The proteasome is the major ATP-dependent protease in eukaryotic cells, but limited structural information strongly restricts a mechanistic understanding of its activities. The proteasome regulatory particle, consisting of the lid and base subcomplexes, recognizes and processes poly-ubiquitinated substrates. We used electron microscopy and a newly-developed heterologous expression system for the lid to delineate the complete subunit architecture of the regulatory particle. Our studies reveal the spatial arrangement of ubiquitin receptors, deubiquitinating enzymes, and the protein unfolding machinery at subnanometer resolution, outlining the substrate’s path to degradation. Unexpectedly, the ATPase subunits within the base unfoldase are arranged in a spiral staircase, providing insight into potential mechanisms for substrate translocation through the central pore. Large conformational rearrangements of the lid upon holoenzyme formation suggest allosteric regulation of deubiquitination. We provide a structural basis for the ability of the proteasome to degrade a diverse set of substrates and thus regulate vital cellular processes.
26S proteasome; 19S regulatory particle; ubiquitin recognition; deubiquitination; AAA+ ATPase; cryoEM; UPS; PCI domain
The 26S proteasome, the central eukaryotic protease, comprises a core particle capped by a 19S regulatory particle (RP). The RP is divisible into base and lid subcomplexes. Lid biogenesis and incorporation into the RP remain poorly understood. We report several lid intermediates, including the free Rpn12 subunit and a lid particle (LP) containing the remaining eight subunits, LP2. Rpn12 binds LP2 in vitro, and each requires the other for assembly into 26S proteasomes. Stable Rpn12 incorporation depends on all other lid subunits, indicating Rpn12 distinguishes LP2 from smaller lid subcomplexes. The highly conserved C-terminus of Rpn12 bridges the lid and base, mediating both stable binding to LP2 and lid-base joining. Our data suggest a hierarchical assembly mechanism where Rpn12 binds LP2 only upon correct assembly of all other lid subunits, and the Rpn12 tail then helps drive lid-base joining. Rpn12 incorporation thus links proper lid assembly to subsequent assembly steps.
The 26S proteasome is responsible for a large fraction of the regulated protein degradation in eukaryotic cells. The enzyme complex is composed of a 20S proteolytic core particle (CP) capped on one or both ends with a 19S regulatory particle (RP). The RP recognizes and unfolds substrates and translocates them into the CP. The RP can be further divided into lid and base subcomplexes. The base contains a ring of six AAA+ ATPases (Rpts) that directly abuts the CP and is responsible for unfolding substrates and driving them into the CP for proteolysis. Although 120 arrangements of the six different ATPases within the ring are possible in principle, they array themselves in one specific order. The high sequence and structural similarity between the Rpt subunits presents special challenges for their ordered association and incorporation into the assembling proteasome. In this review, we discuss recent advances in our understanding of proteasomal RP base biogenesis, with emphasis on potential specificity determinants in ring arrangement, and the implications of the ATPase ring arrangement for proteasome assembly.
Proteasome; ATPase; multiprotein complex assembly; proteolysis; ubiquitin; RP assembly chaperone (RAC)
The 26S proteasome consists of the 20S proteasome (core particle) and the 19S regulatory particle made of the base and lid substructures, and it is mainly localized in the nucleus in yeast. To examine how and where this huge enzyme complex is assembled, we performed biochemical and microscopic characterization of proteasomes produced in two lid mutants, rpn5-1 and rpn7-3, and a base mutant ΔN rpn2, of the yeast Saccharomyces cerevisiae. We found that, although lid formation was abolished in rpn5-1 mutant cells at the restrictive temperature, an apparently intact base was produced and localized in the nucleus. In contrast, in ΔN rpn2 cells, a free lid was formed and localized in the nucleus even at the restrictive temperature. These results indicate that the modules of the 26S proteasome, namely, the core particle, base, and lid, can be formed and imported into the nucleus independently of each other. Based on these observations, we propose a model for the assembly process of the yeast 26S proteasome.
The central protease of eukaryotes, the 26S proteasome, has a 20S proteolytic core particle (CP) and an attached 19S regulatory particle (RP). The RP is further subdivided into lid and base subcomplexes. Little is known about RP assembly. Here we show that four conserved assembly factors govern biogenesis of the yeast RP base. Nas2 forms a complex with the Rpt4 and Rpt5 ATPases and enhances 26S proteasome formation in vivo and in vitro. Other RP subcomplexes contain Hsm3, which is related to mammalian proteasome subunit S5b. Hsm3 also contributes to base assembly. Larger Hsm3-containing complexes include two additional proteins, Nas6 and Rpn14, which function as assembly chaperones as well. Specific deletion combinations affecting these four factors cause severe perturbations to RP assembly. Our results demonstrate that proteasomal RP biogenesis requires multiple, functionally overlapping chaperones and suggest a model in which subunits form specific subcomplexes that then assemble into the base.
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.
Schizosaccharomyces pombe Rpn10 (SpRpn10) is a proteasomal ubiquitin (Ub) receptor located within the 19 S regulatory particle where it binds to subunits of both the base and lid subparticles. We have solved the structure of full-length SpRpn10 by determining the crystal structure of the von Willebrand factor type A domain and characterizing the full-length protein by NMR. We demonstrate that the single Ub-interacting motif (UIM) of SpRpn10 forms a 1:1 complex with Lys48-linked diUb, which it binds selectively over monoUb and Lys63-linked diUb. We further show that the SpRpn10 UIM binds to SpRpn12, a subunit of the lid subparticle, with an affinity comparable with Lys48-linked diUb. This is the first observation of a UIM binding other than a Ub fold and suggests that SpRpn12 could modulate the activity of SpRpn10 as a proteasomal Ub receptor.
Biophysics; NMR; Proteasome; Signal Transduction; Ubiquitin; X-ray Crystallography
Functional redundancy of Rpn5 in budding yeast allows its participation and function in two distinct but structurally related PCI complexes: the proteasome lid and the CSN. As a lid subunit, Rpn5 stabilizes proteasome integrity; as a CSN subunit, Rpn5 is required for enzymatic hydrolysis of Rub1/Nedd8 from cullins.
Subunit composition and architectural structure of the 26S proteasome lid is strictly conserved between all eukaryotes. This eight-subunit complex bears high similarity to the eukaryotic translation initiation factor 3 and to the COP9 signalosome (CSN), which together define the proteasome CSN/COP9/initiation factor (PCI) troika. In some unicellular eukaryotes, the latter two complexes lack key subunits, encouraging questions about the conservation of their structural design. Here we demonstrate that, in Saccharomyces cerevisiae, Rpn5 plays dual roles by stabilizing proteasome and CSN structures independently. Proteasome and CSN complexes are easily dissected, with Rpn5 the only subunit in common. Together with Rpn5, we identified a total of six bona fide subunits at roughly stoichiometric ratios in isolated, affinity-purified CSN. Moreover, the copy of Rpn5 associated with the CSN is required for enzymatic hydrolysis of Rub1/Nedd8 conjugated to cullins. We propose that multitasking by a single subunit, Rpn5 in this case, allows it to function in different complexes simultaneously. These observations demonstrate that functional substitution of subunits by paralogues is feasible, implying that the canonical composition of the three PCI complexes in S. cerevisiae is more robust than hitherto appreciated.
Eukaryotic proteasome consists of a core particle (CP), which degrades unfolded protein, and a regulatory particle (RP), which is responsible for recognition, ATP-dependent unfolding and translocation of polyubiquitinated substrate protein. In the archaea Methanocaldococcus jannaschii, the RP is a homohexameric complex of proteasome-activating nucleotidase (PAN). Here we report the crystal structures of essential elements of the archaeal proteasome: the CP, the ATPase domain of PAN, and a distal subcomplex that is likely the first to encounter substrate. The distal subcomplex contains a coiled-coil segment and an OB-fold domain, both of which appear to be conserved in the eukaryotic proteasome. The OB domains of PAN form a hexameric ring with a 13-Å pore, which likely constitutes the outermost constriction of the substrate translocation channel. These studies reveal structural codes and architecture of the complete proteasome, identify potential substrate-binding sites, and uncover unexpected asymmetry in the RP of archaea and eukaryotes.
In Arabidopsis seedlings and cauliflower
florets, Rpn6 (a proteasome non-ATPase regulatory subunit) was found in
two distinct protein complexes of ∼800 and 500 kDa, respectively. The
large complex likely represents the proteasome 19S regulator particle
(RP) because it displays the expected subunit composition and all
characteristics. The small complex, designated PR500, shares at least
three subunits with the “lid” subcomplex of 19S RP and is loosely
associated with an hsp70 protein. In Arabidopsis
COP9 signalosome mutants, PR500 was specifically absent or reduced to
an extent that correlates with the severity of the mutations.
Furthermore, PR500 was also diminished in response to potential
protein-misfolding stresses caused by the heat shock and canavanine
treatment. Immunofluorescence studies suggest that PR500 has a distinct
localization pattern and is enriched in specific nuclear foci. We
propose that PR500 may be evolved in higher plants to cope with the
frequently encountered environmental stresses.
The 'lid' subcomplex of the 26S proteasome and the COP9 signalosome (CSN complex) share a common architecture consisting of six subunits harbouring a so-called PCI domain (proteasome, CSN, eIF3) at their C-terminus, plus two subunits containing MPN domains (Mpr1/Pad1 N-terminal). The translation initiation complex eIF3 also contains PCI- and MPN-domain proteins, but seems to deviate from the 6+2 stoichiometry. Initially, the PCI domain was defined as the region of detectable sequence similarity between the components mentioned above.
During an exhaustive bioinformatical analysis of proteasome components, we detected multiple instances of tetratrico-peptide repeats (TPR) in the N-terminal region of most PCI proteins, suggesting that their homology is not restricted to the PCI domain. We also detected a previously unrecognized PCI domain in the eIF3 component eIF3k, a protein whose 3D-structure has been determined recently. By using profile-guided alignment techniques, we show that the structural elements found in eIF3k are most likely conserved in all PCI proteins, resulting in a structural model for the canonical PCI domain.
Our model predicts that the homology domain PCI is not a true domain in the structural sense but rather consists of two subdomains: a C-terminal 'winged helix' domain with a key role in PCI:PCI interaction, preceded by a helical repeat region. The TPR-like repeats detected in the N-terminal region of PCI proteins most likely form an uninterrupted extension of the repeats found within the PCI domain boundaries. This model allows an interpretation of several puzzling experimental results.
The proteasome is the most complex protease known, with a molecular mass of approximately 3 MDa and 33 distinct subunits. Recent studies reported the discovery of four chaperones that promote the assembly of a 19-subunit subcomplex of the proteasome known as the regulatory particle, or RP. These and other findings define a new and highly unusual macromolecular assembly pathway. The RP mediates substrate selection by the proteasome and injects substrates into the core particle (CP) to be degraded. A heterohexameric ring of ATPases, the Rpt proteins, is critical for RP function. These ATPases abut the CP and their C-terminal tails help to stabilize the RP-CP interface. ATPase heterodimers bound to the chaperone proteins are early intermediates in assembly of the ATPase ring. The four chaperones have the common feature of binding the C-domains of Rpt proteins, apparently a remarkable example of convergent evolution; each chaperone binds a specific Rpt subunit. The C-domains are distinct from the C-terminal tails but proximal to them. Some but probably not all of the RP chaperones appear to compete with CP for binding of the Rpt proteins, as a result of the proximity of the tails to the C-domain. This competition may underlie the release mechanism for these chaperones. Genetic studies in yeast point to the importance of the interaction between the CP and the Rpt tails in assembly, and a recent biochemical study in mammals suggests that RP assembly takes place on pre-assembled CP. These results do not exclude a parallel, CP-independent pathway of assembly. Ongoing work should soon clarify the roles of both the CP and the four chaperones in RP assembly.
proteasome; assembly; regulatory particle; AAA protein; chaperone
The 26S proteasome interacts with a number of different proteins, while the TREX-2 complex is an important component of the mRNA export machinery. In animals and yeast, members of the Ubiquitin C-terminal Hydrolase 37 (UCH37) family are found to associate with the 26S proteasome, but this has not been demonstrated in plants. The Arabidopsis UCH1 and UCH2 are orthologous to UCH37. Here, we show that UCH1 and UCH2 interact with the 26S proteasome lid subunits. In addition, the two UCHs also interact with TREX-2 components. Our data suggest that Arabidopsis UCHs may serve as a link between the 26S proteasome lid complex and the TREX-2 complex.
Arabidopsis; deubiquitinating protease; Ubiquitin C-terminal Hydrolase (UCH); 26S proteasome; TREX-2 complex
The 26S proteasome is a large cytoplasmic protease that degrades polyubiquitinated proteins to short peptides in a processive manner. The proteasome 19S regulatory subcomplex tethers the target protein via its polyubiquitin adduct and unfolds the target polypeptide, which is then threaded into the proteolytic site-containing 20S subcomplex. Hul5 is a 19S subcomplex-associated ubiquitin ligase that elongates ubiquitin chains on proteasome-bound substrates. We isolated hul5Δ as a mutation with which fusions of an unstable cyclin to stable reporter proteins accumulate as partially processed products. These products appear transiently in the wild type but are strongly stabilized in 19S ATPase mutants and in the hul5Δ mutant, supporting a role for the ATPase subunits in the unfolding of proteasome substrates before insertion into the catalytic cavity and suggesting a role for Hul5 in the processive degradation of proteins that are stalled on the proteasome.
Csn2 (Trip15/Cops2/Alien) encodes the second subunit of the COP9 signalosome (CSN), an eight-subunit heteromeric complex homologous to the lid subcomplex of the 26S proteasome. CSN is a regulator of SCF (Skp1-cullin-F-box protein)ubiquitin ligases, mostly through the enzymatic activity that deconjugates the ubiquitin-like protein Nedd8 from the SCF Cul1 component. In addition, CSN associates with protein kinase activities targeting p53, c-Jun, and IκB for phosphorylation. Csn2 also interacts with and regulates a subset of nuclear hormone receptors and is considered a novel corepressor. We report that targeted disruption of Csn2 in mice caused arrest of embryo development at the peri-implantation stage. Csn2−/− blastocysts failed to outgrow in culture and exhibited a cell proliferation defect in inner cell mass, accompanied by a slight decrease in Oct4. In addition, lack of Csn2 disrupted the CSN complex and resulted in a drastic increase in cyclin E, supporting a role for CSN in cooperating with the SCF-ubiquitin-proteasome system to regulate protein turnover. Furthermore, Csn2−/− embryos contained elevated levels of p53 and p21, which may contribute to premature cell cycle arrest of the mutant.
In recent years mass spectrometry based techniques have emerged as structural biology tools for the characterization of macromolecular, non-covalent assemblies. Many of these efforts involve preservation of intact protein complexes within the mass spectrometer, providing molecular weight measurements that allow the determination of subunit stoichiometry and real-time monitoring of protein interactions. Attempts have been made to further elucidate subunit architecture through the dissociation of subunits from the intact complex by colliding it into inert gas atoms such as argon or xenon. Unfortunately, the amount of structural information that can be derived from such strategies is limited by the nearly ubiquitous ejection of a single, unfolded subunit. Here, we present results from the gas-phase dissociation of protein-protein complexes upon collision into a surface. Dissociation of a series of tetrameric and pentameric proteins demonstrate that alternative subunit fragments, not observed through multiple collisions with gas atoms, can be generated through surface collision. Evidence is presented for the retention of individual subunit structure, and in some cases, retention of non-covalent interactions between subunits and ligands. We attribute these differences to the rapid large energy input of ion-surface collisions, which leads to the dissociation of subunits prior to the unfolding of individual monomers.
In all eukaryotic cells, 26S proteasome plays an essential role in the process of ATP-dependent protein degradation. In this review, we focus on structure characterization of the 26S proteasome. Although the progress towards a high-resolution structure of the 26S proteasome has been slow, the recently solved structures of various proteasomal subcomplexes have greatly enhanced our understanding of this large machinery. In addition to having an ATP-dependent proteolytic function, the 26S proteasome is also involved in many non-proteolytic cellular activities, which are often mediated by subunits in its 19S regulatory complex. Thus, we include a detailed discussion of the structures of 19S subunits, including proteasomal ATPases, ubiquitin receptors, deubiquitinating enzymes and subunits that contain PCI domain.
26S proteasome; structure
The 26S proteasome is the most downstream element of the ubiquitin-proteasome pathway of protein degradation. It is composed of the 20S core particle (CP) and the 19S regulatory particle (RP). The RP consists of 6 AAA-ATPases and at least 13 non-ATPase subunits. Based on a cryo-EM map of the 26S proteasome, structures of homologs, and physical protein-protein interactions we derive an atomic model of the AAA-ATPase-CP sub-complex. The ATPase order in our model (Rpt1/Rpt2/Rpt6/Rpt3/Rpt4/Rpt5) is in excellent agreement with the recently identified base-precursor complexes formed during the assembly of the RP. Furthermore, the atomic CP-AAA-ATPase model suggests that the assembly chaperone Nas6 facilitates CP-RP association by enhancing the shape complementarity between Rpt3 and its binding CP alpha subunits partners.
Proteasome; Protein degradation; Modeling; assembly; cryo-EM; Proteasome; AAA-ATPase
The small subunit (SSU) processome is a ribosome biogenesis intermediate that assembles from its subcomplexes onto the pre-18S rRNA with yet unknown order and structure. Here, we investigate the architecture of the UtpB subcomplex of the SSU processome, focusing on the interaction between the half-a-tetratricopeptide repeat (HAT) domain of Utp6 and a specific peptide in Utp21. We present a comprehensive map of the interactions within the UtpB subcomplex and further show that the N-terminal domain of Utp6 interacts with Utp18 while the HAT domain interacts with Utp21. Using a panel of point and deletion mutants of Utp6, we show that an intact HAT domain is essential for efficient pre-rRNA processing and cell growth. Further investigation of the Utp6-Utp21 interaction using both genetic and biophysical methods shows that the HAT domain binds a specific peptide ligand in Utp21, the first example of a HAT domain peptide ligand, with a dissociation constant of 10 μM.
Ubiquitin-dependent proteolysis is catalyzed by the 26S proteasome,
a dynamic complex of 32 different proteins whose mode of assembly and
mechanism of action are poorly understood, in part due to the
difficulties encountered in purifying the intact complex. Here we
describe a one-step affinity method for purifying intact 26S
proteasomes, 19S regulatory caps, and 20S core particles from budding
yeast cells. Affinity-purified 26S proteasomes hydrolyze both model
peptides and the ubiquitinated Cdk inhibitor Sic1. Affinity
purifications performed in the absence of ATP or presence of the poorly
hydrolyzable analog ATP-γ-S unexpectedly revealed that a large number
of proteins, including subunits of the skp1-cullin-F-box protein ligase
(SCF) and anaphase-promoting complex (APC) ubiquitin ligases,
copurify with the 19S cap. To identify these proteasome-interacting
proteins, we used a recently developed method that enables the
direct analysis of the composition of large protein complexes (DALPC)
by mass spectrometry. Using DALPC, we identified more than 24 putative
proteasome-interacting proteins, including Ylr421c (Daq1), which we
demonstrate to be a new subunit of the budding yeast 19S cap, and
Ygr232w (Nas6), which is homologous to a subunit of the mammalian 19S
cap (PA700 complex). Additional PIPs include the heat shock proteins
Hsp70 and Hsp82, the deubiquitinating enzyme Ubp6, and proteins
involved in transcriptional control, mitosis, tubulin assembly, RNA
metabolism, and signal transduction. Our data demonstrate that
nucleotide hydrolysis modulates the association of many proteins with
the 26S proteasome, and validate DALPC as a powerful tool for rapidly
identifying stoichiometric and substoichiometric components of large
In studies of protein complexes for which high-resolution structural data are unavailable, it is often still possible to determine both nearest-neighbor relationships between subunits and atomic-resolution details of these interactions. The eukaryotic 26S proteasome, a ~2.5 MDa protein complex with at least 33 different subunits, is a prime example. Important information about quaternary organization and assembly of proteasomes has been gained using a combination of sequence alignments with related proteins of known tertiary structure, molecular modeling, and disulfide engineering to allow oxidative crosslinking between predicted polypeptide neighbors. Here we provide detailed protocols for engineered cysteine crosslinking of yeast proteasome subunits in whole cell extracts, in active 26S proteasome complexes first isolated by native polyacrylamide gel electrophoresis, and in subcomplexes that function as potential assembly intermediates.
disulfide engineering; proteasome; site-directed mutagenesis; nondenaturing polyacrylamide gel electrophoresis (native PAGE); substrate overlay; Saccharomyces cerevisiae; yeast; immunoblotting
The AMPA-type ionotropic glutamate receptors (AMPA-Rs) are tetrameric ligand-gated ion channels that play crucial roles in synaptic transmission and plasticity. Our knowledge about the ultrastructure and subunit assembly mechanisms of intact AMPA-Rs was very limited. However, the new studies using single particle EM and X-ray crystallography are revealing important insights. For example, the tetrameric crystal structure of the GluA2cryst construct provided the atomic view of the intact receptor. In addition, the single particle EM structures of the subunit assembly intermediates revealed the conformational requirement for the dimer-to-tetramer transition during the maturation of AMPA-Rs. These new data in the field provide new models and interpretations. In the brain, the native AMPA-R complexes contain auxiliary subunits that influence subunit assembly, gating, and trafficking of the AMPA-Rs. Understanding the mechanisms of the auxiliary subunits will become increasingly important to precisely describe the function of AMPA-Rs in the brain. The AMPA-R proteomics studies continuously reveal a previously unexpected degree of molecular heterogeneity of the complex. Because the AMPA-Rs are important drug targets for treating various neurological and psychiatric diseases, it is likely that these new native complexes will require detailed mechanistic analysis in the future. The current ultrastructural data on the receptors and the receptor-expressing stable cell lines that were developed during the course of these studies are useful resources for high throughput drug screening and further drug designing. Moreover, we are getting closer to understanding the precise mechanisms of AMPA-R-mediated synaptic plasticity.
AMPA-type ionotropic glutamate receptors; AMPA receptors; Glutamate receptors; Ionotropic glutamate receptors; Biochemistry; Ultrastructure; Subunit assembly mechanism; Subunit assembly; Subunit; Biosynthesis; Biosynthetic intermediates; Electron microscopy; EM; Single particle; Single particle EM; 3D EM; Density map; Comparing EM density and X-ray structure; Cryo-negative stain single particle EM; Cryo-EM; Image reconstruction; Vesicle trafficking; Endoplasmic reticulum; Glycosylation; X-linked mental retardation; Alzheimer’s disease; Amyotrophic lateral sclerosis; Limbic encephalitis; Rasmussen’s encephalitis; GluR1; GluR2; GluR3; GluR4; GluA1; GluA2; GluA3; GluA4; Stragazin; TARP; Cornichon; CNIH; CKAMP44; SOL-1; Lurcher; Membrane biochemistry; Synaptic plasticity; Synaptic transmission; Postsynaptic density; Cell biology
In Saccharomyces cerevisiae, more than 180 assembly factors associate with preribosomes to enable folding of pre-rRNA, recruitment of ribosomal proteins, and processing of pre-rRNAs to produce mature ribosomes. To examine the molecular architecture of preribosomes and to connect this structure to functions of each assembly factor, assembly subcomplexes have been purified from preribosomal particles. The Nop7-subcomplex contains three assembly factors: Nop7, Erb1, and Ytm1, each of which is necessary for conversion of 27SA3 pre-rRNA to 27SBS pre-rRNA. However, interactions among these three proteins and mechanisms of their recruitment and function in pre-rRNPs are poorly understood. Here we show that Ytm1, Erb1, and Nop7 assemble into preribosomes in an interdependent manner. We identified which domains within Ytm1, Erb1, and Nop7 are necessary for their interaction with each other and are sufficient for recruitment of each protein into preribosomes. Dominant negative effects on growth and ribosome biogenesis caused by overexpressing truncated Ytm1, Erb1, or Nop7 constructs, and recessive phenotypes of the truncated proteins revealed not only interaction domains but also other domains potentially important for each protein to function in ribosome biogenesis. Our data suggest a model for the architecture of the Nop7-subcomplex and provide potential functions of domains of each protein.
In eukaryotic cells, the vast majority of proteins in the cytosol and nucleus are degraded via the proteasome-ubiquitin pathway. The 26S proteasome is a huge protein degradation machine of 2.5 MDa, built of approximately 35 different subunits. It contains a proteolytic core complex, the 20S proteasome and one or two 19S regulatory complexes which associate with the termini of the barrel-shaped 20S core. The 19S regulatory complex serves to recognize ubiquitylated target proteins and is implicated to have a role in their unfolding and translocation into the interior of the 20S complex where they are degraded into oligopeptides. While much progress has been made in recent years in elucidating the structure, assembly and enzymatic mechanism of the 20S complex, our knowledge of the functional organization of the 19S regulator is rather limited. Most of its subunits have been identified, but specific functions can be assigned to only a few of them.
The COP9/signalosome complex is highly conserved in evolution and possesses significant structural similarity to the 19S regulatory lid complex of the proteasome. It also shares limited similarity to the translation initiation factor eIF3. The signalosome interacts with multiple cullins in mammalian cells. In the fission yeast Schizosaccharomyces pombe, the Csn1 subunit is required for the removal of covalently attached Nedd8 from Pcu1, one of three S. pombe cullins. It remains unclear whether this activity is required for all the functions ascribed to the signalosome. We previously identified Csn1 and Csn2 as signalosome subunits in S. pombe. csn1 and csn2 null mutants are DNA damage sensitive and exhibit slow DNA replication. Two further putative subunits, Csn4 and Csn5, were identified from the S. pombe genome database. Herein, we characterize null mutations of csn4 and csn5 and demonstrate that both genes are required for removal of Nedd8 from the S. pombe cullin Pcu1 and that their protein products associate with Csn1 and Csn2. However, neither csn4 nor csn5 null mutants share the csn1 and csn2 mutant phenotypes. Our data suggest that the subunits of the signalosome cannot be considered as a distinct functional unit and imply that different subunits of the signalosome mediate distinct functions.