The COP9 signalosome-like complex in S. cerevisiae and links to other PCI complexes

The COP9 signalosome (CSN), the lid subcomplex of the proteasome and translational initiation factor 3 (eIF3) share structural similarities and are often referred to as the PCI family of complexes. In multicellular eukaryotes, the CSN is highly conserved as an 8-subunit complex but in Saccharomyces cerevisiae the complex is rather divergent. We further characterize the composition and properties of the CSN in budding yeast and its interactions with these related complexes. Using the generalized profile method we identified CSN candidates, four with PCI domains: Csn9, Csn10, Pci8/Csn11, and Csn12, and one with an MPN domain, Csn5/Rri1. These proteins and an additional interactor, Csi1, were tested for pairwise interactions by yeast two-hybrid and were found to form a cluster surrounding Csn12. Csn5 and Csn12 cofractionate in a complexed form with an apparent molecular weight of roughly 250kDa. However, Csn5 migrates as a monomer in Deltacsn12 supporting the pivotal role of Csn12 in stabilizing the complex. Confocal fluorescence microscopy detects GFP-tagged Csn5 preferentially in the nucleus, whereas in absence of Csn12, Csn10, Pci8/Csn11, or Csi1, Csn5 is delocalized throughout the cell, indicating that multiple subunits are required for nuclear localization of Csn5. Two CSN subunits, Csn9 and Csi1, interact with the proteasome lid subunit Rpn5. Pci8/Csn11 has previously been shown to interact with eIF3. Together, these results point to a network of interactions between these three structurally similar, yet functionally diverse, complexes.     

Consequences of COP9 signalosome and 26S proteasome interaction

The COP9 signalosome (CSN) occurs in all eukaryotic cells. It is a regulatory particle of the ubiquitin (Ub)/26S proteasome system. The eight subunits of the CSN possess sequence homologies with the polypeptides of the 26S proteasome lid complex and just like the lid, the CSN consists of six subunits with PCI (proteasome, COP9 signalosome, initiation factor 3) domains and two components with MPN (Mpr-Pad1-N-terminal) domains. Here we show that the CSN directly interacts with the 26S proteasome and competes with the lid, which has consequences for the peptidase activity of the 26S proteasome in vitro. Flag-CSN2 was permanently expressed in mouse B8 fibroblasts and Flag pull-down experiments revealed the formation of an intact Flag-CSN complex, which is associated with the 26S proteasome. In addition, the Flag pull-downs also precipitated cullins indicating the existence of super-complexes consisting of the CSN, the 26S proteasome and cullin-based Ub ligases. Permanent expression of a chimerical subunit (Flag-CSN2-Rpn6) consisting of the N-terminal 343 amino acids of CSN2 and of the PCI domain of S9/Rpn6, the paralog of CSN2 in the lid complex, did not lead to the assembly of an intact complex showing that the PCI domain of CSN2 is important for complex formation. The consequence of permanent Flag-CSN2 overexpression was de-novo assembly of the CSN complex connected with an accelerated degradation of p53 and stabilization of c-Jun in B8 cells. The possible role of super-complexes composed of the CSN, the 26S proteasome and of Ub ligases in the regulation of protein stability is discussed.     

The proteasome regulatory particle subunit Rpn6 is required for Drosophila development and interacts physically with signalosome subunit Alien/CSN2

The eukaryotic 26S proteasome plays a central role in ubiquitin-dependent intracellular protein metabolism. The multimeric holoenzyme is composed of two major subcomplexes, known as the 20S proteolytic core particle and the 19S regulatory particle (RP). The RP can be further dissected into two multisubunit complexes, the lid and the base complex. The lid complex shares striking similarities with another multiprotein complex, the COP9 signalosome. Several subunits of both complexes contain the characteristic PCI domain, a structural motif important for complex assembly. The COP9 signalosome was shown to act as a versatile regulator in numerous pathways. To help define the molecular interactions of the signalosome during Drosophila development, we performed a yeast two-hybrid screen to identify proteins that physically interact with subunit 2 of the complex, namely Alien/CSN2. Here, we report that Drosophila Rpn6, a non-ATPase subunit of the RP lid complex, interacts with Alien/CSN2 via its PCI domain. The temporal and spatial expression patterns of Rpn6 and alien/CSN2 overlap on a large scale during development providing additional evidence for their interaction in vivo. Analyses of an Rpn6 P element insertion mutant and newly generated Rpn6 alleles reveal that Rpn6 is essential for Drosophila development.     

COP9 signalosome components play a role in the mating pheromone response of S. cerevisiae

A family of genetically and structurally homologous complexes, the proteasome lid, Cop9 signalosome (CSN) and eukaryotic translation initiation factor 3, mediate different regulatory pathways. The CSN functions in numerous eukaryotes as a regulator of development and signaling, yet until now no evidence for a complex has been found in Saccharomyces cerevisiae. We identified a group of proteins, including a homolog of Csn5/Jab1 and four uncharacterized PCI components, that interact in a manner suggesting they form a complex analogous to the CSN in S. cerevisiae. These newly identified subunits play a role in adaptation to pheromone signaling. Deletants for individual subunits enhance pheromone response and increase mating efficiency. Overexpression of individual subunits or a human homolog mitigates sst2-induced pheromone sensitivity. Csi1, a novel CSN interactor, exhibits opposite phenotypes. Deletants also accumulate Cdc53/cullin in a Rub1-modified form; however, this role of the CSN appears to be distinct from that in the mating pathway.     

Subunit interaction maps for the regulatory particle of the 26S proteasome and the COP9 signalosome.

The 26S proteasome plays a major role in eukaryotic protein breakdown, especially for ubiquitin-tagged proteins. Substrate specificity is conferred by the regulatory particle (RP), which can dissociate into stable lid and base subcomplexes. To help define the molecular organization of the RP, we tested all possible paired interactions among subunits from Saccharomyces cerevisiae by yeast two-hybrid analysis. Within the base, a Rpt4/5/3/6 interaction cluster was evident. Within the lid, a structural cluster formed around Rpn5/11/9/8. Interactions were detected among synonymous subunits (Csn4/5/7/6) from the evolutionarily related COP9 signalosome (CSN) from Arabidopsis, implying a similar quaternary arrangement. No paired interactions were detected between lid, base or core particle subcomplexes, suggesting that stable contacts between them require prior assembly. Mutational analysis defined the ATPase, coiled-coil, PCI and MPN domains as important for RP assembly. A single residue in the vWA domain of Rpn10 is essential for amino acid analog resistance, for degrading a ubiquitin fusion degradation substrate and for stabilizing lid-base association. Comprehensive subunit interaction maps for the 26S proteasome and CSN support the ancestral relationship of these two complexes.     

PCI proteins eIF3e and eIF3m define distinct translation initiation factor 3 complexes

Background  

PCI/MPN domain protein complexes comprise the 19S proteasome lid, the COP9 signalosome (CSN), and eukaryotic translation initiation factor 3 (eIF3). The eIF3 complex is thought to be composed of essential core subunits required for global protein synthesis and non-essential subunits that may modulate mRNA specificity. Interactions of unclear significance were reported between eIF3 subunits and PCI proteins contained in the CSN.     

Solution Structure of Yeast Rpn9: INSIGHTS INTO PROTEASOME LID ASSEMBLY*

The regulatory particle (RP) of the 26 S proteasome functions in preparing polyubiquitinated substrates for degradation. The lid complex of the RP contains an Rpn8-Rpn11 heterodimer surrounded by a horseshoe-shaped scaffold formed by six proteasome-COP9/CSN-initiation factor (PCI)-containing subunits. The PCI domains are essential for lid assembly, whereas the detailed molecular mechanisms remain elusive. Recent cryo-EM studies at near-atomic resolution provided invaluable information on the RP architecture in different functional states. Nevertheless, atomic resolution structural information on the RP is still limited, and deeper understanding of RP assembly mechanism requires further studies on the structures and interactions of individual subunits or subcomplexes. Herein we report the high-resolution NMR structures of the PCI-containing subunit Rpn9 from Saccharomyces cerevisiae. The 45-kDa protein contains an all-helical N-terminal domain and a C-terminal PCI domain linked via a semiflexible hinge. The N-terminal domain mediates interaction with the ubiquitin receptor Rpn10, whereas the PCI domain mediates interaction with the neighboring PCI subunit Rpn5. The Rpn9-Rpn5 interface highlights two structural motifs on the winged helix module forming a hydrophobic center surrounded by ionic pairs, which is a common pattern for all PCI-PCI interactions in the lid. The results suggest that divergence in surface composition among different PCI pairs may contribute to the modulation of lid assembly.     

Association of the mammalian proto-oncoprotein Int-6 with the three protein complexes eIF3, COP9 signalosome and 26S proteasome

The mammalian Int-6 protein has been characterized as a subunit of the eIF3 translation initiation factor and also as a transforming protein when its C-terminal part is deleted. It includes a protein domain, which also exists in various subunits of eIF3, of the 26S proteasome and of the COP9 signalosome (CSN). By performing a two-hybrid screen with Int-6 as bait, we have isolated subunits belonging to all three complexes, namely eIF3-p110, Rpt4, CSN3 and CSN6. The results of transient expression experiments in COS7 cells confirmed the interaction of Int-6 with Rpt4, CSN3 and CSN6, but also showed that Int-6 is able to bind another subunit of the CSN: CSN7a. Immunoprecipitation experiments performed with the endogenous proteins showed that Int-6 binds the entire CSN, but in low amount, and also that Int-6 is associated with the 26S proteasome. Taken together these results show that the Int-6 protein can bind the three complexes with various efficiencies, possibly exerting a regulatory activity in both protein translation and degradation.     

1H, 13C and 15N resonance assignments of Rpn9, a regulatory subunit of 26S proteasome from Saccharomyces cerevisiae

The 26S proteasome is an essential molecular machine for specific protein degradation in eukaryotic cells. The 26S proteasome is formed by a central 20S core particle capped by two 19S regulatory particle (RP) at both ends. The Rpn9 protein is a non-ATPase subunit located in the lid complex of the 19S RP, and is identified to be essential for efficient assembly of yeast 26S proteasome. Bioinformatics analysis of Saccharomyces cerevisiae Rpn9 suggested it contains a PCI domain at the C-terminal region. However, high-resolution structures of either the PCI domain or the full-length Rpn9 still remain elusive. Herein, we report the chemical shift assignments of ¹H, ¹³C and ¹⁵N atoms of the individual N- and C-domains, as well as full-length S. cerevisiae Rpn9, which provide the basis for further structural and functional studies of Rpn9 using solution NMR technique.     

Post-transcriptional fine-tuning of COP9 signalosome subunit biosynthesis is regulated by the c-Myc/Lin28B/let-7 pathway

The COP9 signalosome (CSN) complex controls protein degradation via the ubiquitin (Ub) proteasome system (UPS) in eukaryotes. In mammalian cells, the multimeric CSN is composed of eight subunits (CSN1 - CSN8). It regulates cullin-RING Ub ligases (CRLs), which target essential regulatory proteins for ubiquitination and subsequent degradation. Thereby, the CSN cooperates with the UPS in a variety of essential cellular functions, including DNA repair, cell cycle and differentiation. Although functions of the CSN have been elucidated, mechanisms and regulatory principles of its de novo formation are completely unknown. Here, we show that there is a fundamental mechanism that allows a coordinated expression of all CSN subunits, a prerequisite for CSN assembly. CSN subunit mRNAs are targets of miRNAs of the let-7 family suppressing CSN subunit expression in human cells. Factors that reduce or block let-7 miRNAs induce the coordinated expression of CSN subunits. For instance, over-expression of CSN1 specifically traps let-7a-1 miRNA and elevates CSN subunit levels by two- to fourfold in a coordinated manner. CSN subunit expression is also increased by specific miRNA inhibitors or by interferon (IFN)-mediated induction of STAT1 and c-Myc reducing levels of let-7 miRNAs. Activation of STAT1 by IFNα or IFNγ induces c-Myc, which increases CSN subunit expression via the Lin28B/let-7 regulatory pathway. By contrast, a let-7a-1 mimic reduces CSN subunit expression. Our data show that let-7 miRNAs control the fine-tuning and coordinated expression of subunits for CSN de novo formation, presumably a general regulatory principle for other Zomes complexes as well.     

1H, 13C and 15N resonance assignments of the VWA domain of Saccharomyces cerevisiae Rpn10, a regulatory subunit of 26S proteasome

Rpn10 is a ubiquitin receptor of the 26S proteasome, and plays an important role in poly-ubiquitinated proteins recognition in the ubiquitin–proteasome protein degradation pathway. It is located in the 19S regulatory particle and interacts with several subunits of both lid and base complexes. Bioinformatics analysis of yeast Rpn10 suggests that it contains a von Willebrand (VWA domain) and a C-terminal tail containing a Ub-interacting motif. Studies of Saccharomyces cerevisiae Rpn10 suggested that its VWA domain might participate in interactions with subunit from both lid and base subcomplexes of the 19S regulatory particle. Herein, we report the chemical shift assignments of ¹H, ¹³C and ¹⁵N atoms of the VWA domain of S. cerevisiae Rpn10, which provide the basis for further structural and functional studies of Rpn10 by solution NMR technique.     

The COP9 signalosome (CSN): an evolutionary conserved proteolysis regulator in eukaryotic development

The COP9 signalosome (CSN) is a multiprotein complex of the ubiquitin-proteasome pathway. CSN is typically composed of eight subunits, each of which is related to one of the eight subunits that form the lid of the 26S proteasome regulatory particle. CSN was first identified in Arabidopsis where it is required for the repression of photomorphogenic seedling development in the dark. CSN or CSN-related complexes have by now been reported from most eukaryotic model organisms and CSN has been implicated in a vast array of biological processes. It is widely accepted that CSN directly interacts with cullin-containing E3 ubiquitin ligases, and that CSN is required for their proper function. The requirement of CSN for proper E3 function may at least in part be explained by the observation that CSN subunit 5 (CSN5) is the isopeptidase that deconjugates the essential ubiquitin-like Nedd8 modification from the E3 cullin subunit. In addition to its interaction with E3s, CSN may also regulate proteolysis by its association with protein kinases and deubiquitylating enzymes. This review provides a summary of the role of CSN in regulating protein degradation and in eukaryotic development.     

Dissection of the assembly pathway of the proteasome lid in Saccharomyces cerevisiae

The 26S proteasome is a highly conserved multisubunit protease that degrades ubiquitinated proteins in eukaryotic cells. It comprises a 20S core particle and two 19S regulatory particles that are further divided into the lid and base complexes. The lid is a nine subunits complex that is structurally related to the COP9 signalosome and the eukaryotic initiation factor 3. Although the assembly pathway of the 20S and the base are well described, that of the lid is still unclear. In this study, we dissected the lid assembly using yeast lid mutant cells, rpn7-3, Δrpn9, and rpn12-1. Using mass spectrometry, we identified a number of lid subassemblies, such as Rpn3-Rpn7 pair and a lid-like complex lacking Rpn12, in the mutants. Our analysis suggests that the assembly of the lid is a highly ordered and multi-step process; first, Rpn5, 6, 8, 9, and 11 are assembled to form a core module, then a second module, consisting of Rpn3, 7, and Sem1, is attached, followed by the incorporation of Rpn12 to form the lid complex.     

Isolation of the Schizosaccharomyces pombe proteasome subunit Rpn7 and a structure-function study of the proteasome-COP9-initiation factor domain

Proper assembly of the 26 S proteasome is required to efficiently degrade polyubiquitinated proteins. Many proteasome subunits contain the proteasome-COP9-initiation factor (PCI) domain, thus raising the possibility that the PCI domain may play a role in mediating proteasome assembly. We have previously characterized the PCI protein Yin6, a fission yeast ortholog of the mammalian Int6 that has been implicated in breast oncogenesis, and demonstrated that it binds and regulates the assembly of the proteasome. In this study, we isolated another PCI proteasome subunit, Rpn7, as a high copy suppressor that rescued the proteasome defects in yin6 null cells. To better define the function of the PCI domain, we aligned protein sequences to identify a conserved leucine residue that is present in nearly all known PCI domains. Replacing it with aspartate in yeast Rpn7, Yin6, and Rpn5 inactivated these proteins, and mutant human Int6 mislocalized in HeLa cells. Rpn7 and Rpn5 bind Rpn9 with high affinity, but their mutant versions do not. Our data suggest that this leucine may interact with several hydrophobic amino acid residues to influence the spatial arrangement either within the N-terminal tandem alpha-helical repeats or between these repeats and the more C-terminal winged helix subdomain. Disruption of such an arrangement in the PCI domain may substantially inactivate many PCI proteins and block their binding to other proteins.     

Arabidopsis eIF3e (INT-6) associates with both eIF3c and the COP9 signalosome subunit CSN7

The Arabidopsis COP9 signalosome is a multisubunit repressor of photomorphogenesis that is conserved among eukaryotes. This complex may have a general role in development. As a step in dissecting the biochemical mode of action of the COP9 signalosome, we determined the sequence of proteins that copurify with this complex. Here we describe the association between components of the COP9 signalosome (CSN1, CSN7, and CSN8) and two subunits of eukaryotic translation initiation factor 3 (eIF3), eIF3e (p48, known also as INT-6) and eIF3c (p105). To obtain a biochemical marker for Arabidopsis eIF3, we cloned the Arabidopsis ortholog of the eIF3 subunit eIF3b (PRT1). eIF3e coimmunoprecipitated with CSN7, and eIF3c coimmunoprecipitated with eIF3e, eIF3b, CSN8, and CSN1. eIF3e directly interacted with CSN7 and eIF3c. However, eIF3e and eIF3b cofractionated by gel filtration chromatography in a complex that was larger than the COP9 signalosome. Whereas eIF3, as detected through eIF3b, localized solely to the cytoplasm, eIF3e, like CSN7, was also found in the nucleus. This suggests that eIF3e and eIF3c are probably components of multiple complexes and that eIF3e and eIF3c associate with subunits of the COP9 signalosome, even though they are not components of the COP9 signalosome core complex. This interaction may allow for translational control by the COP9 signalosome.     

COP9 Signalosome Interacts ATP-dependently with p97/Valosin-containing Protein (VCP) and Controls the Ubiquitination Status of Proteins Bound to p97/VCP

Ubiquitinated proteins can alternatively be delivered directly to the proteasome or via p97/VCP (valosin-containing protein). Whereas the proteasome degrades ubiquitinated proteins, the homohexameric ATPase p97/VCP seems to control the ubiquitination status of recruited substrates. The COP9 signalosome (CSN) is also involved in the ubiquitin/proteasome system (UPS) as exemplified by regulating the neddylation of ubiquitin E3 ligases. Here, we show that p97/VCP colocalizes and directly interacts with subunit 5 of the CSN (CSN5) in vivo and is associated with the entire CSN complex in an ATP-dependent manner. Furthermore, we provide evidence that the CSN and in particular the isopeptidase activity of its subunit CSN5 as well as the associated deubiquitinase USP15 are required for proper processing of polyubiquitinated substrates bound to p97/VCP. Moreover, we show that in addition to NEDD8, CSN5 binds to oligoubiquitin chains in vitro. Therefore, CSN and p97/VCP could form an ATP-dependent complex that resembles the 19 S proteasome regulatory particle and serves as a key mediator between ubiquitination and degradation pathways.     

Base-CP proteasome can serve as a platform for stepwise lid formation

26S proteasome, a major regulatory protease in eukaryotes, consists of a 20S proteolytic core particle (CP) capped by a 19S regulatory particle (RP). The 19S RP is divisible into base and lid sub-complexes. Even within the lid, subunits have been demarcated into two modules: module 1 (Rpn5, Rpn6, Rpn8, Rpn9 and Rpn11), which interacts with both CP and base sub-complexes and module 2 (Rpn3, Rpn7, Rpn12 and Rpn15) that is attached mainly to module 1. We now show that suppression of RPN11 expression halted lid assembly yet enabled the base and 20S CP to pre-assemble and form a base-CP. A key role for Regulatory particle non-ATPase 11 (Rpn11) in bridging lid module 1 and module 2 subunits together is inferred from observing defective proteasomes in rpn11–m1, a mutant expressing a truncated form of Rpn11 and displaying mitochondrial phenotypes. An incomplete lid made up of five module 1 subunits attached to base-CP was identified in proteasomes isolated from this mutant. Re-introducing the C-terminal portion of Rpn11 enabled recruitment of missing module 2 subunits. In vitro, module 1 was reconstituted stepwise, initiated by Rpn11–Rpn8 heterodimerization. Upon recruitment of Rpn6, the module 1 intermediate was competent to lock into base-CP and reconstitute an incomplete 26S proteasome. Thus, base-CP can serve as a platform for gradual incorporation of lid, along a proteasome assembly pathway. Identification of proteasome intermediates and reconstitution of minimal functional units should clarify aspects of the inner workings of this machine and how multiple catalytic processes are synchronized within the 26S proteasome holoenzymes.     

Integrity of the Saccharomyces cerevisiae Rpn11 Protein Is Critical for Formation of Proteasome Storage Granules (PSG) and Survival in Stationary Phase

Decline of proteasome activity has been reported in mammals, flies and yeasts during aging. In the yeast Saccharomyces cerevisiae, the reduction of proteolysis in stationary phase is correlated with disassembly of the 26S proteasomes into their 20S and 19S subcomplexes. However a recent report showed that upon entry into the stationary phase, proteasome subunits massively re-localize from the nucleus into mobile cytoplasmic structures called proteasome storage granules (PSGs). Whether proteasome subunits in PSG are assembled into active complexes remains an open question that we addressed in the present study. We showed that a particular mutant of the RPN11 gene (rpn11-m1), encoding a proteasome lid subunit already known to exhibit proteasome assembly/stability defect in vitro, is unable to form PSGs and displays a reduced viability in stationary phase. Full restoration of long-term survival and PSG formation in rpn11-m1 cells can be achieved by the expression in trans of the last 45 amino acids of the C-terminal domain of Rpn11, which was moreover found to co-localize with PSGs. In addition, another rpn11 mutant leading to seven amino acids change in the Rpn11 C-terminal domain, which exhibits assembled-26S proteasomes, is able to form PSGs but with a delay compared to the wild type situation. Altogether, our findings indicate that PSGs are formed of fully assembled 26S proteasomes and suggest a critical role for the Rpn11 protein in this process.     

COP9信号传导体与调节盖子复合体的进化

COP9信号传导体和26S蛋白酶体的调节盖子复合体皆为含有8个亚基的蛋白复合体,在真核生物体中普遍存在,它们的相应亚基在大小和氨基酸序列上具有一一对应关系。从NCBI站点的所有数据库中获得了裂殖酵母、酿酒酵母、线虫、果蝇、哺乳动物和拟南芥等多种生物的复合体的亚基序列共8组。COP9信号传导体与调节盖子复合体相应亚基之间的氨基酸序列一致性大于12％,它们均具有一些保守的区域,而且保守位点分布均匀,表明它们来自于同一祖先。在基于氨基酸序列构建的系统发育树中,各组序列分别形成两个分支：一个分支由COP9信号传导体亚基和相似蛋白组成,另一分支由相应的调节盖子复合体亚基和相似蛋白构成。各个分支中单细胞生物的序列位于动、植物序列的根部,表明COP9信号传导体与调节盖子复合体的基因重复发生在真核单细胞生物和多细胞生物分化以前,并且二者的亚基基因沿各自的方向独立进化。几乎所有编码两个蛋白复合体的基因在基因组中均为单拷贝,第Ⅴ、Ⅵ组的亚基保守程度最高,暗示着它们在复合体中起着关键的作用。对COP9信号传导体和调节盖子复合体的相应亚基基因两两之间进行dN／dS的相关性分析,分别鉴定出21和15对亚基编码序列间具有显著的Pearson相关关系,推测其相应亚基间可能通过承担相互关联的重要的生物学功能而协同进化。     

Characterization of the human ortholog of Mov34 reveals eight N-terminal residues important for MPN domain stability

Eukaryotic MPN domain proteins are components of the complexes proteasome lid, COP9-signalosome (CSN), and translation initiation factor 3 (eIF3). The proteasome lid Rpn11 and COP9-signalosome Csn5 subunits, which contain the conserved JAMM motif involved in zinc ion coordination, show catalytic isopeptidase activity. Homology modeling indicates that the MPN domain of Mov34 cannot coordinate a zinc ion in the same manner as catalytically active MPN domains. In this work, we show that the MPN domain of Mov34 is highly resistant to proteolysis and the major product comprises residues 9-186, which includes the conserved MPN domain. Two clones containing the MPN domain region (MPN1-177 and MPN1-186) including the eight N-terminal residues show a less pronounced band in the 220 nm region of the CD, indicating lower alpha-helical content relative to the clones lacking these residues (MPN9-177 and MPN9-186). However, clones lacking residues 1-8 show lower expression levels and thermal stability, indicating that residues 1-8 are required for proper folding and stability of this particular MPN domain.