共查询到20条相似文献,搜索用时 15 毫秒
1.
Lisa M. Farmer Adam J. Book Kwang-Hee Lee Ya-Ling Lin Hongyong Fu Richard D. Vierstra 《The Plant cell》2010,22(1):124-142
The ubiquitin (Ub)/26S proteasome system (UPS) directs the turnover of numerous regulatory proteins, thereby exerting control over many aspects of plant growth, development, and survival. The UPS is directed in part by a group of Ub-like/Ub-associated (UBL/UBA) proteins that help shuttle ubiquitylated proteins to the 26S proteasome for breakdown. Here, we describe the collection of UBL/UBA proteins in Arabidopsis thaliana, including four isoforms that comprise the RADIATION SENSITIVE23 (RAD23) family. The nuclear-enriched RAD23 proteins bind Ub conjugates, especially those linked internally through Lys-48, via their UBA domains, and associate with the 26S proteasome Ub receptor RPN10 via their N-terminal UBL domains. Whereas homozygous mutants individually affecting the four RAD23 genes are without phenotypic consequences (rad23a, rad23c, and rad23d) or induce mild phyllotaxy and sterility defects (rad23b), higher-order mutant combinations generate severely dwarfed plants, with the quadruple mutant displaying reproductive lethality. Both the synergistic effects of a rad23b-1 rpn10-1 combination and the response of rad23b plants to mitomycin C suggest that RAD23b regulates cell division. Taken together, RAD23 proteins appear to play an essential role in the cell cycle, morphology, and fertility of plants through their delivery of UPS substrates to the 26S proteasome. 相似文献
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3.
Ubiquitylation marks proteins for destruction by the 26S proteasome. These signals are deciphered and targeted by distinct direct and indirect pathways involving a set of evolutionarily conserved ubiquitin receptors. Although biochemical and structural studies have revealed the mechanistic complexity of these substrate recognition pathways, conclusive evidence of the in vivo relevance of their substrate recognition function is currently not available. We recently showed that the structural elements involved in substrate recognition are not responsible for the important roles of the ubiquitin receptor RPN10 in vegetative and reproductive growth or for the abundance of the two-capped proteasomes (RP2-CP). Moreover, Arabidopsis plants subjected to severe knockdown or knockout any of the major ubiquitin receptors displayed wild-type phenotypes. Our results clearly suggest a functional redundancy of the major Arabidopsis ubiquitin receptors, and this evolved multiplicity is probably used to secure the substrates delivery. Based on the reduced abundance of RP2-CP in rpn10-2 and a role of RPN10 in lid-base association, a structural role of RPN10 in 26S proteasome stability is likely to be more relevant in vivo. Further efforts using structural and functional analyses in higher-order mutants to identify the specific biological functions of substrate recognition for the major Arabidopsis ubiquitin receptors are described here. 相似文献
4.
《Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms》2023,1866(2):194925
A moonlighting protein is one, which carries out multiple, often wholly unrelated, functions. The RAD23 protein is a fascinating example of this, where the same polypeptide and the embedded domains function independently in both nucleotide excision repair (NER) and protein degradation via the ubiquitin-proteasome system (UPS). Hence, through direct binding to the central NER component XPC, RAD23 stabilizes XPC and contributes to DNA damage recognition. Conversely, RAD23 also interacts directly with the 26S proteasome and ubiquitylated substrates to mediate proteasomal substrate recognition. In this function, RAD23 activates the proteolytic activity of the proteasome and engages specifically in well-characterized degradation pathways through direct interactions with E3 ubiquitin-protein ligases and other UPS components. Here, we summarize the past 40 years of research into the roles of RAD23 in NER and the UPS. 相似文献
5.
Elsasser S Chandler-Militello D Müller B Hanna J Finley D 《The Journal of biological chemistry》2004,279(26):26817-26822
The selective recognition of ubiquitin conjugates by proteasomes is a key step in protein degradation. The receptors that mediate this step have yet to be clearly defined although specific candidates exist. Here we show that the proteasome directly recognizes ubiquitin chains through a specific subunit, Rpn10, and also recognizes chains indirectly through Rad23, a reversibly bound proteasome cofactor. Both binding events can be observed in purified biochemical systems. A block substitution in the chain-binding ubiquitin interacting motif of RPN10 when combined with a null mutation in RAD23 results in a synthetic defect in protein degradation consistent with the view that the direct and indirect recognition modes function to some extent redundantly in vivo. Rad23 and the deubiquitinating enzyme Ubp6 both bind proteasome subunit Rpn1 through N-terminal ubiquitin-like domains. Surprisingly, Rad23 and Ubp6 do not compete with each other for proteasome binding. Thus, Rpn1 may act as a scaffold to assemble on the proteasome multiple proteins that act to either bind or hydrolyze multiubiquitin chains. 相似文献
6.
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
7.
Saeki Y Saitoh A Toh-e A Yokosawa H 《Biochemical and biophysical research communications》2002,293(3):986-992
Rpn10, a subunit of the 26S proteasome, has been proposed to act as a receptor for multiubiquitin chains in ubiquitin-dependent proteolysis. However, studies on RPN10-deleted mutants in yeasts have suggested the presence of other multiubiquitin chain-binding factors functioning in ubiquitin-dependent proteolysis. Here, we report that a mutant with a triple deletion of RAD23, DSK2, and RPN10 genes accumulates large amounts of polyubiquitinated proteins, as is the case with a mutant with RAD23 and DSK2 deletions under restrictive conditions. Dsk2, Rad23, and Rpn10 have different capacities to bind multiubiquitin chains. Another ubiquitin-like protein, Ddi1, has similar activity to those of Rad23 and Dsk2. Taken together, the results suggest that ubiquitin-like proteins, Rad23, Dsk2, possibly Ddi1, and Rpn10 play cooperative roles in ubiquitin-dependent proteolysis, serving as multiubiquitin chain-binding proteins. 相似文献
8.
Ubiquitin receptors connect substrate ubiquitylation to proteasomal degradation. HHR23a binds proteasome subunit 5a (S5a) through a surface that also binds ubiquitin. We report that UIM2 of S5a binds preferentially to hHR23a over polyubiquitin, and we provide a model for the ternary complex that we expect represents one of the mechanisms used by the proteasome to capture ubiquitylated substrates. Furthermore, we demonstrate that hHR23a is surprisingly adept at sequestering the ubiquitin moieties of a polyubiquitin chain, and provide evidence that it and the ubiquitylated substrate are committed to each other after binding. 相似文献
9.
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
10.
Background
The proteasome is a multi-subunit protein machine that is the final destination for cellular proteins that have been marked for degradation via an ubiquitin (Ub) chain appendage. These ubiquitylated proteins either bind directly to the intrinsic proteasome ubiqutin chain receptors Rpn10, Rpn13, or Rpt5, or are shuttled to the proteasome by Rad23, Dsk2, or Ddi1. The latter proteins share an Ub association domain (UBA) for binding poly-Ub chains and an Ub-like-domain (UBL) for binding to the proteasome. It has been proposed that shuttling receptors dock on the proteasome via Rpn1, but the precise nature of the docking site remains poorly defined.Results
To shed light on the recruitment of shuttling receptors to the proteasome, we performed both site-directed mutagenesis and genetic screening to identify mutations in Rpn1 that disrupt its binding to UBA-UBL proteins. Here we demonstrate that delivery of Ub conjugates and docking of Ddi1 (and to a lesser extent Dsk2) to the proteasome are strongly impaired by an aspartic acid to alanine point mutation in the highly-conserved D517 residue of Rpn1. Moreover, degradation of the Ddi1-dependent proteasome substrate, Ufo1, is blocked in rpn1-D517A yeast cells. By contrast, Rad23 recruitment to the proteasome is not affected by rpn1-D517A.Conclusions
These studies provide insight into the mechanism by which the UBA-UBL protein Ddi1 is recruited to the proteasome to enable Ub-dependent degradation of its ligands. Our studies suggest that different UBA-UBL proteins are recruited to the proteasome by distinct mechanisms. 相似文献11.
《Journal of molecular biology》2014,426(24):4049-4060
Rad23 was identified as a DNA repair protein, although a role in protein degradation has been described. The protein degradation function of Rad23 contributes to cell cycle progression, stress response, endoplasmic reticulum proteolysis, and DNA repair. Rad23 binds the proteasome through a UbL (ubiquitin-like) domain and contains UBA (ubiquitin-associated) motifs that bind multiubiquitin chains. These domains allow Rad23 to function as a substrate shuttle-factor. This property is shared by structurally similar proteins (Dsk2 and Ddi1) and is conserved among the human and mouse counterparts of Rad23. Despite much effort, the regulation of Rad23 interactions with ubiquitinated substrates and the proteasome is unknown. We report here that Rad23 is extensively phosphorylated in vivo and in vitro. Serine residues in UbL are phosphorylated and influence Rad23 interaction with proteasomes. Replacement of these serine residues with acidic residues, to mimic phosphorylation, reduced proteasome binding. We reported that when UbL is overexpressed, it can compete with Rad23 for proteasome interaction and can inhibit substrate turnover. This effect is not observed with UbL containing acidic substitutions, consistent with results that phosphorylation inhibits interaction with the proteasome. Loss of both Rad23 and Rpn10 caused pleiotropic defects that were suppressed by overexpressing either Rad23 or Rpn10. Rad23 bearing a UbL domain with acidic substitutions failed to suppress rad23Δ rpn10Δ, confirming the importance of regulated Rad23/proteasome binding. Strikingly, threonine 75 in human HR23B also regulates interaction with the proteasome, suggesting that phosphorylation is a conserved mechanism for controlling Rad23/proteasome interaction. 相似文献
12.
Mayor T Graumann J Bryan J MacCoss MJ Deshaies RJ 《Molecular & cellular proteomics : MCP》2007,6(11):1885-1895
The ubiquitin proteasome system (UPS) comprises hundreds of different conjugation/deconjugation enzymes and multiple receptors that recognize ubiquitylated proteins. A formidable challenge to deciphering the biology of ubiquitin is to map the networks of substrates and ligands for components of the UPS. Several different receptors guide ubiquitylated substrates to the proteasome, and neither the basis for specificity nor the relative contribution of each pathway is known. To address how broad of a role the ubiquitin receptor Rpn10 (S5a) plays in turnover of proteasome substrates, we implemented a method to perform quantitative analysis of ubiquitin conjugates affinity-purified from experimentally perturbed and reference cultures of Saccharomyces cerevisiae that were differentially labeled with 14N and 15N isotopes. Shotgun mass spectrometry coupled with relative quantification using metabolic labeling and statistical analysis based on q values revealed ubiquitylated proteins that increased or decreased in level in response to a particular treatment. We first identified over 225 candidate UPS substrates that accumulated as ubiquitin conjugates upon proteasome inhibition. To determine which of these proteins were influenced by Rpn10, we evaluated the ubiquitin conjugate proteomes in cells lacking either the entire Rpn10 (rpn10delta) (or only its UIM (ubiquitin-interacting motif) polyubiquitin-binding domain (uimdelta)). Twenty-seven percent of the UPS substrates accumulated as ubiquitylated species in rpn10delta cells, whereas only one-fifth as many accumulated in uimdelta cells. These findings underscore a broad role for Rpn10 in turnover of ubiquitylated substrates but a relatively modest role for its ubiquitin-binding UIM domain. This approach illustrates the feasibility of systems-level quantitative analysis to map enzyme-substrate networks in the UPS. 相似文献
13.
Rad23 is a member of a novel class of proteins that contain unprocessed ubiquitin-like (UbL) domains. We showed recently that a small fraction of Rad23 can form an interaction with the 26S proteasome. Similarly, a small fraction of Rpn10 is a component of the proteasome. Rpn10 can bind multiubiquitin chains in vitro, but genetic studies have not clarified its role in vivo. We report here that the loss of both Rad23 and Rpn10 results in pleiotropic defects that are not observed in either single mutant. rad23Delta rpn10Delta displays slow growth, cold sensitivity, and a pronounced G2/M phase delay, implicating overlapping roles for Rad23 and Rpn10. Although rad23Delta rpn10Delta displays similar sensitivity to DNA damage as a rad23Delta single mutant, deletion of RAD23 in rpn10Delta significantly increased sensitivity to canavanine, a phenotype associated with an rpn10Delta single mutant. A mutant Rad23 that is unable to bind the proteasome ((DeltaUbL)rad23) does not suppress the canavanine or cold-sensitive defects of rad23Delta rpn10Delta, demonstrating that Rad23/proteasome interaction is related to these effects. Finally, the accumulation of multiubiquitinated proteins and the stabilization of a specific proteolytic substrate in rad23Delta rpn10Delta suggest that proteasome function is altered. 相似文献
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Yeast Cycloheximide-resistant crl Mutants Are Proteasome Mutants Defective in Protein Degradation
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Uwe-M. Gerlinger Roland Gückel Michael Hoffmann Dieter H. Wolf Wolfgang Hilt 《Molecular biology of the cell》1997,8(12):2487-2499
In 1988 McCusker and Haber generated a series of mutants which are resistant to the minimum inhibitory concentration of the protein synthesis inhibitor cycloheximide. These cycloheximide-resistant, temperature-sensitive (crl) mutants, in addition, exhibited other pleiotropic phenotypes, e.g., incorrect response to starvation, hypersensitivity against amino acid analogues, and other protein synthesis inhibitors. Temperature sensitivity of one of these mutants, crl3–2, had been found to be suppressed by a mutation, SCL1–1, which resided in an α-type subunit of the 20S proteasome. We cloned the CRL3 gene by complementation and found CRL3 to be identical to the SUG1/CIM3 gene coding for a subunit of the 19S cap complex of the 26S proteasome. Another mutation, crl21, revealed to be allelic with the 20S proteasomal gene PRE3. crl3–2 and crl21 mutant cells show significant defects in proteasome-dependent proteolysis, whereas the SCL1–1 suppressor mutation causes partial restoration of crl3–2-induced proteolytic defects. Notably, cycloheximide resistance was also detected for other proteolytically deficient proteasome mutants (pre1–1, pre2–1, pre3–1, pre4–1). Moreover, proteasomal genes were found within genomic sequences of 9 of 13 chromosomal loci to which crl mutations had been mapped. We therefore assume that most if not all crl mutations reside in the proteasome and that phenotypes found are a result of defective protein degradation. 相似文献
16.
Abhishek Chandra Li Chen Huiyan Liang Kiran Madura 《The Journal of biological chemistry》2010,285(11):8330-8339
A major fraction of intracellular protein degradation is mediated by the proteasome. Successful degradation of these substrates requires ubiquitination and delivery to the proteasome followed by protein unfolding and disassembly of the multiubiquitin chain. Enzymes, such as Rpn11, dismantle multiubiquitin chains, and mutations can affect proteasome assembly and activity. We report that different rpn11 mutations can affect proteasome interaction with ubiquitinated proteins. Moreover, proteasomes are unstable in rpn11-1 and do not form productive interactions with multiubiquitinated proteins despite high levels in cell extracts. However, increased levels of ubiquitinated proteins were found associated with shuttle factors. In contrast to rpn11-1, proteasomes expressing a catalytically inactive mutant (rpn11AXA) were more stable and bound very high amounts of ubiquitinated substrates. Expression of the carboxyl-terminal domain of Rpn11 partially suppressed the growth and proteasome stability defects of rpn11-1. These results indicate that ubiquitinated substrates are preferentially delivered to intact proteasome. 相似文献
17.
Miguel A. Aguileta Jelena Korac Thomas M. Durcan Jean-Fran?ois Trempe Michael Haber Kalle Gehring Suzanne Elsasser Oliver Waidmann Edward A. Fon Koraljka Husnjak 《The Journal of biological chemistry》2015,290(12):7492-7505
Mutations in the Park2 gene, encoding the RING-HECT hybrid E3 ubiquitin ligase parkin, are responsible for a common familial form of Parkinson disease. By mono- and polyubiquitinating target proteins, parkin regulates various cellular processes, including degradation of proteins within the 26 S proteasome, a large multimeric degradation machine. In our attempt to further elucidate the function of parkin, we have identified the proteasomal ubiquitin receptor Rpn13/ADRM1 as a parkin-interacting protein. We show that the N-terminal ubiquitin-like (Ubl) domain of parkin binds directly to the pleckstrin-like receptor for ubiquitin (Pru) domain within Rpn13. Using mutational analysis and NMR, we find that Pru binding involves the hydrophobic patch surrounding Ile-44 in the parkin Ubl, a region that is highly conserved between ubiquitin and Ubl domains. However, compared with ubiquitin, the parkin Ubl exhibits greater than 10-fold higher affinity for the Pru domain. Moreover, knockdown of Rpn13 in cells increases parkin levels and abrogates parkin recruitment to the 26 S proteasome, establishing Rpn13 as the major proteasomal receptor for parkin. In contrast, silencing Rpn13 did not impair parkin recruitment to mitochondria or parkin-mediated mitophagy upon carbonyl cyanide m-chlorophenyl hydrazone-induced mitochondrial depolarization. However, it did delay the clearance of mitochondrial proteins (TIM23, TIM44, and TOM20) and enhance parkin autoubiquitination. Taken together, these findings implicate Rpn13 in linking parkin to the 26 S proteasome and regulating the clearance of mitochondrial proteins during mitophagy. 相似文献
18.
Decline of proteasome activity has been reported in mammals, flies and yeasts during aging. In the yeast Saccharomyces cerevisiae, the reduction of proteolysis in stationary phase is correlated with disassembly of the 26S proteasomes into their 20S and 19S subcomplexes. However a recent report showed that upon entry into the stationary phase, proteasome subunits massively re-localize from the nucleus into mobile cytoplasmic structures called proteasome storage granules (PSGs). Whether proteasome subunits in PSG are assembled into active complexes remains an open question that we addressed in the present study. We showed that a particular mutant of the RPN11 gene (rpn11-m1), encoding a proteasome lid subunit already known to exhibit proteasome assembly/stability defect in vitro, is unable to form PSGs and displays a reduced viability in stationary phase. Full restoration of long-term survival and PSG formation in rpn11-m1 cells can be achieved by the expression in trans of the last 45 amino acids of the C-terminal domain of Rpn11, which was moreover found to co-localize with PSGs. In addition, another rpn11 mutant leading to seven amino acids change in the Rpn11 C-terminal domain, which exhibits assembled-26S proteasomes, is able to form PSGs but with a delay compared to the wild type situation. Altogether, our findings indicate that PSGs are formed of fully assembled 26S proteasomes and suggest a critical role for the Rpn11 protein in this process. 相似文献
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20.
A protein that exemplifies the intimate link between the ubiquitin/proteasome system (UPS) and DNA repair is the yeast nucleotide excision repair (NER) protein Rad23 and its human orthologs hHR23A and hHR23B. Rad23, which was originally identified as an important factor involved in the recognition of DNA lesions, also plays a central role in targeting ubiquitylated proteins for proteasomal degradation, an activity that it shares with other ubiquitin receptors like Dsk2 and Ddi1. Although the finding that Rad23 serves as a ubiquitin receptor explains to a large extent its importance in proteasomal degradation, the precise mode of action of Rad23 in NER and the possible link with the UPS is less clear. In this review, we discuss our present knowledge on the functions of Rad23 in protein degradation and DNA repair and speculate on the importance of the dual roles of Rad23 for the cell's ability to cope with stress conditions. 相似文献