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1.
The pleiotropic role of the 26S proteasome subunit RPN10 in Arabidopsis growth and development supports a substrate-specific function in abscisic acid signaling 总被引:12,自引:0,他引:12
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Smalle J Kurepa J Yang P Emborg TJ Babiychuk E Kushnir S Vierstra RD 《The Plant cell》2003,15(4):965-980
The 26S proteasome is an essential protease complex responsible for removing most short-lived intracellular proteins, especially those modified with polyubiquitin chains. We show here that an Arabidopsis mutant expressing an altered RPN10 subunit exhibited a pleiotropic phenotype consistent with specific changes in 26S proteasome function. rpn10-1 plants displayed reduced seed germination, growth rate, stamen number, genetic transmission through the male gamete, and hormone-induced cell division, which can be explained partially by a constitutive downregulation of the key cell cycle gene CDKA;1. rpn10-1 also was more sensitive to abscisic acid (ABA), salt, and sucrose stress and to DNA-damaging agents and had decreased sensitivity to cytokinin and auxin. Most of the phenotypes can be explained by a hypersensitivity to ABA, which is reflected at the molecular level by the selective stabilization of the short-lived ABA-signaling protein ABI5. Collectively, these results indicate that RPN10 affects a number of regulatory processes in Arabidopsis likely by directing specific proteins to the 26S proteasome for degradation. A particularly important role may be in regulating the responses to signals promulgated by ABA. 相似文献
2.
The RPN1 subunit of the 26S proteasome in Arabidopsis is essential for embryogenesis 总被引:3,自引:0,他引:3
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Brukhin V Gheyselinck J Gagliardini V Genschik P Grossniklaus U 《The Plant cell》2005,17(10):2723-2737
The 26S proteasome plays a central role in the degradation of regulatory proteins involved in a variety of developmental processes. It consists of two multisubunit protein complexes: the proteolytic core protease and the regulatory particle (RP). The function of most RP subunits is poorly understood. Here, we describe mutants in the Arabidopsis thaliana RPN1 subunit, which is encoded by two paralogous genes, RPN1a and RPN1b. Disruption of RPN1a caused embryo lethality, while RPN1b mutants showed no obvious abnormal phenotype. Embryos homozygous for rpn1a arrested at the globular stage with defects in the formation of the embryonic root, the protoderm, and procambium. Cyclin B1 protein was not degraded in these embryos, consistent with cell division defects. Double mutant plants (rpn1a/RPN1a rpn1b/rpn1b) produced embryos with a phenotype indistinguishable from that of the rpn1a single mutant. Thus, despite their largely overlapping expression patterns in flowers and developing seeds, the two isoforms do not share redundant functions during gametogenesis and embryogenesis. However, complementation of the rpn1a mutation with the coding region of RPN1b expressed under the control of the RPN1a promoter indicates that the two RPN1 isoforms are functionally equivalent. Overall, our data indicate that RPN1 activity is essential during embryogenesis, where it might participate in the destruction of a specific set of protein substrates. 相似文献
3.
Jasmina Kurepa Songhu Wang Yan Li David Zaitlin Andrew J. Pierce Jan A. Smalle 《Plant physiology》2009,150(1):178-189
Although the final size of plant organs is influenced by environmental cues, it is generally accepted that the primary size determinants are intrinsic factors that regulate and coordinate cell proliferation and cell expansion. Here, we show that optimal proteasome function is required to maintain final shoot organ size in Arabidopsis (Arabidopsis thaliana). Loss of function of the subunit regulatory particle AAA ATPase (RPT2a) causes a weak defect in 26S proteasome activity and leads to an enlargement of leaves, stems, flowers, fruits, seeds, and embryos. These size increases are a result of increased cell expansion that compensates for a reduction in cell number. Increased ploidy levels were found in some but not all enlarged organs, indicating that the cell size increases are not caused by a higher nuclear DNA content. Partial loss of function of the regulatory particle non-ATPase (RPN) subunits RPN10 and RPN12a causes a stronger defect in proteasome function and also results in cell enlargement and decreased cell proliferation. However, the increased cell volumes in rpn10-1 and rpn12a-1 mutants translated into the enlargement of only some, but not all, shoot organs. Collectively, these data show that during Arabidopsis shoot development, the maintenance of optimal proteasome activity levels is important for balancing cell expansion with cell proliferation rates. 相似文献
4.
Jasmina Kurepa Consolée Karangwa Liliana Sfichi Duke Jan A. Smalle 《Plant cell reports》2010,29(3):249-259
The 26S proteasome (26SP), the central protease of the ubiquitin-dependent proteolysis pathway, controls the regulated proteolysis
of functional proteins and the removal of misfolded and damaged proteins. In Arabidopsis, cellular and stress response phenotypes
of a number of mutants with partially impaired 26SP function have been reported. Here, we describe the responses of proteasome
mutants to protein synthesis inhibitors. We show that the rpt2a-3, rpn10-1 and rpn12a-1 mutants are hypersensitive to the antibiotic hygromycin B, and tolerant to the translation inhibitor cycloheximide (CHX)
and herbicide l-phosphinothricin (PPT). In addition to the novel mechanism for herbicide tolerance, our data suggests that the combination
of hygromycin B, CHX and PPT growth-response assays could be used as a facile diagnostic tool to detect altered 26SP function
in plant mutants and transgenic lines. 相似文献
5.
Functional Characterization of Rpn3 Uncovers a Distinct 19S Proteasomal Subunit Requirement for Ubiquitin-Dependent Proteolysis of Cell Cycle Regulatory Proteins in Budding Yeast
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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. 相似文献
6.
Structure and functional analyses of the 26S proteasome subunits from plants – Plant 26S proteasome 总被引:4,自引:0,他引:4
Fu Hongyong Girod Pierre-Alain Doelling Jed H. van Nocker Steven Hochstrasser Mark Finley Daniel Vierstra Richard D. 《Molecular biology reports》1999,26(1-2):137-146
As initial steps to define how the 26S proteasome degrades ubiquitinated proteins in plants, we have characterized many of the subunits that comprise the proteolytic complex from Arabidopsis thaliana. A set of 23 Arabidopsis genes encoding the full complement of core particle (CP) subunits and a collection encoding 12 out of 18 known eukaryotic regulatory particle (RP) subunits, including six AAA-ATPase subunits, were identified. Several of these 26S proteasome genes could complement yeast strains missing the corresponding orthologs. Using this ability of plant subunits to functionally replace yeast counterparts, a parallel structure/function analysis was performed with the RP subunit RPN10/MCB1, a putative receptor for ubiquitin conjugates. RPN10 is not essential for yeast viability but is required for amino acid analog tolerance and degradation of proteins via the ubiquitin-fusion degradation pathway, a subpathway within the ubiquitin system. Surprisingly, we found that the C-terminal motif required for conjugate recognition by RPN10 is not essential for in vivo functions. Instead, a domain near the N-terminus is required. We have begun to exploit the moss Physcomitrella patens as a model to characterize the plant 26S proteasome using reverse genetics. By homologous recombination, we have successfully disrupted the RPN10 gene. Unlike yeast rpn10 strains which grow normally, Physcomitrella rpn10 strains are developmentally arrested, being unable to initiate gametophorogenesis. Further analysis of these mutants revealed that RPN10 is likely required for a developmental program triggered by plant hormones. 相似文献
7.
Accumulating evidence shows that proper degradation of proteins that affect defense responses in a positive or negative manner is critical in plant immunity. However, the role of plant degradation systems such as the 26S proteasome in plant immunity is not well understood. Loss‐of‐function mutations in EDR2 (ENHANCED DISEASE RESISTANCE 2) lead to increased resistance to the adapted biotrophic powdery mildew pathogen Golovinomyces cichoracearum. To study the molecular interactions between powdery mildew pathogen and Arabidopsis, we performed a screen for suppressors of edr2 and found that mutation in the gene that encodes RPN1a, a subunit of the 26S proteasome, suppressed edr2‐associated disease resistance phenotypes. In addition, RPN1a is required for edr1‐ and pmr4‐mediated powdery mildew resistance and mildew‐induced cell death. Furthermore, we show that rpn1a displayed enhanced susceptibility to the fungal pathogen G. cichoracearum and to virulent and avirulent bacterial Pto DC3000 strains, which indicated that rpn1a has defects in basal defense and resistance (R) protein‐mediated defense. RPN1a–GFP localizes to both the nucleus and cytoplasm. Accumulation of RPN1a is affected by salicylic acid (SA) and the rpn1a mutant has defects in SA accumulation upon Pto DC3000 infection. Further analysis revealed that two other subunits of the 26S proteasome, RPT2a and RPN8a are also involved in edr2‐mediated disease resistance. Based on these results, we conclude that RPN1a is required for basal defense and R protein‐mediated defense. Our data provide evidence that some subunits of the 26S proteasome are involved in innate immunity in Arabidopsis. 相似文献
8.
Rpn7 is one of the lid subunits of the 26 S proteasome regulatory particle. The RPN7 gene is known to be essential, but its function remains to be elucidated. To explore the function of Rpn7, we isolated and characterized temperature-sensitive rpn7 mutants. All of the rpn7 mutants obtained accumulated poly-ubiquitinated proteins when grown at the restrictive temperature. The N-end rule substrate (Ub-Arg-beta-galactosidase), the UFD pathway substrate (Ub-Pro-beta-galactosidase), and cell cycle regulators (Pds1 and Clb2) were found to be stabilized in experiments using one of the rpn7 mutants termed rpn7-3 at the restrictive temperature, indicating its defect in the ubiquitin-proteasome pathway. Subsequent analysis of the structure of the 26 S proteasome in rpn7-3 cells suggested that the defect was in the assembly of the 26 S holoenzyme. The most striking characteristic of the proteasome of the rpn7-3 mutant was that a lid subcomplex affinity-purified from the rpn7-3 cells grown at the restrictive temperature contained only 5 of the 8 lid components, a phenomenon that has not been reported in the previously isolated lid mutants. From these results, we concluded that Rpn7 is required for the integrity of the 26 S complex by establishing a correct lid structure. 相似文献
9.
The ubiquitin/26S proteasome-dependent proteolysis of response regulators is a critical element of many plant hormone signaling pathways. We have recently shown that cytokinin signaling requires the AXR1 component of the related to ubiquitin (RUB) protein modification pathway to promote the proteasome-dependent degradation of the cytokinin response inhibitor ARR5. Here, we show that ARR5 also accumulates in the 26S proteasome mutant rpn12a-1, and leads to a marked resistance to cytokinins. Collectively, these results suggest that proteasome-dependent proteolysis of feedback inhibitors such as ARR5 is essential for the maintenance of optimal responsivity and plasticity in cytokinin signaling. 相似文献
10.
The 19S regulatory particle of the yeast 26S proteasome consists of six related ATPases (Rpt proteins) and at least 11 non-ATPase proteins (Rpn proteins). RPN12 (formerly NIN1) encodes an Rpn component of the 19S regulatory particle and is essential for growth. To determine which subunit(s) of the 26S proteasome interact(s) with Rpn12, we attempted to screen for mutations that cause synthetic lethality in the presence of the rpn12-1 (formerly nin1-1) mutation. Among the candidates recovered was a new allele of RPT1 (formerly CIM5). This mutant allele was designated rpt1-2; on its own this mutation caused no phenotypic change, whereas the rpn12-1 rpt1-2 double mutant was lethal, suggesting a strong interaction between Rpn12 and Rpt1. The site of the rpt1-2 mutation was determined by DNA sequencing of the RPT1 locus retrieved from the mutant, and a single nucleotide alteration was found. This changes amino acid 446 of the RPT1 product from alanine to valine. The alanine residue is conserved in all Rpt proteins, except Rpt5, but no function has yet been assigned to the region that contains it. We propose that this region is necessary for Rpt1 to interact with Rpn12. The terminal phenotype of the rpn12-1 rpt1-2 double mutant was not cell cycle specific, suggesting that in the double mutant cells the function of the 26S proteasome is completely eliminated, thereby inducing multiple defects in cellular functions. 相似文献
11.
The 19S regulatory particle of the yeast 26S proteasome consists of six related ATPases (Rpt proteins) and at least 11 non-ATPase
proteins (Rpn proteins). RPN12 (formerly NIN1) encodes an Rpn component of the 19S regulatory particle and is essential for growth. To determine which subunit(s) of the
26S proteasome interact(s) with Rpn12, we attempted to screen for mutations that cause synthetic lethality in the presence
of the rpn12-1 (formerly nin1-1) mutation. Among the candidates recovered was a new allele of RPT1 (formerly CIM5). This mutant allele was designated rpt1-2; on its own this mutation caused no phenotypic change, whereas the rpn12-1 rpt1-2 double mutant was lethal, suggesting a strong interaction between Rpn12 and Rpt1. The site of the rpt1-2 mutation was determined by DNA sequencing of the RPT1 locus retrieved from the mutant, and a single nucleotide alteration was found. This changes amino acid 446 of the RPT1 product from alanine to valine. The alanine residue is conserved in all Rpt proteins, except Rpt5, but no function has yet
been assigned to the region that contains it. We propose that this region is necessary for Rpt1 to interact with Rpn12. The
terminal phenotype of the rpn12-1 rpt1-2 double mutant was not cell cycle specific, suggesting that in the double mutant cells the function of the 26S proteasome
is completely eliminated, thereby inducing multiple defects in cellular functions.
Received: 1 February 1999 / Accepted: 5 May 1999 相似文献
12.
Zanlin Yu Nurit Livnat-Levanon Oded Kleifeld Wissam Mansour Mark?A. Nakasone Carlos?A. Castaneda Emma?K. Dixon David Fushman Noa Reis Elah Pick Michael?H. Glickman 《Bioscience reports》2015,35(3)
26S proteasome, a major regulatory protease in eukaryotes, consists of a 20S proteolytic core particle (CP) capped by a 19S regulatory particle (RP). The 19S RP is divisible into base and lid sub-complexes. Even within the lid, subunits have been demarcated into two modules: module 1 (Rpn5, Rpn6, Rpn8, Rpn9 and Rpn11), which interacts with both CP and base sub-complexes and module 2 (Rpn3, Rpn7, Rpn12 and Rpn15) that is attached mainly to module 1. We now show that suppression of RPN11 expression halted lid assembly yet enabled the base and 20S CP to pre-assemble and form a base-CP. A key role for Regulatory particle non-ATPase 11 (Rpn11) in bridging lid module 1 and module 2 subunits together is inferred from observing defective proteasomes in rpn11–m1, a mutant expressing a truncated form of Rpn11 and displaying mitochondrial phenotypes. An incomplete lid made up of five module 1 subunits attached to base-CP was identified in proteasomes isolated from this mutant. Re-introducing the C-terminal portion of Rpn11 enabled recruitment of missing module 2 subunits. In vitro, module 1 was reconstituted stepwise, initiated by Rpn11–Rpn8 heterodimerization. Upon recruitment of Rpn6, the module 1 intermediate was competent to lock into base-CP and reconstitute an incomplete 26S proteasome. Thus, base-CP can serve as a platform for gradual incorporation of lid, along a proteasome assembly pathway. Identification of proteasome intermediates and reconstitution of minimal functional units should clarify aspects of the inner workings of this machine and how multiple catalytic processes are synchronized within the 26S proteasome holoenzymes. 相似文献
13.
The proteolytic function of the Arabidopsis 26S proteasome is required for specifying leaf adaxial identity 总被引:6,自引:0,他引:6
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Polarity formation is central to leaf morphogenesis, and several key genes that function in adaxial-abaxial polarity establishment have been identified and characterized extensively. We previously reported that Arabidopsis thaliana ASYMMERTIC LEAVES1 (AS1) and AS2 are important in promoting leaf adaxial fates. We obtained an as2 enhancer mutant, asymmetric leaves enhancer3 (ae3), which demonstrated pleiotropic plant phenotypes, including a defective adaxial identity in some leaves. The ae3 as2 double mutant displayed severely abaxialized leaves, which were accompanied by elevated levels of leaf abaxial promoting genes FILAMENTOUS FLOWER, YABBY3, KANADI1 (KAN1), and KAN2 and a reduced level of the adaxial promoting gene REVOLUTA. We identified AE3, which encodes a putative 26S proteasome subunit RPN8a. Furthermore, double mutant combinations of as2 with other 26S subunit mutations, including rpt2a, rpt4a, rpt5a, rpn1a, rpn9a, pad1, and pbe1, all displayed comparable phenotypes with those of ae3 as2, albeit with varying phenotypic severity. Since these mutated genes encode subunits that are located in different parts of the 26S proteasome, it is possible that the proteolytic function of the 26S holoenzyme is involved in leaf polarity formation. Together, our findings reveal that posttranslational regulation is essential in proper leaf patterning. 相似文献
14.
The assembly pathway of the 19S regulatory particle of the yeast 26S proteasome 总被引:1,自引:0,他引:1
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Isono E Nishihara K Saeki Y Yashiroda H Kamata N Ge L Ueda T Kikuchi Y Tanaka K Nakano A Toh-e A 《Molecular biology of the cell》2007,18(2):569-580
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 DeltaN 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 DeltaN 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. 相似文献
15.
The 26S proteasome is a 2-MDa complex with a central role in protein turn over. The 26S proteasome is comprised of one 20S core particle and two 19S regulatory particles (RPs). The RPN12a protein, a non-ATPase subunit of the 19S RP, was previously shown to be involved in cytokinin signaling in Arabidopsis. To further investigate cellular roles of RPN12a, RNAi transgenic plants of RPN12a were constructed. As expected, the 35S:RNAi-RPN12a plants showed cytokinin signaling defective phenotypes, including abnormal formation of leaves and inflorescences. Furthermore, RNAi knock-down transgenic plants exhibited additional unique phenotypes, including concave and heart-shape cotyledons, triple cotyledons, irregular and clustered guard cells, and defects in phyllotaxy, all of which are typical for defective cytokinin signaling. We next examined the mRNA level of cytokinin signaling components, including type-A ARRs, type-B ARRs, and CRFs. The expression of type-A ARRs, encoding negative regulators of cytokinin signaling, was markedly reduced in 35S:RNAi-RPN12a transgenic plants relative to that in wild type plants, while type-B ARRs and CRFs were unaffected. Our results also indicate that in vivo stability of the ARR5 protein, a negative regulator of cytokinin signaling, is mediated by the 26S proteasome complex. These results suggest that RPN12a participates in feedback inhibitory mechanism of cytokinin signaling through modulation of the abundance of ARR5 protein in Arabidopsis. 相似文献
16.
26S proteasome regulatory particle mutants have increased oxidative stress tolerance 总被引:1,自引:0,他引:1
The 26S proteasome (26SP) is a multi-subunit, multi-catalytic protease that is responsible for most of the cytosolic and nuclear protein turnover. The 26SP is composed of two sub-particles, the 19S regulatory particle (RP) that binds and unfolds protein targets, and the 20S core particle (20SP) that degrades proteins into small peptides. Most 26SP targets are conjugated to a poly-ubiquitin (Ub) chain that serves as a degradation signal. However, some targets, such as oxidized proteins, do not require a poly-Ub tag for proteasomal degradation, and recent studies have shown that the main protease in this Ub-independent pathway is free 20SP. It is currently unknown how the ratio of 26SP- to 20SP-dependent proteolysis is controlled. Here we show that loss of function of the Arabidopsis RP subunits RPT2a, RPN10 and RPN12a leads to decreased 26SP accumulation, resulting in reduced rates of Ub-dependent proteolysis. In contrast, all three RP mutants have increased 20SP levels and thus enhanced Ub-independent protein degradation. As a consequence of this shift in proteolytic activity, mutant seedlings are hypersensitive to stresses that cause protein misfolding, and have increased tolerance to treatments that promote protein oxidation. Taken together, the data show that plant cells increase 20SP-dependent proteolysis when 26SP activity is impaired. 相似文献
17.
18.
Ubiquitination is involved in diverse cellular processes in higher plants. In this report, we describe Arabidopsis thaliana PUB22 and PUB23, two homologous U-box-containing E3 ubiquitin (Ub) ligases. The PUB22 and PUB23 genes were rapidly and coordinately induced by abiotic stresses but not by abscisic acid. PUB22- and PUB23-overexpressing transgenic plants were hypersensitive to drought stress. By contrast, loss-of-function pub22 and pub23 mutant plants were significantly more drought-tolerant, and a pub22 pub23 double mutant displayed even greater drought tolerance. These results indicate that PUB22 and PUB23 function as negative regulators in the water stress response. Yeast two-hybrid, in vitro pull-down, and in vivo coimmunoprecipitation experiments revealed that PUB22 and PUB23 physically interacted with RPN12a, a subunit of the 19S regulatory particle (RP) in the 26S proteasome. Bacterially expressed RPN12a was effectively ubiquitinated in a PUB-dependent fashion. RPN12a was highly ubiquitinated in 35S:PUB22 plants, but not in pub22 pub23 double mutant plants, consistent with RPN12a being a substrate of PUB22 and PUB23 in vivo. In water-stressed wild-type and PUB-overexpressing plants, a significant amount of RPN12a was dissociated from the 19S RP and appeared to be associated with small-molecular-mass protein complexes in cytosolic fractions, where PUB22 and PUB23 are localized. Overall, our results suggest that PUB22 and PUB23 coordinately control a drought signaling pathway by ubiquitinating cytosolic RPN12a in Arabidopsis. 相似文献
19.
Keisuke Fukunaga Akio Toh-e Yasushi Saeki 《Biochemical and biophysical research communications》2010,396(4):1048-917
The 26S proteasome is a highly conserved multisubunit protease that degrades ubiquitinated proteins in eukaryotic cells. It comprises a 20S core particle and two 19S regulatory particles that are further divided into the lid and base complexes. The lid is a nine subunits complex that is structurally related to the COP9 signalosome and the eukaryotic initiation factor 3. Although the assembly pathway of the 20S and the base are well described, that of the lid is still unclear. In this study, we dissected the lid assembly using yeast lid mutant cells, rpn7-3, Δrpn9, and rpn12-1. Using mass spectrometry, we identified a number of lid subassemblies, such as Rpn3-Rpn7 pair and a lid-like complex lacking Rpn12, in the mutants. Our analysis suggests that the assembly of the lid is a highly ordered and multi-step process; first, Rpn5, 6, 8, 9, and 11 are assembled to form a core module, then a second module, consisting of Rpn3, 7, and Sem1, is attached, followed by the incorporation of Rpn12 to form the lid complex. 相似文献
20.
Junko Takeuchi Masahiro Fujimuro Hideyosi Yokosawa Keiji Tanaka Akio Toh-e 《Molecular and cellular biology》1999,19(10):6575-6584
We have isolated the RPN9 gene by two-hybrid screening with, as bait, RPN10 (formerly SUN1), which encodes a multiubiquitin chain receptor residing in the regulatory particle of the 26S proteasome. Rpn9 is a nonessential subunit of the regulatory particle of the 26S proteasome, but the deletion of this gene results in temperature-sensitive growth. At the restrictive temperature, the Deltarpn9 strain accumulated multiubiquitinated proteins, indicating that the RPN9 function is needed for the 26S proteasome activity at a higher temperature. We analyzed the proteasome fractions separated by glycerol density gradient centrifugation by native polyacrylamide gel electrophoresis and found that a smaller amount of the 26S proteasome was produced in the Deltarpn9 cells and that the 26S proteasome was shifted to lighter fractions than expected. The incomplete proteasome complexes were found to accumulate in the Deltarpn9 cells. Furthermore, Rpn10 was not detected in the fractions containing proteasomes of the Deltarpn9 cells. These results indicate that Rpn9 is needed for incorporating Rpn10 into the 26S proteasome and that Rpn9 participates in the assembly and/or stability of the 26S proteasome. 相似文献