The ubiquitin-specific protease family from Arabidopsis. AtUBP1 and 2 are required for the resistance to the amino acid analog canavanine

Ubiquitin-specific proteases (UBPs) are a family of unique hydrolases that specifically remove polypeptides covalently linked via peptide or isopeptide bonds to the C-terminal glycine of ubiquitin. UBPs help regulate the ubiquitin/26S proteolytic pathway by generating free ubiquitin monomers from their initial translational products, recycling ubiquitins during the breakdown of ubiquitin-protein conjugates, and/or by removing ubiquitin from specific targets and thus presumably preventing target degradation. Here, we describe a family of 27 UBP genes from Arabidopsis that contain both the conserved cysteine (Cys) and histidine boxes essential for catalysis. They can be clustered into 14 subfamilies based on sequence similarity, genomic organization, and alignments with their closest relatives from other organisms, with seven subfamilies having two or more members. Recombinant AtUBP2 functions as a bona fide UBP: It can release polypeptides attached to ubiquitins via either alpha- or epsilon-amino linkages by an activity that requires the predicted active-site Cys within the Cys box. From the analysis of T-DNA insertion mutants, we demonstrate that the AtUBP1 and 2 subfamily helps confer resistance to the arginine analog canavanine. This phenotype suggests that the AtUBP1 and 2 enzymes are needed for abnormal protein turnover in Arabidopsis.     

In vivo disassembly of free polyubiquitin chains by yeast Ubp14 modulates rates of protein degradation by the proteasome.

Degradation of many eukaryotic proteins requires their prior ligation to polyubiquitin chains, which target substrates to the 26S proteasome, an abundant cellular protease. We describe a yeast deubiquitinating enzyme, Ubp14, that specifically disassembles unanchored ('free') ubiquitin chains in vitro, a specificity shared by mammalian isopeptidase T. Correspondingly, deletion of the UBP14 gene from yeast cells results in a striking accumulation of free ubiquitin chains, which correlates with defects in ubiquitin-dependent proteolysis. Increasing the steady-state levels of ubiquitin chains in wild-type cells (by expressing a derivative of ubiquitin with an altered C-terminus) inhibits protein degradation to a degree comparable with that observed in ubp14delta cells. Inhibition of degradation is also seen when an active site mutant of Ubp14 is overproduced in vivo. Surprisingly, overproduction of wild-type Ubp14 can inhibit degradation of some proteins as well. Finally, Ubp14 and human isopeptidase T are shown to be functional homologs by complementation analysis. We propose that Ubp14 and isopeptidase T facilitate proteolysis in vivo by preventing unanchored ubiquitin chains from competitively inhibiting polyubiquitin-substrate binding to the 26S proteasome.     

Structure and mechanisms of the proteasome-associated deubiquitinating enzyme USP14

The ubiquitin-specific processing protease (UBP) family of deubiquitinating enzymes plays an essential role in numerous cellular processes. Mammalian USP14 (Ubp6 in yeast) is unique among known UBP enzymes in that it is activated catalytically upon specific association with the 26S proteasome. Here, we report the crystal structures of the 45-kDa catalytic domain of USP14 in isolation and in a complex with ubiquitin aldehyde, which reveal distinct structural features. In the absence of ubiquitin binding, the catalytic cleft leading to the active site of USP14 is blocked by two surface loops. Binding by ubiquitin induces a significant conformational change that translocates the two surface loops thereby allowing access of the ubiquitin C-terminus to the active site. These structural observations, in conjunction with biochemical characterization, identify important regulatory mechanisms for USP14.     

At UBP3 and At UBP4 are two closely related Arabidopsis thaliana ubiquitin-specific proteases present in the nucleus

The ubiquitin-specific proteases (UBPs) are a class of enzymes vital to the ubiquitin pathway. These enzymes cleave ubiquitin at its C-terminus from two types of substrates containing (i) ubiquitin in an α-amino linkage, as found in the primary ubiquitin translation products, polyubiquitin and ubiquitin-ribosomal fusion proteins, or (ii) ubiquitin in an ?-amino linkage, as found in multiubiquitin chains either unattached or conjugated to cellular proteins. We have isolated cDNAs for two Arabidopsis thaliana genes, AtUBP3 and AtUBP4, which encode UBPs that are 93% identical. These two cDNAs represent the only two members of this subgroup and encode the smallest UBPs described to date in any organism. Using in vivo assays in Escherichia coli that allow the coexpression of a UBP with a putative substrate, we have shown that AtUBP3 and AtUBP4 can specifically deubiquitinate the artificial substrate Ub-X-β-gal but cannot act upon the natural α-amino-linked ubiquitin fusions Arabidopsis Ub-CEP52 and Arabidopsis polyubiquitin. Affinity-purified antibody prepared against AtUBP3 expressed in E. coli recognizes both AtUBP3 and AtUBP4. AtUBP3 and/or AtUBP4 are present in all Arabidopsis organs examined and at multiple developmental stages. Subcellular localization studies show that AtUBP3 and/or AtUBP4 are present in nuclear extracts. Possible physiological roles for these UBPs are discussed.     

At UBP3 and At UBP4 are two closely related Arabidopsis thaliana ubiquitin-specific proteases present in the nucleus

The ubiquitin-specific proteases (UBPs) are a class of enzymes vital to the ubiquitin pathway. These enzymes cleave ubiquitin at its C-terminus from two types of substrates containing (i) ubiquitin in an α-amino linkage, as found in the primary ubiquitin translation products, polyubiquitin and ubiquitin-ribosomal fusion proteins, or (ii) ubiquitin in an ɛ-amino linkage, as found in multiubiquitin chains either unattached or conjugated to cellular proteins. We have isolated cDNAs for two Arabidopsis thaliana genes, AtUBP3 and AtUBP4, which encode UBPs that are 93% identical. These two cDNAs represent the only two members of this subgroup and encode the smallest UBPs described to date in any organism. Using in vivo assays in Escherichia coli that allow the coexpression of a UBP with a putative substrate, we have shown that AtUBP3 and AtUBP4 can specifically deubiquitinate the artificial substrate Ub-X-β-gal but cannot act upon the natural α-amino-linked ubiquitin fusions Arabidopsis Ub-CEP52 and Arabidopsis polyubiquitin. Affinity-purified antibody prepared against AtUBP3 expressed in E. coli recognizes both AtUBP3 and AtUBP4. AtUBP3 and/or AtUBP4 are present in all Arabidopsis organs examined and at multiple developmental stages. Subcellular localization studies show that AtUBP3 and/or AtUBP4 are present in nuclear extracts. Possible physiological roles for these UBPs are discussed. Received: 14 November 1996 / Accepted: 6 February 1997     

UFD4 lacking the proteasome-binding region catalyses ubiquitination but is impaired in proteolysis

The ubiquitin system recognizes degradation signals of protein substrates through E3-E2 ubiquitin ligases, which produce a substrate-linked multi-ubiquitin chain. Ubiquitinated substrates are degraded by the 26S proteasome, which consists of the 20S protease and two 19S particles. We previously showed that UBR1 and UFD4, two E3 ligases of the yeast Saccharomyces cerevisiae, interact with specific proteasomal subunits. Here we advance this analysis for UFD4 and show that it interacts with RPT4 and RPT6, two subunits of the 19S particle. The 201-residue amino-terminal region of UFD4 is essential for its binding to RPT4 and RPT6. UFD4(DeltaN), which lacks this N-terminal region, adds ubiquitin to test substrates with apparently wild-type activity, but is impaired in conferring short half-lives on these substrates. We propose that interaction of a targeted substrate with the 26S proteasome involves contacts of specific proteasomal subunits with the substrate-bound ubiquitin ligase, with the substrate-linked multi-ubiquitin chain and with the substrate itself. This multiple-site binding may function to slow down dissociation of the substrate from the proteasome and to facilitate the unfolding of substrate through ATP-dependent movements of the chaperone subunits of the 19S particle.     

Lysine 63‐linked polyubiquitin chain may serve as a targeting signal for the 26S proteasome

Recruitment of substrates to the 26S proteasome usually requires covalent attachment of the Lys48‐linked polyubiquitin chain. In contrast, modifications with the Lys63‐linked polyubiquitin chain and/or monomeric ubiquitin are generally thought to function in proteasome‐independent cellular processes. Nevertheless, the ubiquitin chain‐type specificity for the proteasomal targeting is still poorly understood, especially in vivo. Using mass spectrometry, we found that Rsp5, a ubiquitin‐ligase in budding yeast, catalyzes the formation of Lys63‐linked ubiquitin chains in vitro. Interestingly, the 26S proteasome degraded well the Lys63‐linked ubiquitinated substrate in vitro. To examine whether Lys63‐linked ubiquitination serves in degradation in vivo, we investigated the ubiquitination of Mga2‐p120, a substrate of Rsp5. The polyubiquitinated p120 contained relatively high levels of Lys63‐linkages, and the Lys63‐linked chains were sufficient for the proteasome‐binding and subsequent p120‐processing. In addition, Lys63‐linked chains as well as Lys48‐linked chains were detected in the 26S proteasome‐bound polyubiquitinated proteins. These results raise the possibility that Lys63‐linked ubiquitin chain also serves as a targeting signal for the 26S proteaseome in vivo.     

Proteasome Inhibitors Which Induce Neurite Outgrowth From PC12h Cells Cause Different Subcellular Accumulations of Multi-Ubiquitin Chains

The effects of two proteasome inhibitors on neurite outgrowth from PC12h cells were investigated in terms of the mean length of the neurites and the frequency of occurrence of cells with long neurites. Benzyloxycarbonyl-leucyl-leucyl-leucinal (ZLLLal) and benzyloxycarbonyl-isoleucyl-t-butyl-glutamyl-leucinal (PSI) caused a significant elongation of PC12h cell neurites. Since ZLLLal is known to inhibit both calpain and proteasome activity, we examined the effects of benzyloxycarbonyl-leucyl-leucinal (ZLLal) which inhibits calpain activity to the same degree as ZLLLal, but which inhibits proteasome activity only weakly. ZLLal did not induce the significant elongation of neurites at any of the concentrations we studied. These results show that the inhibition of proteasome activity causes neurite elongation. We also quantified subcellular levels of multi-ubiquitin chains and free ubiquitin after treatments with PSI, ZLLLal and ZLLal. Treatment with ZLLal had no effects on levels of water- and urea-soluble multi-ubiquitin chains or of free ubiquitin either in the nucleus or in the cytoplasm. PSI and ZLLLal induced a large accumulation of water- and urea-soluble multi-ubiquitin chains and free ubiquitin in the nucleus. Similarly, PSI and ZLLLal increased cytoplasmic levels of urea-soluble multi-ubiquitin chains. On the contrary, PSI and ZLLLal had no effect on levels of water-soluble multi-ubiquitin chains or free ubiquitin in the cytoplasm. This is the first study to demonstrate subcellular differences in the accumulation of multi-ubiquitin chains and free ubiquitin during the neurite elongation induced by proteasome inhibitors.     

Ubiquitin-specific proteases from Arabidopsis thaliana: cloning of AtUBP5 and analysis of substrate specificity of AtUBP3, AtUBP4, and AtUBP5 using Escherichia coli in vivo and in vitro assays

A cDNA for a new ubiquitin-specific protease (UBP), AtUBP5, was identified from Arabidopsis thaliana flower mRNA using an oligonucleotide made against the conserved UBP cysteine (Cys) box. The 924-amino-acid AtUBP5 contains the regions characteristic of all UBPs and has 35% identity and 53% similarity overall to a mammalian UBP (Unp), resulting from additional significant similarity outside these regions. AtUBP5 has 48% identity and 58% similarity overall to two uncharacterized Arabidopsis genomic sequences but is distinct outside the UBP conserved regions from two other previously published Arabidopsis UBPs, AtUBP3 and -4. Using in vivo Escherichia coli assays, which allow co-expression of GSTAtUBPs and substrates, we show that all three UBPs were active. AtUBP5 was active without 311 amino acids N-terminal to the active site cysteine, or without 233 nonconserved amino acids between the Cys and His boxes, or without both, indicating the core region was sufficient. In in vivo and in vitro assays, GSTAtUBP3, -4, and -5 exhibited preference for specific Ub-Ub linkages, suggesting accessibility and/or conformation is important and demonstrating that these enzymes cleave post-translationally. A chimeric UBP consisting of the AtUBP5 Cys box with AtUBP3 amino acids was active and exhibited AtUBP3 specificity, indicating that the modular nature of UBPs and specificity for cleavage sites is not determined by the Cys box.     

The ubiquitin-specific protease subfamily UBP3/UBP4 is essential for pollen development and transmission in Arabidopsis

Deubiquitinating enzymes are essential to the ubiquitin (Ub)/26S proteasome system where they release Ub monomers from the primary translation products of poly-Ub and Ub extension genes, recycle Ubs from polyubiquitinated proteins, and reverse the effects of ubiquitination by releasing bound Ubs from individual targets. The Ub-specific proteases (UBPs) are one large family of deubiquitinating enzymes that bear signature cysteine and histidine motifs. Here, we genetically characterize a UBP subfamily in Arabidopsis (Arabidopsis thaliana) encoded by paralogous UBP3 and UBP4 genes. Whereas homozygous ubp3 and ubp4 single mutants do not display obvious phenotypic abnormalities, double-homozygous mutant individuals could not be created due to a defect in pollen development and/or transmission. This pollen defect was rescued with a transgene encoding wild-type UBP3 or UBP4, but not with a transgene encoding an active-site mutant of UBP3, indicating that deubiquitination activity of UBP3/UBP4 is required. Nuclear DNA staining revealed that ubp3 ubp4 pollen often fail to undergo mitosis II, which generates the two sperm cells needed for double fertilization. Substantial changes in vacuolar morphology were also evident in mutant grains at the time of pollen dehiscence, suggesting defects in vacuole and endomembrane organization. Even though some ubp3 ubp4 pollen could germinate in vitro, they failed to fertilize wild-type ovules even in the absence of competing wild-type pollen. These studies provide additional evidence that the Ub/26S proteasome system is important for male gametogenesis in plants and suggest that deubiquitination of one or more targets by UBP3/UBP4 is critical for the development of functional pollen.     

Uch2/Uch37 is the major deubiquitinating enzyme associated with the 26S proteasome in fission yeast

Conjugation of proteins to ubiquitin plays a central role for a number of cellular processes including endocytosis, DNA repair and degradation by the 26S proteasome. However, ubiquitination is reversible as a number of deubiquitinating enzymes mediate the disassembly of ubiquitin-protein conjugates. Some deubiquitinating enzymes are associated with the 26S proteasome contributing to and regulating the particle's activity. Here, we characterise fission yeast Uch2 and Ubp6, two proteasome associated deubiquitinating enzymes. The human orthologues of these enzymes are known as Uch37 and Usp14, respectively. We report that the subunit Uch2/Uch37 is the major deubiquitinating enzyme associated with the fission yeast 26S proteasome. In contrast, the activity of Ubp6 appears to play a more regulatory and/or structural role involving the proteasome subunits Mts1/Rpn9, Mts2/Rpt2 and Mts3/Rpn12, as Ubp6 becomes essential when activity of these subunits is compromised by conditional mutations. Finally, when the genes encoding Uch2/Uch37 and Ubp6 are disrupted, the cells are viable without showing obvious signs of impaired ubiquitin-dependent proteolysis, indicating that other deubiquitinating enzymes may remedy for the redundancy of these enzymes.     

The Arabidopsis mitochondrial membrane‐bound ubiquitin protease UBP27 contributes to mitochondrial morphogenesis

Mitochondria are essential organelles with dynamic morphology and function. Post‐translational modifications (PTMs), which include protein ubiquitination, are critically involved in animal and yeast mitochondrial dynamics. How PTMs contribute to plant mitochondrial dynamics is just beginning to be elucidated, and mitochondrial enzymes involved in ubiquitination have not been reported from plants. In this study, we identified an Arabidopsis mitochondrial localized ubiquitin protease, UBP27, through a screen that combined bioinformatics and fluorescent fusion protein targeting analysis. We characterized UBP27 with respect to its membrane topology and enzymatic activities, and analysed the mitochondrial morphological changes in UBP27T‐DNA insertion mutants and overexpression lines. We have shown that UBP27 is embedded in the mitochondrial outer membrane with an N_in–C_out orientation and possesses ubiquitin protease activities in vitro. UBP27 demonstrates similar sub‐cellular localization, domain structure, membrane topology and enzymatic activities with two mitochondrial deubiquitinases, yeast ScUBP16 and human HsUSP30, which indicated that these proteins are functional orthologues in eukaryotes. Although loss‐of‐function mutants of UBP27 do not show obvious phenotypes in plant growth and mitochondrial morphology, UBP27 overexpression can change mitochondrial morphology from rod to spherical shape and reduce the mitochondrial association of dynamin‐related protein 3 (DRP3) proteins, large GTPases that serve as the main mitochondrial fission factors. Thus, our study has uncovered a plant ubiquitin protease that plays a role in mitochondrial morphogenesis possibly through modulation of the function of organelle division proteins.     

Structure and functional analyses of the 26S proteasome subunits from plants – Plant 26S proteasome

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.     

Arabidopsis ubiquitin-specific protease 6 (AtUBP6) interacts with calmodulin

Calmodulin (CaM), a key Ca(2+) sensor in eukaryotes, regulates diverse cellular processes by interacting with many proteins. To identify Ca(2+)/CaM-mediated signaling components, we screened an Arabidopsis expression library with horseradish peroxidase-conjugated Arabidopsis calmodulin2 (AtCaM2) and isolated a homolog of the UBP6 deubiquitinating enzyme family (AtUBP6) containing a Ca(2+)-dependent CaM-binding domain (CaMBD). The CaM-binding activity of the AtUBP6 CaMBD was confirmed by CaM mobility shift assay, phosphodiesterase competition assay and site-directed mutagenesis. Furthermore, expression of AtUBP6 restored canavanine resistance to the Deltaubp6 yeast mutant. This is the first demonstration that Ca(2+) signaling via CaM is involved in ubiquitin-mediated protein degradation and/or stabilization in plants.     

The multiubiquitin-chain-binding protein Mcb1 is a component of the 26S proteasome in Saccharomyces cerevisiae and plays a nonessential, substrate-specific role in protein turnover.

The 26S proteasome is an essential proteolytic complex that is responsible for degrading proteins conjugated with ubiquitin. It has been proposed that the recognition of substrates by the 26S proteasome is mediated by a multiubiquitin-chain-binding protein that has previously been characterized in both plants and animals. In this study, we identified a Saccharomyces cerevisiae homolog of this protein, designated Mcb1. Mcb1 copurified with the 26S proteasome in both conventional and nickel chelate chromatography. In addition, a significant fraction of Mcb1 in cell extracts was present in a low-molecular-mass form free of the 26S complex. Recombinant Mcb1 protein bound multiubiquitin chains in vitro and, like its plant and animal counterparts, exhibited a binding preference for longer chains. Surprisingly, (delta)mcb1 deletion mutants were viable, grew at near-wild-type rates, degraded the bulk of short-lived proteins normally, and were not sensitive to UV radiation or heat stress. These data indicate that Mcb1 is not an essential component of the ubiquitin-proteasome pathway in S.cerevisiae. However, the (delta)mcb1 mutant exhibited a modest sensitivity to amino acid analogs and had increased steady-state levels of ubiquitin-protein conjugates. Whereas the N-end rule substrate, Arg-beta-galactosidase, was degraded at the wild-type rate in the (delta)mcb1 strain, the ubiquitin fusion degradation pathway substrate, ubiquitin-Pro-beta-galactosidase, was markedly stabilized. Collectively, these data suggest that Mcb1 is not the sole factor involved in ubiquitin recognition by the 26S proteasome and that Mcb1 may interact with only a subset of ubiquitinated substrates.     

Multiubiquitin chain binding subunit MCB1 (RPN10) of the 26S proteasome is essential for developmental progression in Physcomitrella patens.

The 26S proteasome, a multisubunit complex, is the primary protease of the ubiquitin-mediated proteolytic system in eukaryotes. We have recently characterized MCB1 (RPN10), a subunit of the 26S complex that has affinity for multiubiquitin chains in vitro and as a result may function as a receptor for ubiquitinated substrates. To define the role of MCB1 further, we analyzed its function in Physcomitrella patens by generating MCB1 gene disruptions using homologous recombination. PpMCB1, which is 50 to 75% similar to orthologs from other eukaryotes, is present in the 26S proteasome complex and has a similar affinity for multiubiquitin chains, using a conserved hydrophobic domain within the C-terminal half of the polypeptide. Unlike yeast Deltamcb1 strains, which grow normally, P. patens Deltamcb1 strains are viable but are under developmental arrest, generating abnormal caulonema that are unable to form buds and gametophores. Treatment with auxin and cytokinin restored bud formation and subsequent partial development of gametophores. Complementation of a Deltamcb1 strain with mutated versions of PpMCB1 revealed that the multiubiquitin chain binding site is not essential for the wild-type phenotype. These results show that MCB1 has an important function in the 26S proteasome of higher order eukaryotes in addition to its ability to bind multiubiquitin chains, and they provide further support for a role of the ubiquitin/26S proteasome proteolytic pathway in plant developmental processes triggered by hormones.     

泛素/26S蛋白酶体途径与显花植物自交不亲和反应

植物的生长和发育离不开短命调控蛋白的有选择性降解, 其中一种重要的降解方式就是泛素/26S蛋白酶体途径。在这个途径中, 泛素(ubiquitin)和26S蛋白酶体起着至关重要的作用, 需要被降解的蛋白会通过E1-E2-E3酶接合反应由Ub进行标记, 随后标记蛋白会被26S蛋白酶体识别并降解。自交不亲和反应也正是通过此途径实现的, ARC1(arm repeat containing 1)和SCFs (skp1-cul1-F-box-proteins)作为E3s分别在孢子体自交不亲和和配子体自交不亲和反应中起作用。本文综述了就泛素/26S蛋白酶体途径的组成及其在自交不亲和反应中的作用。     

泛素/26S蛋白酶体途径与显花植物自交不亲和反应

植物的生长和发育离不开短命调控蛋白的有选择性降解,其中一种重要的降解方式就是泛素,26S蛋白酶体途径。在这个途径中,泛素（ubiquitin）和26S蛋白酶体起着至关重要的作用,需要被降解的蛋白会通过E1-E2-E3酶接合反应由Ub进行标记,随后标记蛋白会被26s蛋白酶体识别并降解。自交不亲和反应也正是通过此途径实现的,ARC1（arm repeat containing 1）和SCFs（skp1-cul1-F-box-proteins）作为E3s分别在孢子体自交不亲和和配子体自交不亲和反应中起作用。本文综述了就泛素/26S蛋白酶体途径的组成及其在自交不亲和反应中的作用。     

The regulation of proteasome degradation by multi-ubiquitin chain binding proteins

The 26S proteasome is a large multi-protein complex that functions to degrade proteins tagged with multi-ubiquitin chains. There are several mechanisms employed by the cell to ensure the efficient delivery of multi-ubiquitinated substrate proteins to the 26S proteasome. This is not only important to ensure the degradation of damaged and misfolded proteins, but also the regulated turnover of critical cell regulators. This discussion will concentrate on what is known about the recognition and delivery of ubiquitinated substrate proteins to the 26S proteasome.