed of
Fig. 1. The assembly pathway of the 26S proteasome. The 33 different subunits are incorporated into the 26S proteasome in a precise and highly specialized manner. The assembly of the CP and the base is regulated by proteasome-dedicated chaperones. In contrast, the specific chaperones for the lid assembly have not been identified. See main text for details.
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nine non-ATPase subunits, Rpn3, 5–9, 11,12, and 15/Sem1. The major function of the lid is to deubiquitinate the captured substrates, which is catalyzed by the metalloisopeptidase Rpn11 (Verma et al., 2002; Yao and Cohen, 2002). Some prokaryotic species also have proteasomes, but the subunit organization is much less complex. The a- and b-rings of prokaryotic CPs are a homoheptamer of a single type of a-subunit and bsubunit, respectively. The RP is a homohexameric ring of a single ATPase subunit. The assembly of the prokaryotic proteasome is therefore not a difficult task and can be reconstituted in Escherichia coli (Zühl et al., 1997; Zwickl et al., 1999). In contrast, the assembly of eukaryotic proteasomes requires high precision and accuracy, ensuring that every subunit occupies a particular, predetermined place within the newly synthesized proteasome. Recent studies have identified many chaperones dedicated to this highly complex proteasome assembly. Here, we summarize the current understanding of the assembly mechanism of the proteasome. 20S core particle assembly by dedicated assembly chaperones CP assembly is mediated by at least five conserved extrinsic chaperones PAC1–4 and UMP1/POMP in mammals and Pba1–4 and Ump1 in yeast (Hirano et al., 2005, 2006; Hoyt et al., 2008; Kusmierczyk et al., 2008; Le Tallec et al., 2007; Murata et al., 2009; Ramos et al., 1998; Yashiroda et al., 2008) (Fig. 1). The assembly of the CP starts with the a-ring formation supported by two heterodimeric chaperone complexes PAC1-PAC2 and PAC3-PAC4 in mammals and Pba1-Pba2 and Pba3-Pba4 in yeast. The precise mechanism of a-ring formation is unknown, but since knockdown of PAC3 and PAC4 causes marked reduction in completed a-ring structures in mammalian cells, it is suggested that PAC3-PAC4 is required for a-ring formation (Hirano et al., 2006). At the very least, the role of PAC1-PAC2 is to prevent dimerization of a-rings, which would cause stalling of CP biogenesis, since knockdown of PAC1 and PAC2 causes accumulation of a-ring dimers (Hirano et al., 2005). A structural analysis of the Pba1-Pba2CP complex revealed that Pba1 and Pba2 have HbYX motifs at their carboxyl termini and bind to the outer surface of the a-ring, which should prevent attachment of the RP to the a-ring prior to finishing CP assembly (Hirano et al., 2005; Stadtmueller et al., 2012). After the assembly of the a-ring is completed, b-subunits are incorporated onto the a-ring. This process is assisted both by propeptides and C-terminal tails of b-subunits as well as by the assembly chaperones. Each b-subunit is recruited onto the a-ring in a defined order of b2, b3, b4, b5, b6, b1, and b7, as revealed in knockdown experiments of each b-subunit in mammalian cells (Hirano et al., 2008). b2 is the first subunit to be assembled onto the a-ring. This b2 recruitment is dependent on another dedicated chaperone, Ump1. The dislocation of the PAC3-PAC4 complex from the a-ring occurs when b3 is incorporated. A structural analysis of the yeast Pba3-Pba4-a5 complex revealed steric hindrance between Pba3-Pba4 and b4, further confirming the release of PAC3-4/Pba3-4 early on during b-subunit assembly (Yashiroda et al., 2008). Half-CPs dimerize upon incorporation of b7 as the last subunit and form mature CPs, followed by the cleavage of b-subunit propeptides and the degradation of UMP1 and PAC1-PAC2 (Hirano et al., 2008; Li et al., 2007; Marques et al., 2007). Zygote-specific CP assembly chaperone During the maternal-to-zygotic transition (MZT) after fertilization, massive maternal proteins are degraded by the UPS. This process is important for the replacement of oogenic proteins by proteins newly synthesized from the zygotic genome. We recently identified a molecule named ZPAC for zygotespecific proteasome assembly chaperone, which is specifically expressed in mouse gonads (Shin et al., 2013). In zygotes, the expression of ZPAC as well as the proteasome activity is transiently increased at the MZT. ZPAC directly binds to Ump1 and is specifically associated with precursor forms of the CP. Knockdown of ZPAC in zygotes reduces the CP amount and causes accumulation of precursor CPs, resulting in early developmental arrest of embryos. The exact mechanism in which ZPAC helps CP assembly is unknown, but deletion of ZPAC results in a decrease of Ump1, and vice versa, suggesting a role of ZPAC in stabilizing Ump1 proteins. This finding demonstrates that a cell-type specific assembly strategy could occur and raises the possibility that other assembly chaperones working in a defined cell-type or only under particular conditions may exist. Please cite this article in press as: Sahara K, et al., The mechanism for molecular assembly of the proteasome, Advances in Biological Regulation (2013), http://dx.doi.org/10.1016/j.jbior.2013.09.010
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Base assembly is also mediated by dedicated chaperones The RP consists of the base and the lid subcomplexes. The base and the lid are assembled independently, and subsequently associate with each other to form the RP. The assembly of the base is facilitated by another set of dedicated chaperones called p28/gankyrin, p27, S5b, and Rpn14 in mammals and Nas6, Nas2, Hsm3, and Rpn14 in yeast (Funakoshi et al., 2009; Kaneko et al., 2009; La Tallec et al., 2009; Park et al., 2009; Roelofs et al., 2009; Saeki et al., 2009) (Fig. 1). In mammalian and yeast cells, the base subunits and these chaperones are assembled into three modules, the p28(Nas6)-Rpt3-Rpt6-Rpn14 module, the p27(Nas2)-Rpt4-Rpt5 module and the S5b(Hsm3)-Rpt1Rpt2-Rpn1 module. These three modules come together to form the heterohexameric ATPase ring plus Rpn1 complex, which then assembles with the Rpn2-Rpn13 complex. The base chaperones then dislocate from the subunits during the assembly. These base assembly chaperones seem to define the order of assembly of the three modules, at least in mammalian cells. They also play an important role in preventing premature association of the base with the CP by binding to the carboxyl termini of the Rpt subunits. In addition, two other molecules have been shown to be involved in the base assembly. Ubp6, a proteasome-associated deubiquitinating enzyme in yeast, is included in the Hsm3-Rpt1-Rpt2-Rpn1 module and removes polyubiquitinated proteins from the module by deubiquinating them, thus promoting assembly of the base complex (Sakata et al., 2011). Another molecule is the ADPribosyltransferase tankyrase in drosophila. Tankyrase mediates ADP-ribosylation of PI31, which has been shown to regulate proteasome activity by an unknown mechanism. ADP-ribosylation of PI31 increases PI31 affinity for p27 and S5b, and thereby sequesters p27 and S5b away from the base subunits, thus promoting RP assembly (Cho-Park and Steller, 2013). This finding might provide a link between proteasome activity and cellular metabolic status. Stepwise assembly of the lid occurs without dedicated chaperones Recent studies in yeast suggest that the lid assembly is also a stepwise process (Fig. 1). The lid assembly starts with the assembly of two modules: the Rpn5-6-8-9-11 module and the Rpn3-7-15 module. These two modules associate with each other, forming a lid subcomplex without Rpn12. Cryo-EM analyses revealed that the C-terminal helices of Rpn3, 5–9, and 11 form a helical bundle that directs the ordered self-assembly of the lid subcomplex (Estrin et al., 2013). Although a genetic analysis suggests that Hsp90 has a role in the lid assembly, any dedicated chaperones for the lid have not been identified so far. Together with the observation that the lid subcomplex is reconstituted by mixing purified recombinant lid subunits and Hsp90 (Lander et al., 2012), it is suggested that the lid assembly occurs without any dedicated chaperones (Fukunaga et al., 2010). Rpn12 is subsequently recruited to the newly formed, incomplete lid subcomplex, which then enables the completed lid subcomplex to join together with the base (Tomko and Hochstrasser, 2011). The TRC/GET pathway participates in CP assembly Recently we identified the components of the guided entry of tail-anchored proteins (GET) pathway as factors involved in CP assembly by genetic analysis using budding yeast (Akahane et al., 2013). The GET pathway is responsible for the insertion of most of tail-anchored (TA) proteins into the endoplasmic reticulum (ER) (Schuldiner et al, 2008; Stefanovic and Hedge, 2007). Its mammalian counterpart is called the transmembrane recognition complex (TRC) pathway. The TRC/GET pathway comprises ribosome-bound Bag6-TRC35-Ubl4A/Sgt2-Get4-Get5, cytosolic TRC40/Get3 homodimer, and an ER-localized receptor complex WRB-CAML/Get1-Get2. This pathway starts with the capture of a C-terminal hydrophobic stretch of a TA protein by the BAG6/Sgt2 complex at the ribosome. Subsequently, the TA proteins are transferred to TRC40/GET3 and inserted into the ER with the help of the WRB-CAML/Get1-Get2 complex (Hedge and Keenan, 2011; Vilardi et al., 2011; Yamamoto and Sakisaka, 2012) (Fig. 2). Deletion of either GET1, GET2, or GET3 results in synthetic growth defects when crossed with deletion of genes involved in proteasome assembly such as UMP1, NAS6, and HSM3 in yeast. In Please cite this article in press as: Sahara K, et al., The mechanism for molecular assembly of the proteasome, Advances in Biological Regulation (2013), http://dx.doi.org/10.1016/j.jbior.2013.09.010
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Fig. 2. The TRC pathway. The TRC pathway delivers TA proteins into the ER membrane in 3 steps. Firstly, translated TA proteins at the ribosomes are captured by pre-targeting factor (Bag6-TRC35-Ubl4A in mammals, and Sgt2-Get4-Get5 in yeast). Secondly, TA proteins are then transferred to the TRC40/Get3 homodimer and recruited onto the ER membrane. Lastly, the delivered TA proteins are inserted into the ER via the receptor complex (WRB-CAML in mammals, and Get1-Get2 in yeast).
mammalian cells, knockdown of either TRC40 (Get3 homolog) or Bag6 (the most upstream component of the TRC pathway) causes the accumulation of CP assembly intermediates representing inefficient bsubunit assembly on the a-ring. The notable finding is that the association of CP intermediates with the ER membrane was reduced compared to control cells. These observations are consistent with the previous report showing an association of Ump1 and CP intermediates with the ER (Fricke et al., 2007) and strongly suggest that CP assembly occurs more efficiently on the ER than in the cytosol. Considering that all the components of the TRC/GET pathway are involved in CP assembly and that there is no physical interaction between the components of the TRC/GET pathway and CP subunits/chaperones, it is likely that a particular TA protein that is delivered to the ER by the TRC/GET pathway recruits CP intermediates and supports the accurate assembly of the CP. The finding suggests a possible link between CP biogenesis and ER membrane integrity (Fig. 3). It is suggested that nearly 400 TA proteins are encoded in the human genome (Kalbfleisch et al, 2007), and further studies should be focused on the identification of the responsible TA protein that plays a role in CP assembly. Bag6 is required for base assembly Interestingly, Bag6-knockdown causes not only a defect in CP assembly but also in base assembly. Bag6 directly interacts with Rpt5 and promotes the incorporation of Rpt4 into the p27-Rpt5 complex, thus facilitating the formation of the p27-Rpt4-Rpt5 module, which is required for efficient base assembly (Fig. 3). Bag6 is a multi-functional molecule. One of its important roles is as a ribosome-associating chaperone that recognizes aggregation-prone substrates in mammalian cells and regulates protein triage, in which proteins undergo either productive folding or degradation by the UPS (Kawahara et al., 2013; Lee & Ye, 2013). Therefore, it could be possible that an excess expression of misfolded proteins in cells affects proteasome assembly by engaging Bag6 to a function as protein quality control and thus Please cite this article in press as: Sahara K, et al., The mechanism for molecular assembly of the proteasome, Advances in Biological Regulation (2013), http://dx.doi.org/10.1016/j.jbior.2013.09.010
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Fig. 3. Involvement of the TRC pathway and Bag6 in proteasome assembly. The TRC pathway may be involved in the formation of the 20S proteasome. TRC40 and Bag6 play an important role in this function. TA proteins, the substrate of this pathway, may be directly involved in this function. Bag6 also plays a role in the formation of the base complex through assisting the formation of the p27Rpt4-Rpt5 module.
reducing the amount of Bag6 available for proteasome assembly. The finding may provide a link between protein homeostasis and proteasome biogenesis and raise the possibility that age-dependent accumulation of misfolded proteins in cells affects proteasome assembly and might be one of the causes of age-dependent decline in proteasome activity (Gregersen et al., 2006; Tonoki et al., 2009). Concluding remarks A growing line of evidence shows the involvement of the proteasome in various human diseases such as cancers, neurodegeneration, autoinflammation, aging, and autoimmunity. Increased proteasome activity is found in cancers, and treatment using proteasome inhibitors is now accepted as one of the most effective antineoplastic strategies (Dick and Fleming, 2010; Ruschak et al, 2011). On the other hand, decline in proteasome activity with age is linked to longevity in Drosophila melanogaster and Caenorhabditis elegans (Tonoki et al., 2009; Vilchez et al., 2012). These observations suggest the importance of maintaining the right balance of proteasome amount and activity in human cells. Proteasome assembly is an important process that determines the amount of active proteasomes. Recent progress in the study of proteasome assembly should provide suggestions on how we can manipulate proteasome activity and will help in understanding and treating human diseases. Please cite this article in press as: Sahara K, et al., The mechanism for molecular assembly of the proteasome, Advances in Biological Regulation (2013), http://dx.doi.org/10.1016/j.jbior.2013.09.010
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