Identification of the 19S regulatory particle subunits from the rice 26S proteasome.

The 26S proteasome, a protein complex consisting of a 20S proteasome and a pair of 19S regulatory particles (RP), is involved in ATP-dependent proteolysis in eukaryotes. In yeast, the RP contains six different ATPase subunits and, at least, 11 non-ATPase subunits. In this study, we identified the rice homologs of yeast RP subunit genes from the rice expressed sequence tag (EST) library. The complete nucleotide sequences of the homologs for five ATPase subunits, OsRpt1, OsRpt2, OsRpt4, OsRpt5 and OsRpt6, and five non-ATPase subunits, OsRpn7, OsRpn8, OsRpn10, OsRpn11 and OsRpn12, and the partial sequences of one ATPase subunit, OsRpt3, and six non-ATPase subunits, OsRpn1, OsRpn2, OsRpn3, OsRpn5, OsRpn6 and OsRpn9, were determined. Gene homologs of four ATPase subunits, OsRpt1, OsRpt2, OsRpt4 and OsRpt5, and three non- ATPase subunits, OsRpn1, OsRpn2 and OsRpn9, were found to be encoded by duplicated genes. The rice RP was purified by immunoaffinity chromatography with a Protein A column immobilized antibody against rice 20S proteasome, and the subunit composition was determined. The homologs obtained from the rice EST library were identified as genes encoding subunits of RP purified from rice, including the both products of duplicated genes by using electrospray ionization quadrupole time-of-flight mass spectrometry. Post-translational modifications and processing in rice RP subunits were also identified. Various types of RP complex with different subunit compositions are present in rice cells, suggesting the multiple functions of rice proteasome.     

Stable incorporation of ATPase subunits into 19 S regulatory particle of human proteasome requires nucleotide binding and C-terminal tails

The 26 S proteasome is a large multi-subunit protein complex that degrades ubiquitinated proteins in eukaryotic cells. Proteasome assembly is a complex process that involves formation of six- and seven-membered ring structures from homologous subunits. Here we report that the assembly of hexameric Rpt ring of the 19 S regulatory particle (RP) requires nucleotide binding but not ATP hydrolysis. Disruption of nucleotide binding to an Rpt subunit by mutation in the Walker A motif inhibits the assembly of the Rpt ring without affecting heterodimer formation with its partner Rpt subunit. Coexpression of the base assembly chaperones S5b and PAAF1 with mutant Rpt1 and Rpt6, respectively, relieves assembly inhibition of mutant Rpts by facilitating their interaction with adjacent Rpt dimers. The mutation in the Walker B motif which impairs ATP hydrolysis does not affect Rpt ring formation. Incorporation of a Walker B mutant Rpt subunit abrogates the ATPase activity of the 19 S RP, suggesting that failure of the mutant Rpt to undergo the conformational transition from an ATP-bound to an ADP-bound state impairs conformational changes in the other five wild-type Rpts in the Rpt ring. In addition, we demonstrate that the C-terminal tails of Rpt subunits possessing core particle (CP)-binding affinities facilitate the cellular assembly of the 19 S RP, implying that the 20 S CP may function as a template for base assembly in human cells. Taken together, these results suggest that the ATP-bound conformational state of an Rpt subunit with the exposed C-terminal tail is competent for cellular proteasome assembly.     

Transfer of Ho endonuclease and Ufo1 to the proteasome by the UbL-UbA shuttle protein, Ddi1, analysed by complex formation in vitro

The F-box protein, Ufo1, recruits Ho endonuclease to the SCF(Ufo1) complex for ubiquitylation. Both ubiquitylated Ho and Ufo1 are transferred by the UbL-UbA protein, Ddi1, to the 19S Regulatory Particle (RP) of the proteasome for degradation. The Ddi1-UbL domain binds Rpn1 of the 19S RP, the Ddi1-UbA domain binds ubiquitin chains on the degradation substrate. Here we used complex reconstitution in vitro to identify stages in the transfer of Ho and Ufo1 from the SCF(Ufo1) complex to the proteasome. We report SCF(Ufo1) complex at the proteasome formed in the presence of Ho. Subsequently Ddi1 is recruited to this complex by interaction between the Ddi1-UbL domain and Ufo1. The core of Ddi1 binds both Ufo1 and Rpn1; this interaction confers specificity of SCF(Ufo1) for Ddi1. The substrate-shield model predicts that Ho would protect Ufo1 from degradation and we find that Ddi1 binds Ho, Ufo1, and Rpn1 simultaneously forming a complex for transfer of Ho to the 19S RP. In contrast, in the absence of Ho, Rpn1 displaces Ufo1 from Ddi1 indicating a higher affinity of the Ddi1-UbL for the 19S RP. However, at high Rpn1 levels there is synergistic binding of Ufo1 to Ddi1 that is dependent on the Ddi1-UbA domain. Our interpretation is that in the absence of substrate, the Ddi1-UbL binds Rpn1 while the Ddi1-UbA binds ubiquitin chains on Ufo1. This would promote degradation of Ufo1 and disassembly of SCF(Ufo1) complexes.     

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.     

Mass spectrometric analysis of expression of ATPase subunits encoded by duplicated genes in the 19S regulatory particle of rice 26S proteasome

The 26S proteasome consisting of a 20S proteasome and a pair of 19S regulatory particles (RP) plays important roles in degradation of the ubiquitinated protein in eukaryotic cells. The RP consists of six different ATPase subunits and, at least, 11 non-ATPase subunits. In rice, we previously identified duplicated genes encoding four ATPase subunits, OsRpt1, OsRpt2, OsRpt4, and OsRpt5. In this study, the genomic sequences of all rice ATPase subunits were identified from the rice genome database and the genomic structure of ATPase subunit genes was determined. The rice RP was purified, and the ATPase subunit isoforms encoded by three pairs of duplicated genes, OsRpt2a/OsRpt2b, OsRpt4a/OsRpt4b, and OsRpt5a/OsRpt5b, were identified in RP by using electrospray ionization quadrupole time-of-flight mass spectrometry. The relative amounts and the expression patterns of these ATPase subunit isoforms in the bran were found to be different from those of the callus, suggesting the presence of multiform 19S regulatory particles engaged in the tissue-specific protein metabolism.     

NOXA, a sensor of proteasome integrity, is degraded by 26S proteasomes by an ubiquitin-independent pathway that is blocked by MCL-1

Ubiquitin (Ub)-mediated proteasome-dependent proteolysis is critical in regulating multiple biological processes including apoptosis. We show that the unstructured BH3-only protein, NOXA, is degraded by an Ub-independent mechanism requiring 19S regulatory particle (RP) subunits of the 26S proteasome, highlighting the possibility that other unstructured proteins reported to be degraded by 20S proteasomes in vitro may be bona fide 26S proteasome substrates in vivo. A lysine-less NOXA (NOXA-LL) mutant, which is not ubiquitinated, is degraded at a similar rate to wild-type NOXA. Myeloid cell leukemia 1, but not other anti-apoptotic BCL-2 family proteins, stabilizes NOXA by interaction with the NOXA BH3 domain. Depletion of 19S RP subunits, but not alternate proteasome activator REG subunits, increases NOXA half-life in vivo. A NOXA-LL mutant, which is not ubiquitinated, also requires an intact 26S proteasome for degradation. Depletion of the 19S non-ATPase subunit, PSMD1 induces NOXA-dependent apoptosis. Thus, disruption of 26S proteasome function by various mechanisms triggers the rapid accumulation of NOXA and subsequent cell death strongly implicating NOXA as a sensor of 26S proteasome integrity.     

Sem1p is a novel subunit of the 26 S proteasome from Saccharomyces cerevisiae

The 26 S proteasome, which catalyzes degradation of polyubiquitinated proteins, is composed of the 20 S proteasome and the 19 S regulatory particle (RP). The RP is composed of the lid and base subcomplexes and regulates the catalytic activity of the 20 S proteasome. In this study, we carried out affinity purification of the lid and base subcomplexes from the tagged strains of Saccharomyces cerevisiae, and we found that the lid contains a small molecular mass protein, Sem1. The Sem1 protein binds with the 26 S proteasome isolated from a mutant with deletion of SEM1 but not with the 26 S proteasome from the wild type. The lid lacking Sem1 is unstable at a high salt concentration. The 19 S RP was immunoprecipitated together with Sem1 by immunoprecipitation using hemagglutinin epitope-tagged Sem1 as bait. Degradation of polyubiquitinated proteins in vivo or in vitro is impaired in the Sem1-deficient 26 S proteasome. In addition, genetic interaction between SEM1 and RPN10 was detected. The human Sem1 homologue hDSS1 was found to be a functional homologue of Sem1 and capable of interacting with the human 26 S proteasome. The results suggest that Sem1, possibly hDSS1, is a novel subunit of the 26 S proteasome and plays a role in ubiquitin-dependent proteolysis.     

Structural basis for specific recognition of Rpt1p, an ATPase subunit of 26 S proteasome, by proteasome-dedicated chaperone Hsm3p

The 26 S proteasome is a 2.5-MDa molecular machine that degrades ubiquitinated proteins in eukaryotic cells. It consists of a proteolytic core particle and two 19 S regulatory particles (RPs) composed of 6 ATPase (Rpt) and 13 non-ATPase (Rpn) subunits. Multiple proteasome-dedicated chaperones facilitate the assembly of the proteasome, but little is known about the detailed mechanisms. Hsm3, a 19 S RP dedicated chaperone, transiently binds to the C-terminal domain of the Rpt1 subunit and forms a tetrameric complex, Hsm3-Rpt1-Rpt2-Rpn1, during maturation of the ATPase ring of 19 S RP. To elucidate the structural basis of Hsm3 function, we determined the crystal structures of Hsm3 and its complex with the C-terminal domain of the Rpt1 subunit (Rpt1C). Hsm3 has a C-shaped structure that consists of 11 HEAT repeats. The structure of the Hsm3-Rpt1C complex revealed that the interacting surface between Hsm3 and Rpt1 is a hydrophobic core and a complementary charged surface. Mutations in the Hsm3-Rpt1 surface resulted in the assembly defect of the 26 S proteasome. Furthermore, a structural model of the Hsm3-Rpt ring complex and an in vitro binding assay suggest that Hsm3 can bind Rpt2 in addition to Rpt1. Collectively, our results provide the structural basis of the molecular functions of Hsm3 for the RP assembly.     

Identification of a specific motif of the DSS1 protein required for proteasome interaction and p53 protein degradation

Deleted in Split hand/Split foot 1 (DSS1) was previously identified as a novel 12-O-tetradecanoylphorbol-13-acetate (TPA)-inducible gene with possible involvement in early event of mouse skin carcinogenesis. The mechanisms by which human DSS1 (HsDSS1) exerts its biological effects via regulation of the ubiquitin-proteasome system (UPS) are currently unknown. Here, we demonstrated that HsDSS1 regulates the human proteasome by associating with it in the cytosol and nucleus via the RPN3/S3 subunit of the 19S regulatory particle (RP). Molecular anatomy of HsDSS1 revealed an RPN3/S3-interacting motif (R3IM), located at amino acid residues 15 to 21 of the NH(2) terminus. Importantly, negative charges of the R3IM motif were demonstrated to be required for proteasome interaction and binding to poly-ubiquitinated substrates. Indeed, the R3IM motif of HsDSS1 protein alone was sufficient to replace the ability of intact HsDSS1 protein to pull down proteasome complexes and protein substrates with high-molecular mass ubiquitin conjugates. Interestingly, this interaction is highly conserved throughout evolution from humans to nematodes. Functional study, lowering the levels of the endogenous HsDSS1 using siRNA, indicates that the R3IM/proteasome complex binds and targets p53 for ubiquitin-mediated degradation via gankyrin-MDM2/HDM2 pathway. Most significantly, this work indicates that the R3IM motif of HsDSS1, in conjunction with the complexes of 19S RP and 20S core particle (CP), regulates proteasome interaction through RPN3/S3 molecule, and utilizes a specific subset of poly-ubiquitinated p53 as a substrate.     

Molecular characterization of NbPAF encoding the alpha6 subunit of the 20S proteasome in Nicotiana benthamiana

The 26S proteasome involved in degradation of proteins covalently modified with polyubiquitin consists of the 20S proteasome and 19S regulatory complex. The NbPAF gene encoding the alpha6 subunit of the 20S proteasome was identified from Nicotiana benthamiana. NbPAF exhibits high sequence homology with the corresponding genes from Arabidopsis, human and yeast. The deduced amino acid sequence of NbPAF reveals that this protein contains the proteasome alpha-type subunits signature and nuclear localization signal at the N-terminus. The genomic Southern blot analysis suggests that the N. benthamiana genome contains one copy of NbPAF. The NbPAF mRNA was detected abundantly in flowers and weakly in roots and stems, but it was almost undetectable in mature leaves. In response to stresses, accumulation of the NbPAF mRNA was stimulated by methyl jasmonate, NaCl and salicylic acid, but not by abscisic acid and cold treatment in leaves. The NbPAF-GFP fusion protein was localized in the cytoplasm and nucleus.     

Functional Characterization of Rpn3 Uncovers a Distinct 19S Proteasomal Subunit Requirement for Ubiquitin-Dependent Proteolysis of Cell Cycle Regulatory Proteins in Budding Yeast

By selectively eliminating ubiquitin-conjugated proteins, the 26S proteasome plays a pivotal role in a large variety of cellular regulatory processes, particularly in the control of cell cycle transitions. Access of ubiquitinated substrates to the inner catalytic chamber within the 20S core particle is mediated by the 19S regulatory particle (RP), whose subunit composition in budding yeast has been recently elucidated. In this study, we have investigated the cell cycle defects resulting from conditional inactivation of one of these RP components, the essential non-ATPase Rpn3/Sun2 subunit. Using temperature-sensitive mutant alleles, we show that rpn3 mutations do not prevent the G(1)/S transition but cause a metaphase arrest, indicating that the essential Rpn3 function is limiting for mitosis. rpn3 mutants appear severely compromised in the ubiquitin-dependent proteolysis of several physiologically important proteasome substrates. Thus, RPN3 function is required for the degradation of the G(1)-phase cyclin Cln2 targeted by SCF; the S-phase cyclin Clb5, whose ubiquitination is likely to involve a combination of E3 (ubiquitin protein ligase) enzymes; and anaphase-promoting complex targets, such as the B-type cyclin Clb2 and the anaphase inhibitor Pds1. Our results indicate that the Pds1 degradation defect of the rpn3 mutants most likely accounts for the metaphase arrest phenotype observed. Surprisingly, but consistent with the lack of a G(1) arrest phenotype in thermosensitive rpn3 strains, the Cdk inhibitor Sic1 exhibits a short half-life regardless of the RPN3 genotype. In striking contrast, Sic1 turnover is severely impaired by a temperature-sensitive mutation in RPN12/NIN1, encoding another essential RP subunit. While other interpretations are possible, these data strongly argue for the requirement of distinct RP subunits for efficient proteolysis of specific cell cycle regulators. The potential implications of these data are discussed in the context of possible Rpn3 function in multiubiquitin-protein conjugate recognition by the 19S proteasomal regulatory particle.     

Isolation and characterization of a novel 530-kDa protein complex (PC530) capable of associating with the 20S proteasome from starfish oocytes

A novel protein complex called PC530 was purified concomitantly with proteasomes from oocytes of the starfish, Asterina pectinifera, by chromatography with DEAE-cellulose, phosphocellulose, Mono Q, and Superose 6 columns. The molecular mass of this complex was estimated to be 530 kDa by Ferguson plot analysis and about 500 kDa by Superose 6 gel filtration. Since the 1500-kDa proteasome fractions contain the PC530 subunits as well as the 20S proteasomal subunits, and also since the purified PC530 and the 20S proteasome were cross-linked with a bifunctional cross-linking reagent, it is thought that PC530 is able to associate with the 20S proteasome. The PC530 comprises six main subunits with molecular masses of 105, 70, 50, 34, 30, and 23 kDa. The 70-kDa subunit showed a sequence similarity to the S3/p58/Sun2/Rpn3p subunit of the 26S proteasome, whereas the other subunits showed little or no appreciable similarity to the mammalian and yeast regulatory subunits. These results indicate that starfish oocytes contain a novel 530-kDa protein complex capable of associating with the 20S proteasome, which is distinctly different from PA700 or the 19S regulatory complex in molecular size and subunit composition.     

Nob1p, a new essential protein, associates with the 26S proteasome of growing saccharomyces cerevisiae cells

Nob1p, which interacts with Nin1p/Rpn12, a subunit of the 19S regulatory particle (RP) of the yeast 26S proteasome, has been identified by two-hybrid screening. NOB1 was found to be an essential gene, encoding a protein of 459 amino acid residues. Nob1p was detected in growing cells but not in cells in the stationary phase. During the transition to the stationary phase, Nob1p was degraded, at least in part, by the 26S proteasome. Nob1p was found only in proteasomal fractions in a glycerol gradient centrifugation profile and immuno-coprecipitated with Rpt1, which is an ATPase component of the yeast proteasomes. These results suggest that association of Nob1p with the proteasomes is essential for the function of the proteasomes in growing cells.     

Use of RNA interference and complementation to study the function of the Drosophila and human 26S proteasome subunit S13

The S13 subunit (also called Pad1, Rpn11, and MPR1) is a component of the 19S complex, a regulatory complex essential for the ubiquitin-dependent proteolytic activity of the 26S proteasome. To address the functional role of S13, we combined double-stranded RNA interference (RNAi) against the Drosophila proteasome subunit DmS13 with expression of wild-type and mutant forms of the homologous human gene, HS13. These studies show that DmS13 is essential for 26S function. Loss of the S13 subunit in metazoan cells leads to increased levels of ubiquitin conjugates, cell cycle defects, DNA overreplication, and apoptosis. In vivo assays using short-lived proteasome substrates confirmed that the 26S ubiquitin-dependent degradation pathway is compromised in S13-depleted cells. In complementation experiments using Drosophila cell lines expressing HS13, wild-type HS13 was found to fully rescue the knockdown phenotype after DmS13 RNAi treatment, while an HS13 containing mutations (H113A-H115A) in the proposed isopeptidase active site was unable to rescue. A mutation within the conserved MPN/JAMM domain (C120A) abolished the ability of HS13 to rescue the Drosophila cells from apoptosis or DNA overreplication. However, the C120A mutant was found to partially restore normal levels of ubiquitin conjugates. The S13 subunit may possess multiple functions, including a deubiquitinylating activity and distinct activities essential for cell cycle progression that require the conserved C120 residue.     

Assembly of the 26S proteasome is regulated by phosphorylation of the p45/Rpt6 ATPase subunit

We investigated whether the assembly/disassembly of the 26S proteasome is regulated by phosphorylation/dephosphorylation. The regulatory complex disassembled from the 26S proteasome was capable of phosphorylating the p45/Sug1/Rpt6 subunit, suggesting that the protein kinase is activated upon dissociation of the 26S proteasome or that the phosphorylation site of p45 becomes susceptible to the protein kinase. In addition, the p45-phosphorylated regulatory complex was found to be incorporated into the 26S proteasome. When the 26S proteasome was treated with alkaline phosphatase, it was dissociated into the 20S proteasome and the regulatory complex. Furthermore, the p45 subunit and the C3/alpha2 subunit were cross-linked with DTBP, whereas these subunits were not cross-linked by dephosphorylating the 26S proteasome. These results indicate that the 26S proteasome is disassembled into the constituent subcomplexes by dephosphorylation and that it is assembled by phosphorylation of p45 by a protein kinase, which is tightly associated with the regulatory complex. It was also revealed that the p45 subunit is directly associated with the 20S proteasome alpha-subunit C3 in a phosphorylation-dependent manner.     

Genetic analyses of the Arabidopsis 26S proteasome regulatory particle reveal its importance during light stress and a specific role for the N-Terminus of RPT2 in development.

The 26S proteasome subunit RPT2 is a component of the hexameric ring of AAA-ATPases that forms the base of the 19S regulatory particle (RP). This subunit has specific roles in the yeast and mammalian proteasomes by helping promote assembly of the RP with the 20S core protease (CP) and gate the CP to prevent indiscriminate degradation of cytosolic and nuclear proteins. In plants, this subunit plays an important role in diverse processes that include shoot and root apical meristem maintenance, cell size regulation, trichome branching, and stress responses. Recently, we reported that mutants in RPT2 and several other RP subunits have reduced histone levels, suggesting that at least some of the pleiotropic phenotypes observed in these plants result from aberrant nucleosome assembly. Here, we expand our genetic analysis of RPT2 in Arabidopsis to shed additional light on the roles of the N- and C-terminal ends. We also present data showing that plants bearing mutations in RP subunit genes have their seedling phenotypes exacerbated by prolonged light exposure.     

Mapping the Structural Topology of the Yeast 19S Proteasomal Regulatory Particle Using Chemical Cross-linking and Probabilistic Modeling

Structural characterization of proteasome complexes is an essential step toward understanding the ubiquitin-proteasome system. Currently, high resolution structures are not available for the 26S proteasome holocomplex as well as its subcomplex, the 19S regulatory particle (RP). Here we have employed a novel integrated strategy combining chemical cross-linking with multistage tandem mass spectrometry to define the proximity of subunits within the yeast 19S RP to elucidate its topology. This has resulted in the identification of 174 cross-linked peptides of the yeast 19S RP, representing 43 unique lysine-lysine linkages within 24 nonredundant pair-wise subunit interactions. To map the spatial organization of the 19S RP, we have developed and utilized a rigorous probabilistic framework to derive maximum likelihood (ML) topologies based on cross-linked peptides determined from our analysis. Probabilistic modeling of the yeast 19S AAA-ATPase ring (i.e., Rpt1–6) has produced an ML topology that is in excellent agreement with known topologies of its orthologs. In addition, similar analysis was carried out on the 19S lid subcomplex, whose predicted ML topology corroborates recently reported electron microscopy studies. Together, we have demonstrated the effectiveness and potential of probabilistic modeling for unraveling topologies of protein complexes using cross-linking data. This report describes the first study of the 19S RP topology using a new integrated strategy combining chemical cross-linking, mass spectrometry, and probabilistic modeling. Our results have provided a solid foundation to advance our understanding of the 19S RP architecture at peptide level resolution. Furthermore, our methodology developed here is a valuable proteomic tool that can be generalized for elucidating the structures of protein complexes.Basic cellular homeostasis depends on the regulated protein degradation and turnover by the ubiquitin-proteasome system (1, 2). Central to this pathway is the 26S proteasome complex, which is responsible for ubiquitin/ATP-dependent protein degradation (3–5). The 26S holocomplex is a megadalton-sized protein assembly consisting of the 20S catalytic core particle (CP)¹ and the 19S regulatory particle (RP). The eukaryotic 20S CP is composed of two copies of 14 nonidentical subunits (α_1–7 and β_1–7) arranged into four stacked heptameric rings in an order of α₇β₇β₇α₇. The crystal structure and topology of the highly ordered 20S CP has been resolved and is evolutionarily conserved (6). Although α subunits of the 20S CP are essential for the assembly of the complex and its interactions with the regulatory complex, three catalytic β subunits (β1, β2, and β5) harbor various catalytic activities responsible for regulated proteasomal degradation. The 19S RP is composed of 19 subunits, which forms two subcomplexes, the base consisting of six related AAA-ATPase (Rpt1–6) and four non-ATPase (Rpn1, Rpn2, Rpn10, and Rpn13) subunits and the lid containing nine non-ATPase subunits (Rpn3, Rpn5–9, Rpn11, Rpn12, and Rpn15/Sem1) (7, 8). In comparison with the 20S core, the function and structure of the 19S RP is much less well understood. Nevertheless, it is believed that the 19S RP is involved in multiple functions including recognition of polyubiquitinated substrates (9, 10), cleavage of the polyubiquitin chains to recycle ubiquitin (11), unfolding of substrates, assisting in opening the gate of the 20S chamber, and subsequently translocating the unfolded substrates into the catalytic chamber (4, 12–14). The six AAA-ATPase subunits (Rpt1–6), which directly interact with the 20S α-ring, function as a molecular chaperone responsible for protein unfolding and are involved in substrate translocation and modulating gating of the CP (5, 15). Although detailed functions for most of the 19S non-ATPase subunits remain elusive, Rpn11 is known to carry an Mpr1p and Pad1p N-termini (MPN) domain, which harbors an essential deubiquitination activity responsible for cleaving polyubiquitin chains from proteasomal substrates (11, 16). In addition, two proteasome subunits, Rpn10 and Rpn13, have been identified as ubiquitin receptors, which are important in docking ubiquitinated substrates to the proteasome for degradation (4). Moreover, the two largest proteasome subunits, Rpn1 and Rpn2, interact with a variety of proteins including ubiquitin receptors and deubiquitinases and thus may function as scaffolding proteins to assist proteasomal degradation. Thus far, no atomic resolution structures are available for either the 19S RP or the 26S holocomplex. New insights of the overall topology of the 19S RP will illuminate protein interactions within, thus providing evidence for its otherwise unknown functions.Although many studies have been performed to characterize the 19S structure utilizing various techniques including cryo-EM (17, 18) and native mass spectrometry (19), details on spatial interfaces and subunit interconnectivity of the 19S RP remain to be unraveled. During the course of our study, the rough topology of the 19S RP was determined by cryo-EM alone (20) or coupled with other approaches (21); nevertheless more detailed information at the peptide or atomic level is still required. In addition to technological limitations in current approaches, the highly dynamic and heterogeneous nature of the 19S RP may attribute to the difficulty in obtaining its high resolution structure. In recent years, chemical cross-linking coupled with mass spectrometry (XL-MS) has become an attractive alternative for structure analysis of proteins and protein complexes (22, 23). The ability of XL-MS to identify interaction interfaces between proteins allows us to define low resolution protein topology. In addition to protein interaction networks and the site of protein interactions at binding interfaces, cross-linking analysis can reveal information about the spatial distance between cross-linked amino acids on the surface of folded proteins. Although such knowledge only reveals the maximum distance given by the length of the cross-linker and can be influenced by protein conformational flexibility, it can be used as the distance constraint for molecular modeling of protein folds and complex topologies, i.e., the arrangement of the constituents of a complex in space. A recent study by Chen et al. (24) on yeast RNA polymerase II (RNAPII) complex has exemplified the power of XL-MS in elucidating the architecture of large multisubunit complexes. Although effective, cross-linking studies have been challenging because of the low abundance of cross-linked products and the inherent complexity of sequencing interlinked peptides by MS for unambiguous identification. To facilitate MS detection and identification of cross-linked products, we have recently developed a novel homobifunctional amine reactive, low energy MS-cleavable cross-linker, disuccinimidyl sulfoxide (DSSO), and successfully applied it to cross-link the yeast 20S proteasome for rapid, accurate, and simplified determination of protein interaction interfaces within the complex (25). The unique functionality of our cross-linking reagent and specialized bioinformatics tools significantly increase our confidence and speed in the identification of cross-linked products when compared with cross-linking studies using traditional noncleavable reagents. Current cross-linking studies have been focused on protein complexes with known crystal structures, but topological structures of protein complexes based primarily on cross-linking data have not yet been reported. This is due to the lack of computational tools that use cross-linking data to deduce the spatial organization of subunits in a given complex. To define the architecture of the yeast 19S RP, we have characterized the proximity and interconnectivity of the subunits by employing our newly developed cross-linking strategy. The resulting cross-linking information serves as a basis for a rigorous probabilistic analysis to obtain the maximum likelihood (ML) topology. This strategy is developed by first analyzing our cross-linking data for the 19S six-member AAA-ATPase base ring, as the topology ordering of yeast orthologs has been recently determined (14, 26–28). The effectiveness of this new probabilistic platform is supported by the agreement between our derived ML topology of the AAA-ATPase base ring and previous reports. When the same probabilistic approach is applied to the 19S lid subcomplex, the resulting topology is also in agreement with recently proposed models (20, 21). This work represents the first application of probabilistic modeling of protein complexes based solely on cross-link data, establishing a new workflow for future structural analysis of large protein complexes using XL-MS.     

GFP-labelling of 26S proteasomes in living yeast: insight into proteasomal functions at the nuclear envelope/rough ER

26S proteasomes are multisubunit protease complexes that play the central role in the ubiquitin-dependent protein degradation pathway. The proteolytically active core is formed by the 20S proteasome. Regulatory subunits, principally the 19S cap complex, confer the specificity towards ubiquitinated substrates and an ATP-dependence on proteolysis. Green fluorescence protein (GFP)-tagged versions of either an -subunit of the 20S core or an ATPase subunit of the 19S cap complex were functionally incorporated into the protease complex, thus allowing to monitor the subcellular distribution of 26S proteasomes in living yeast. Our localization studies suggest that proteasomal proteolysis mainly occurs at the nuclear envelope (NE)/rough ER. Implications of proteasomal functions at the NE/rough ER are discussed in the context of published work on ER degradation and with regard to possible targeting mechanisms.     

Functional analysis of the proteasome regulatory particle

We have developed S. cerevisiae as a model system for mechanistic studies of the 26S proteasome. The subunits of the yeast 19S complex, or regulatory particle (RP), have been defined, and are closely related to those of mammalian proteasomes. The multiubiquitin chain binding subunit (S5a/Mcb1/Rpn10) was found, surprisingly, to be nonessential for the degradation of a variety of ubiquitin-protein conjugates in vivo. Biochemical studies of proteasomes from rpn10 mutants revealed the existence of two structural subassemblies within the RP, the lid and the base. The lid and the base are both composed of 8 subunits. By electron microscopy, the base and the lid correspond to the proximal and distal masses of the RP, respectively. The base is sufficient to activate the 20S core particle for degradation of peptides, but the lid is required for ubiquitin-dependent degradation. The lid subunits share sequence motifs with components of the COP9/signalosome complex, suggesting that these functionally diverse particles have a common evolutionary ancestry. Analysis of equivalent point mutations in the six ATPases of the base indicate that they have well-differentiated functions. In particular, mutations in one ATPase gene, RPT2, result in an unexpected defect in peptide hydrolysis by the core particle. One interpretation of this result is that Rpt2 participates in gating of the channel through which substrates enter the core particle.