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

2001; Springer Nature; Volume: 20; Issue: 24 Linguagem: Inglês

10.1093/emboj/20.24.7096

1460-2075

Hongyong Fu,

Endoplasmic Reticulum Stress and Disease

Article17 December 2001free access Subunit interaction maps for the regulatory particle of the 26S proteasome and the COP9 signalosome Hongyong Fu Corresponding Author Hongyong Fu Institute of Botany, Academia Sinica, 128, Sec 2, Academy Road, Taipei, Taiwan, 115 Republic of China Search for more papers by this author Noa Reis Noa Reis Department of Biology and Institute for Catalysis (ICST), The Technion, 32000 Haifa, Israel Search for more papers by this author Yenfen Lee Yenfen Lee Institute of Botany, Academia Sinica, 128, Sec 2, Academy Road, Taipei, Taiwan, 115 Republic of China Search for more papers by this author Michael H. Glickman Corresponding Author Michael H. Glickman Department of Biology and Institute for Catalysis (ICST), The Technion, 32000 Haifa, Israel Search for more papers by this author Richard D. Vierstra Corresponding Author Richard D. Vierstra Cellular and Molecular Biology Program and the Department of Horticulture, University of Wisconsin-Madison, Madison, WI, 53706 USA Search for more papers by this author Hongyong Fu Corresponding Author Hongyong Fu Institute of Botany, Academia Sinica, 128, Sec 2, Academy Road, Taipei, Taiwan, 115 Republic of China Search for more papers by this author Noa Reis Noa Reis Department of Biology and Institute for Catalysis (ICST), The Technion, 32000 Haifa, Israel Search for more papers by this author Yenfen Lee Yenfen Lee Institute of Botany, Academia Sinica, 128, Sec 2, Academy Road, Taipei, Taiwan, 115 Republic of China Search for more papers by this author Michael H. Glickman Corresponding Author Michael H. Glickman Department of Biology and Institute for Catalysis (ICST), The Technion, 32000 Haifa, Israel Search for more papers by this author Richard D. Vierstra Corresponding Author Richard D. Vierstra Cellular and Molecular Biology Program and the Department of Horticulture, University of Wisconsin-Madison, Madison, WI, 53706 USA Search for more papers by this author Author Information Hongyong Fu 1, Noa Reis2, Yenfen Lee1, Michael H. Glickman 2 and Richard D. Vierstra 3 1Institute of Botany, Academia Sinica, 128, Sec 2, Academy Road, Taipei, Taiwan, 115 Republic of China 2Department of Biology and Institute for Catalysis (ICST), The Technion, 32000 Haifa, Israel 3Cellular and Molecular Biology Program and the Department of Horticulture, University of Wisconsin-Madison, Madison, WI, 53706 USA *Corresponding authors. E-mail: [email protected] or E-mail: [email protected] or E-mail: [email protected] The EMBO Journal (2001)20:7096-7107https://doi.org/10.1093/emboj/20.24.7096 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Introduction The 26S proteasome is a 2 MDa ATP-dependent protease responsible for the bulk of non-lysosomal proteolysis in eukaryotes, often using the covalent modification of proteins by ubiquitylation to assist in target recognition (Voges et al., 1999). It consists of a 20S proteolytic core particle (CP) and a 19S regulatory particle (RP). The CP is an ATP-independent peptidase, containing post-acidic, post-basic and post-hydrophobic hydrolyzing activities. Its cylindrical shape is created by the assembly of four stacked heptameric rings: the internal rings are each composed of seven related β-subunits and the outer rings are each composed of seven related α-subunits arranged in a α1−7/β1−7/β1−7/α1−7 configuration (Voges et al., 1999). The proteolytic active sites involving the β1, β2 and β5 subunits reside within the central chamber. A small channel formed by the α-subunit rings restricts access to this chamber such that only unfolded proteins may enter. In yeast and probably other eukaryotes, it appears that the channel is gated by flexible N-terminal extensions in the α-subunits to control substrate entry and product exit (Glickman, 2000). Appended to either or both ends of the CP is the RP that confers both ubiquitin and ATP dependence to the 26S proteasome (Voges et al., 1999). Its proposed functions are to recognize and help unfold substrates, open the channel and translocate the substrates into the CP for proteolysis. The RP can be dissociated further into two subcomplexes, the base that directly associates with the CP and a peripheral lid. The base is composed of three non-ATPase subunits (Rpn1, 2 and 10) and six ATPase subunits (Rpt1–6) that are members of the AAA-ATPase family. It is presumed that the Rpt subunits assemble into a six-membered ring similar to the ATP-dependent ClpAP and HslVU proteases, and likewise use ATP hydrolysis to facilitate channel opening and target unfolding (Strickland et al., 2000). The lid is a 400 kDa complex, assembled from at least eight additional Rpn subunits (Rpn3, 5–9, 11 and 12) (Glickman, 2000). Its role in 26S proteasome function remains unclear. At least in vitro, the lid is necessary for proper degradation of polyubiquitylated proteins. Presumably, the lid helps recognize appropriate targets and remove the covalently bound ubiquitins before transport of the target into the CP by the base-related activities. These functions are supported by genetic analyses showing that some lid subunits have substrate-specific roles (e.g. van Nocker et al., 1996; Bailly and Reed, 1999). One RP base subunit, Rpn10, appears to have multiple functions. It was first identified by its ability to bind polyubiquitin chains, thus implicating it as a polyubiquitin-protein receptor (see van Nocker et al., 1996). However, only short-lived proteins degraded by the ubiquitin fusion degradation (UFD) pathway [e.g. ubiquitin-proline β-galactosidase (Ub-Pro-βgal)] are stabilized in yeast rpn10Δ strains, suggesting that Rpn10 has a substrate-specific function (van Nocker et al., 1996). rpn10Δ strains from various species are hypersensitive to amino acid analogs, implicating Rpn10 in abnormal protein removal (van Nocker et al., 1996; Girod et al., 1999). Rpn10 also has a role in RP stability. Loss of Rpn10 weakens association of the lid and base, suggesting that it is necessary for proper contacts between the two subcomplexes (Glickman et al., 1998). Whereas binding of polyubiquitin chains requires a hydrophobic patch in the C-terminal half of Rpn10, the N-terminal portion of the polypeptide appears crucial for in vivo functions that confer amino acid analog resistance and Ub-Pro-βgal degradation (Fu et al., 1998). The RP lid is structurally similar to the newly discovered regulatory complex, the COP9 signalosome (CSN) that is present in most eukaryotes except Saccharomyces cerevisiae (Deng et al., 2000). The CSN contains eight core subunits that assemble into a 450 kDa particle. The core CSN subunits show a remarkable one-to-one sequence correspondence with those of the RP lid, suggesting a common ancestry and architecture (Glickman et al., 1998; Deng et al., 2000). The CSN plays an essential role in a number of developmental processes, including Arabidopsis photomorphogenesis and Drosophila embryogenesis by affecting the turnover of numerous 26S proteasome substrates (Deng et al., 2000). The biochemical function(s) of the CSN is not yet clear. Several activities have been detected, including a protein kinase activity (Bech-Otschir et al., 2001) and a hydrolase activity that removes Nedd8/Rub1, a ubiquitin-related modifier that becomes attached to the SCF E3 ubiquitin ligase complex (Lyapina et al., 2001). Interactions between subunits of the RP and CSN have been reported (see Kim et al., 2001) and mutations in the CSN were found to affect assembly of the RP (Peng et al., 2001), suggesting that the CSN and RP overlap functionally as well as structurally. To help understand how the RP and CSN function, structural resolution of these complexes would be instrumental. At present, such analyses have been hampered by low purification yields, low stability, potential subunit heterogeneity and flexible shape. Electron microscopy has provided crude three-dimensional pictures of the 26S proteasome (Walz et al., 1998), and yielded the first structural comparison of the lid and CSN (Kapelari et al., 2000). Cross-linking and far-western blotting have provided limited insights into the RP base. They identified several interacting pairs including those involving Rpt subunits (Richmond et al., 1997; Gorbea et al., 2000; Hartmann-Petersen et al., 2001). As an alternative strategy to map RP and CSN topology, we exploited the yeast two-hybrid (Y2H) method to help identify interacting subunits. Previous Y2H studies using a limited subset of RP subunits identified a few interacting partners (see Ferrell et al., 2000; Uetz et al., 2000; Hartmann-Petersen et al., 2001; Ito et al., 2001). Here, we completed a comprehensive paired interaction analysis among all 17 principal subunits of the yeast RP and seven CP α-subunits, and among all eight subunits of the CSN. Remarkably similar interactions among synonymous subunits of the CSN and the RP support a related structural organization for these particles. Mutational analysis located structural motifs/residues that are required for contacts between individual RP subunits, and between the lid and base subcomplexes. For Rpn10 in particular, Asp11 appears critical for in vivo protein turnover by the RP and for binding of the lid to the base, probably by participating in a salt bridge that stabilizes a vWA-like protein contact fold. Results Y2H system and tested combinations To help define interactions among the RP subunits, we tested all possible combinations of the 17 yeast RP subunits (Rpt1–6, and Rpn1–3 and 5–12) by a GALl4-based Y2H method (Phizicky and Fields, 1995). Rpn4 is not considered to be part of the RP complex and, indeed, we did not find that Rpn4 interacts with any other RP subunits (data not shown). We assembled a library of Y2H constructions, which included 17 that express RP subunits as C-terminal fusions to the GAL4 binding domain (BD; designated with a BD prefix) and 34 that express RP subunits as C- or N-terminal fusions to the GAL4 activation domain (AD; designated with an AD prefix or suffix, respectively). By using both AD orientations, we hoped to eliminate potential interference by the AD domain. We also attempted to detect interactions using RP subunits fused to the GAL4 BD N-terminus. However, none of these BD fusions succeeded (data not shown), possibly because the N-terminal appendages were detrimental to BD activity. The RP constructions were expressed in all possible paired combinations of GAL4 BD—AD fusions and assessed for subunit interaction by the HIS3 and LacZ reporters using the yeast YRG2 strain. The LacZ reporter appears to be a more stringent detector, since all of the LacZ-positive combinations displayed histidine auxotrophic growth, but not vice versa. In each case, binding activity was compared with a known interacting pair, p53 and SV40 T-antigen, and a non-interacting pair, lamin C and SV40 T-antigen. When each AD and BD fusion was expressed individually, only BD:Rpn3 activated the HIS3 reporter (Figure 1 and data not shown), but it did not elicit significant LacZ activity, suggesting a weak transactivation activity. Paired assortment of the RP subunits gave rise to 136 hetero-interacting combinations and 17 self-interacting combinations. Each of the hetero-interacting combinations was tested in two configurations. Configuration I refers to BD—AD fusions based on an ascending order of Rpt1–6 to Rpn1–12 subunits (e.g. BD:Rpt1— AD:Rpt2) whereas configuration II refers to BD—AD fusions based on a descending order of subunits (e.g. BD:Rpt2—AD:Rpt1). In total, 578 combinations ([(136 × 2) + 17] × 2) were examined. Figure 1.RP subunit interactions of the yeast 26S proteasome as detected by Y2H. The 17 yeast RP subunits were tested for potential interaction in all possible paired BD—AD configurations (I and II, see text) by histidine auxotrophic growth. A subset of non-interacting pairs and all interacting pairs are shown with all tested configurations. Brackets: group interacting pairs within the base and lid. p53—SV40 (SV40 T-antigen) and LAMIN (lamin C)—SV40 represent known interacting and non-interacting protein partners. As shown, BD:Rpn3 can self-activate the HIS3 reporter. Interaction pairs with positive LacZ activity are indicated by a ‘+’ (see Table I). Boxes show interacting pairs involving BD:Rpn3, which were confirmed using different testing configurations and/or by LacZ assay. Download figure Download PowerPoint Rpt subunit interactions suggest a minimal base cluster involving Rpt4/5/3/6 RP subunit pairs that showed a positive reaction when assayed for histidine auxotrophic growth are shown in Figure 1. Surprisingly, none of the lid subunits showed detectable affinity for any of the base subunits, and vice versa. Seventeen positive pairs involved the BD fusion of Rpn3 (Rpn3 × Rpt1–6, Rpn1–3, 5–12), which could not be concluded as interacting due to self-activation by BD:Rpn3 (Figure 1 and data not shown). The remaining interacting pairs involved four subunits within the base and eight subunits within the lid. Within the base complex, the Rpt3/3, Rpt3/5, Rpt3/6 and Rpt4/5 pairs appeared to interact. In most cases, binding was evident using either BD—AD configuration (I or II) and either N- or C-terminal AD fusion (Figure 1). Self-interactions were observed only with Rpt3 in the BD:Rpt3—AD:Rpt3 orientation. Since we presume that the six Rpt subunits assemble as a heteromeric ring (Glickman, 2000), the in vivo significance of this self-interaction is unclear. With the exception of BD:Rpt4—AD:Rpt5, these base interactions were confirmed by assaying LacZ activities that were 2- to 20-fold higher than the negative control (Table I, lamin C—SV40 T-antigen). Collectively, the interactions suggest a minimal base cluster involving Rpt4/5/3/6. Table 1. Yeast RP subunit interaction detected by the two-hybrid method using the LacZ reportera Interacting pair BD/AD configuration Ib BD/AD configuration IIb C-terminalc N-terminalc C-terminalc N-terminalc p53–SV40 308.3 ± 30.4 ndd nd nd Lamin C—SV40 30.1 ± 1.2 nd nd nd Rpn3e 20.7 ± 1.9 Rpt1–Rpt2f 16.3 ± 0.1 nd nd nd Rpt1–Rpn1f 17.0 ± 1.5 nd nd nd Rpt3–Rpt3 194.7 ± 16.6 nd nd nd Rpt3–Rpt5 130.2 ± 7.3 nd 443.0 ± 21.1 104.3 ± 17.8 Rpt3–Rpt6 613.2 ± 10.9 242.0 ± 32.7 381.2 ± 40.4 61.4 ± 10.4 Rpt4–Rpt5 17.7 ± 1.9 66.4 ± 7.6 557.3 ± 25.3 186.3 ± 45.6 Rpn3–Rpn7 1165.9 ± 106.2 645.9 ± 114.4 362.9 ± 30.6 653.7 ± 135.9 Rpn5–Rpn9 144.2 ± 15.2 302.6 ± 30.6 148.1 ± 12.7 76.3 ± 9.9 Rpn8-Rpn11 11.9 ± 0.8 278.4 ± 57.3 89.0 ± 19.3 223.3 ± 45.8 a LacZ activity is expressed in nmol o-nitrophenol/h/mg protein; ± indicates standard error. b In the order of Rpt1–6—Rpn1–12, each pair of 17 × 17 RP subunits (except 17 self-interacting pairs) were constructed separately with a BD or AD domain, respectively, in either an ascending (configuration I) or descending (configuration II) manner (see text). c RP subunits are fused to either the C- or N-terminal end of the GAL4 AD domain. d nd, not determined. e HIS-positive YRG2 cells expressing the BD fusion of Rpn3 alone were assayed for LacZ activity. f Rpt1/2 and Rpt1/Rpn1 are representatives having background LacZ activities. A cluster of lid subunit interactions involves Rpn5, 8, 9 and 11 Within the lid, we identified nine interacting pairs: Rpn3/7, Rpn3/12, Rpn5/6, Rpn5/8, Rpn5/9, Rpn5/11, Rpn8/9, Rpn8/11 and Rpn9/11 (Figure 1). Except for one configuration (BD:Rpn8—AD:Rpn11), three of these partners, Rpn3/7, Rpn5/9 and Rpn8/11, were positive by both the HIS3 and LacZ reporters in all four configurations, suggesting strong affinity (Figure 1 and Table I). Since BD:Rpn3 by itself grew on histidine-minus medium, the Rpn3/7 interaction could not be demonstrated by the HIS3 reporter alone. However, a strong interaction was supported by significantly higher LacZ activities (31- and 56-fold, respectively) for the BD:Rpn3—AD:Rpn7 and BD:Rpn3—Rpn7:AD combinations as compared with BD:Rpn3 alone (Table I). Six pairs, Rpn3/12, Rpn5/6, Rpn5/8, Rpn5/11, Rpn8/9 and Rpn9/11, were detected by HIS3 (Figure 1) but not by the LacZ reporter. Moreover, only some of the four configurations were HIS3 positive, suggesting that the interactions within these combinations were weak or substantially affected by the AD—BD appendages. The exception was the Rpn3/12 pair that showed robust growth in all four combinations, including those containing Rpn3 fusions to AD, which alone did not elicit growth. Most of the lid interaction pairs involved Rpn5, 8, 9 and 11 (seven out of nine), suggesting a localized structural cluster containing these four subunits. Individual interactions between RP and CP subunits were not detected In an attempt to identify which α-subunits interact with which RP subunits, we subjected all seven of the yeast α-subunits to Y2H analysis in combination with the 17 RP subunits. The α-subunits were tested either as C-terminal fusions to BD or N-terminal fusions to AD, whereas the RP subunits were tested as both N- and C-terminal AD fusions and C-terminal BD fusions, resulting in 238 BD:α1–7 × 34 AD—RP fusions and 119 α1–7:AD × 17 BD—RP fusions. When the α-fusions were expressed individually, only BD:α5 activated the HIS3 reporter (data not shown). Except for the 34 pairs involving the BD:α5 construction, none of the combinations appeared to interact. N-terminal coiled-coil domains are involved in Rpt subunit interactions Several motifs have been identified within the RP subunits that may be important for activity, structure and/or protein—protein interactions. To examine their roles in the subunit interactions described above, we tested various site-directed and deletion mutants. All six Rpt subunits contain an N-terminal coiled-coil and an ATPase cassette that defines them as members of the AAA-ATPases superfamily (Glickman et al., 1998). The P-loop present within the ATPase cassette contains an invariant lysine necessary for ATP hydrolysis (Rubin et al., 1998). As shown in Figure 2A, the Rpt3/6 and Rpt4/5 interactions were maintained with the C-terminal deletion mutants of Rpt6 (T6CC) and Rpt4 (T4CC) missing the AAA-ATPase cassette, but abrogated with the N-terminal deletion mutants (T6AAA and T4AAA) missing the coiled-coil, indicating that the coiled-coil is important. For the Rpt4/5 pair, the invariant Lys228 in the P-loop was not required for the interaction. However, substitution of the invariant Lys195 in the P-loop of Rpt6 with either arginine (T6R195) or serine (T6S195) abolished the Rpt3/6interaction, indicating that the P-loop of Rpt6 has a role in the context of the full-length protein (Figure 2A). Figure 2.Detection of structural domains involved in various RP subunit interactions by Y2H using histidine auxotrophic growth. The organization of the various deletions and amino acid substitutions is indicated next to each protein. (A) Coiled-coil domains of Rpt4 (T4) and Rpt6 (T6) and the invariant K195 of Rpt6 are critical for Rpt3/6 or Rpt4/5 interaction. (B) PCI domains are essential for Rpn5/9 and Rpn3/7 interaction. (C) The sequences encompassing the MPN domains are involved in Rpn8/11 interaction. Download figure Download PowerPoint PCI and MPN domains are required for specific lid subunit interactions Defined structural domains in the lid subunits include the PCI domain (proteasome, COP9, eIF3) found in six subunits (Rpn3, 5–7, 9 and 12), the MPN domain (Mpr1, Pad1 N-terminal) found in Rpn8 and 11, putative coiled-coils in Rpn5, 7 and 9, and a potential leucine zipper in Rpn8 (Glickman et al., 1998). Analysis of deletion mutants indicated that the PCI domains and not the coiled-coils are essential for interaction of the Rpn5/9 pair (Figure 2B). Rpn5 and 9 mutants (N5PCI and N9PCI) containing the PCI domain but not the coiled-coil maintained their interaction, whereas Rpn5 and 9 mutants missing the PCI domain but containing the coiled-coil (N9CCN, N9CCC and N5CC) did not (Figure 2B). In fact, BD—AD fusions containing just the PCI domains (N5PCI/N9PCI) interacted, indicating that this domain alone is sufficient for the Rpn5/9 binding. Interaction of PCI domains between Rpn5 and 9 appeared to be subunit specific since the PCI domain from neither Rpn3 (N3PCI) nor Rpn7 (N7PCI) could substitute for that of Rpn9 in its interaction with Rpn5 (Figure 2B). For Rpn3/7 interaction, the PCI domain of Rpn3 was found to be critical, but the motif required in Rpn7 was not obvious. The Rpn3 deletion mutant (N3PCI) containing the PCI maintained its interaction with Rpn7, but a similar deletion in Rpn7 (N7PCI) failed to interact with Rpn3 (Figure 2B). The coiled-coil domain of Rpn7 alone was also insufficient, as the N-terminal half of Rpn7 (N7CC) encompassing this domain did not bind to either full-length Rpn3 or N3PCI. The roles of the MPN and leucine zipper domains were tested in the Rpn8/11 interactions. Alanine substitution mutants of Rpn8, changing key leucines at positions 197 and 204 within the leucine zipper, either singly or in tandem (N8A197, N8A204 and N8A197/204), failed to disrupt its association with Rpn11, indicating that the leucine zipper is not essential (Figure 2C). In contrast, mutants (N8MPN1 and N11MPN1) containing the MPN domain interacted with their wild-type partners whereas the complementary mutants missing their MPN domains (N8MPNΔ and N11MPNΔ) did not, indicating that the MPN domain is involved (Figure 2C). Deletions of Rpn8 (N8MPN2) and Rpn11 (N11MPN2) removing additional sequences near the MPN domains failed to associate with their wild-type partners, as did segments containing just the regions surrounding the MPN domains (N11MPN1 and N8MPN1). Thus, it is possible that the amino acids flanking the C-terminus of the MPN domains contain essential contacts or are needed for proper folding of the MPN domains. Conserved interactions among the synonymous subunits from the Arabidopsis CSN complex Amino acid sequence alignments between subunits of the RP lid and the CSN suggest that these two complexes arose from a common progenitor (Glickman et al., 1998; Deng et al., 2000). In particular, Rpn3, 5, 6, 7, 8, 9, 11 and 12 appear to be synonymous with Csn3, 4, 2, 1, 6, 7, 5 and 8, respectively (Table II). To test whether the synonymous CSN subunits also share protein—protein interactions similar to those of the lid, we subjected all eight proteins from the Arabidopsis CSN to Y2H analysis. Previous studies (see Deng et al., 2000) and our database searches showed that most CSN subunits are encoded by single genes in Arabidopsis. The exceptions are Csn5 and Csn6, which are both encoded by two genes (designated A and B) that share ∼86–88% amino acid sequence identities (Table II). The presence of cDNAs for all the Csn genes in various Arabidopsis expressed sequence tag (EST) collections indicates that all are actively expressed (Table II). Csn7 is unique because it encodes two protein isoforms, Csn7i and Csn7ii. They are derived by alternative splicing of the seventh intron such that the Csn7i protein contains two additional amino acids at its C-terminus and the Csn7ii protein has a 37 amino acid C-terminal extension (data not shown). Two Csn7 proteins differing by several kilodaltons were detected in Arabidopsis, which may reflect these two isoforms (Karniol et al., 1999). The amino acid sequence similarities between orthologous CSN subunits from Arabidopsis and human are ∼45–75% as compared with ∼32–54% similarities between synonymous subunits of the lid and CSN from Arabidopsis (Table II). Table 2. Characterization of Arabidopsis genes encoding the subunits of the COP9 signalosome (CSN) complex Genea Chromosome locationb Peptide length (aa)/mol. wt (kDa) No of EST hits Similarity of AtCSN homologsc Identity/similarity to human CSN and Arabidopsis lid subunitsc Nucleotide Peptide Human CSN Arabidopsis lid AtCSN1 III, cM 96–98 441/50.6 8 49/61 (433) Rpn7 22/34 (312) AtCSN2 II, cM 56–57 439/51.2 4 61/73 (433) Rpn6 24/39 (374) AtCSN3 V, cM 24–26 429/47.7 7 40/48 (407) Rpn3 28/38 (206) AtCSN4 V, cM 90–93 397/45.0 3 52/63 (389) Rpn5 27/40 (328) AtCSN5a I, cM 120–122 358/40.3 1 84 86/90 (357) 68/73 (341) Rpn11 43/53 (206) AtCSN5b I, cM 37–39 357/39.7 11 68/75 (336) Rpn11 42/54 (206) AtCSN6a V, cM 120–121 317/35.7 14 86 88/91 (317) 42/54 (308) Rpn8 28/41 (295) AtCSN6b IV, cM 86–87 317/35.4 4 41/53 (308) Rpn8 28/42 (305) AtCSN7i I, cM 0–3 225/25.5 11 35/45 (211) Rpn9 27/40 (173) AtCSN7ii 260/29.5 11 35/45 (211) Rpn9 27/40 (173) AtCSN8 IV, cM 29 197/22.6 1 34/46 (163) Rpn12 21/32 (71) a Nomenclature is adapted from Deng et al. (2000). b Map locations determined by AtDB's Seq Map (http://www.arabidopsis.org/cgi-bin/maps/Schrom). c Percentage peptide sequence identity/similarity was determined by UW-GCG program BESTFIT; numbers in parentheses are lengths of the best matched polypeptides for each comparison pair; similarities between homologs are compared with both nucleotide (identity) and peptide sequences. The Arabidopsis CSN was tested by Y2H either as C- and N-terminal AD fusions or as C-terminal BD fusions. When each of the AD and BD fusions was expressed alone, only BD:Csn5A and BD:Csn5B self-activated the HIS3 reporter (Figure 3) and only BD:Csn5A slightly activated the LacZ reporter (Table III). The 11 CSN subunits (including isoforms of Csn5, 6 and 7) gave rise to 55 hetero-interacting combinations and 11 self-interacting combinations. When assembled in both ascending (configuration I) and descending (configuration II) configurations, a total of 242 combinations ([(55 × 2) + 11] × 2) were examined. Figure 3.Interactions of Arabidopsis CSN subunits as detected by Y2H using histidine auxotrophic growth. Interacting pairs are shown with all tested configurations. Interactions with positive LacZ activity are indicated by a ‘+’ (see Table III). Boxes show interactions involving self-activated BD:Csn5A or BD:Csn5B, which were confirmed using different testing configurations and/or by LacZ assay. Download figure Download PowerPoint Table 3. Arabidopsis CSN subunit interaction detected by the two-hybrid method using the LacZ reportera Interacting pair BD/AD configuration-Ib BD/AD configuration-IIb C-terminalc N-terminalc C-terminalc N-terminalc p53–SV40 547.1 ± 17.0 ndd nd nd Lamin C–SV40 50.1 ± 2.3 nd nd nd BD:Csn5Ae 109.2 ± 15.3 BD:Csn5Be 53.4 ± 2.2 Csn5B–Csn7if 53.7 ± 11.3 61.4 ± 6.5 16.7 ± 0.4 13.7 ± 0.9 Csn5B–Csn7iif 48.5 ± 4.5 48.4 ± 6.0 16.5 ± 1.6 12.0 ± 0.7 Csn3–Csn8 nd nd nd 1460.6 ± 156.8 Csn4–Csn7i 240.8 ± 19.4 396.6 ± 19.4 369.2 ± 19.5 243.0 ± 100.2 Csn4–Csn7ii 132.3 ± 42.1 296.4 ± 78.5 435.7 ± 32.3 368.7 ± 166.6 Csn5A–Csn6A 711.4 ± 44.1 732.0 ± 8.6 234.4 ± 33.8 341.4 ± 42.2 Csn5A–Csn6B 1089.0 ± 30.9 873.9 ± 21.5 628.6 ± 102.4 844.9 ± 108.7 Csn5B–Csn6A 1019.6 ± 40.7 1605.7 ± 58.1 506.8 ± 35.8 844.6 ± 72.0 Csn5B–Csn6B 524.6 ± 5.9 1692.3 ± 32.0 845.3 ± 67.4 939.3 ± 53.9 Csn5A–Csn8 227.0 ± 28.4 104.0 ± 8.3 95.7 ± 8.6 165.0 ± 49.5 Csn5B–Csn8 47.2 ± 2.9 47.6 ± 3.4 77.1 ± 2.2 130.0 ± 42.9 Csn6A–Csn7i 86.5 ± 8.2 237.42 ± 22.1 435.1 ± 45.0 420.0 ± 68.8 Csn6A–Csn7ii 80.1 ± 5.9 434.2 ± 54.9 476.9 ± 39.2 1116.2 ± 152.5 Csn6B–Csn7i 119.0 ± 12.2 115.6 ± 7.5 769.3 ± 64.4 762.8 ± 89.1 Csn6B–Csn7ii 45.4 ± 4.7 202.2 ± 21.3 899.2 ± 84.1 886.5 ± 309.4 Csn7i–Csn8 50.6 ± 3.4 57.8 ± 13.4 99.6 ± 3.9 370.4 ± 242.9 Csn7ii–Csn8 58.6 ± 5.9 69.7 ± 22.3 225.6 ± 12.2 161.0 ± 60.5 a LacZ activity is expressed in nmol o-nitrophenol/h/mg protein; ± indicates standard error. b In the order of Csn1–8, each pair of 11 × 11 Csn subunits (except 11 self-interacting pairs) were constructed separately with the BD or AD domain, respectively, in either an ascending (configuration I) or descending (configura