Structure of Ubiquitin-Conjugating Enzyme 9 Displays Significant Differences with Other Ubiquitin-Conjugating Enzymes Which May Reflect its Specificity for Sumo Rather Than Ubiquitin

OTUB1 is a deubiquitinating enzyme that cleaves Lys-48–linked polyubiquitin chains and also regulates ubiquitin signaling through a unique, noncatalytic mechanism. OTUB1 binds to a subset of E2 ubiquitin-conjugating enzymes and inhibits their activity by trapping the E2∼ubiquitin thioester and preventing ubiquitin transfer. The same set of E2s stimulate the deubiquitinating activity of OTUB1 when the E2 is not charged with ubiquitin. Previous studies have shown that, in cells, OTUB1 binds to E2-conjugating enzymes of the UBE2D (UBCH5) and UBE2E families, as well as to UBE2N (UBC13). Cellular roles have been identified for the interaction of OTUB1 with UBE2N and members of the UBE2D family, but not for interactions with UBE2E E2 enzymes. We report here a novel role for OTUB1–E2 interactions in modulating E2 protein ubiquitination. We observe that Otub1−/− knockout mice exhibit late-stage embryonic lethality. We find that OTUB1 depletion dramatically destabilizes the E2-conjugating enzyme UBE2E1 (UBCH6) in both mouse and human OTUB1 knockout cell lines. Of note, this effect is independent of the catalytic activity of OTUB1, but depends on its ability to bind to UBE2E1. We show that OTUB1 suppresses UBE2E1 autoubiquitination in vitro and in cells, thereby preventing UBE2E1 from being targeted to the proteasome for degradation. Taken together, we provide evidence that OTUB1 rescues UBE2E1 from degradation in vivo.

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Two Ubiquitin-Conjugating Enzymes, UbcP1/Ubc4 and UbcP4/Ubc11, Have Distinct Functions for Ubiquitination of Mitotic Cyclin

Molecular and Cellular Biology ◽

10.1128/mcb.23.10.3497-3505.2003 ◽

2003 ◽

Vol 23 (10) ◽

pp. 3497-3505 ◽

Cited By ~ 23

Author(s):

Hiroaki Seino ◽

Tsutomu Kishi ◽

Hideo Nishitani ◽

Fumiaki Yamao

Keyword(s):

Fission Yeast ◽

Ubiquitin Proteasome System ◽

High Expression ◽

Mitotic Cyclin ◽

Ubiquitin Conjugating Enzyme ◽

Conjugating Enzymes ◽

Ubiquitin Conjugating Enzymes ◽

Mutant Cells ◽

Conjugating Enzyme

ABSTRACT Cell cycle events are regulated by sequential activation and inactivation of Cdk kinases. Mitotic exit is accomplished by the inactivation of mitotic Cdk kinase, which is mainly achieved by degradation of cyclins. The ubiquitin-proteasome system is involved in this process, requiring APC/C (anaphase-promoting complex/cyclosome) as a ubiquitin ligase. In Xenopus and clam oocytes, the ubiquitin-conjugating enzymes that function with APC/C have been identified as two proteins, UBC4 and UBCx/E2-C. Previously we reported that the fission yeast ubiquitin-conjugating enzyme UbcP4/Ubc11, a homologue of UBCx/E2-C, is required for mitotic transition. Here we show that the other fission yeast ubiquitin-conjugating enzyme, UbcP1/Ubc4, which is homologous to UBC4, is also required for mitotic transition in the same manner as UbcP4/Ubc11. Both ubiquitin-conjugating enzymes are essential for cell division and directly required for the degradation of mitotic cyclin Cdc13. They function nonredundantly in the ubiquitination of CDC13 because a defect in ubcP1/ubc4 + cannot be suppressed by high expression of UbcP4/Ubc11 and a defect in ubcP4/ubc11 + cannot be suppressed by high expression of UbcP1/Ubc4. In vivo analysis of the ubiquitinated state of Cdc13 shows that the ubiquitin chains on Cdc13 were short in ubcP1/ubc4 mutant cells while ubiquitinated Cdc13 was totally reduced in ubcP4/ubc11 mutant cells. Taken together, these results indicate that the two ubiquitin-conjugating enzymes play distinct and essential roles in the degradation of mitotic cyclin Cdc13, with the UbcP4/Ubc11-pathway initiating ubiquitination of Cdc13 and the UbcP1/Ubc4-pathway elongating the short ubiquitin chains on Cdc13.

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Hug and hold tight: Dimerization controls the turnover of the ubiquitin-conjugating enzyme UBE2S

Science Signaling ◽

10.1126/scisignal.abd9892 ◽

2020 ◽

Vol 13 (654) ◽

pp. eabd9892

Author(s):

Anja Bremm

Keyword(s):

Half Life ◽

Anaphase Promoting Complex ◽

Ubiquitin Chain ◽

Precise Control ◽

Ubiquitin Conjugating Enzyme ◽

Conjugating Enzymes ◽

Chain Synthesis ◽

Ubiquitin Conjugating Enzymes ◽

Conjugating Enzyme

Precise control of the activity and abundance of ubiquitin-conjugating enzymes (E2s) ensures fidelity in ubiquitin chain synthesis. In this issue of Science Signaling, Liess et al. demonstrate that the human anaphase-promoting complex (APC/C)–associated E2 UBE2S adopts an autoinhibited dimeric state that increases the half-life of UBE2S by preventing its autoubiquitination-driven turnover.

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A Giant Ubiquitin-conjugating Enzyme Related to IAP Apoptosis Inhibitors

The Journal of Cell Biology ◽

10.1083/jcb.141.6.1415 ◽

1998 ◽

Vol 141 (6) ◽

pp. 1415-1422 ◽

Cited By ~ 166

Author(s):

Hans-Peter Hauser ◽

Michael Bardroff ◽

George Pyrowolakis ◽

Stefan Jentsch

Keyword(s):

Covalent Attachment ◽

Protein Protein Interaction ◽

Small Proteins ◽

The Family ◽

Golgi Compartment ◽

Ubiquitin Conjugating Enzyme ◽

Conjugating Enzymes ◽

And Function ◽

Ubiquitin Conjugating Enzymes ◽

Conjugating Enzyme

Ubiquitin-conjugating enzymes (UBC) catalyze the covalent attachment of ubiquitin to target proteins and are distinguished by the presence of a UBC domain required for catalysis. Previously identified members of this enzyme family are small proteins and function primarily in selective proteolysis pathways. Here we describe BRUCE (BIR repeat containing ubiquitin-conjugating enzyme), a giant (528-kD) ubiquitin-conjugating enzyme from mice. BRUCE is membrane associated and localizes to the Golgi compartment and the vesicular system. Remarkably, in addition to being an active ubiquitin-conjugating enzyme, BRUCE bears a baculovirus inhibitor of apoptosis repeat (BIR) motif, which to this date has been exclusively found in apoptosis inhibitors of the IAP-related protein family. The BIR motifs of IAP proteins are indispensable for their anti–cell death activity and are thought to function through protein–protein interaction. This suggests that BRUCE may combine properties of IAP-like proteins and ubiquitin-conjugating enzymes and indicates that the family of IAP-like proteins is structurally and functionally more diverse than previously expected.

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The Fission Yeast Ubiquitin-Conjugating Enzymes UbcP3, Ubc15, and Rhp6 Affect Transcriptional Silencing of the Mating-Type Region

Eukaryotic Cell ◽

10.1128/ec.1.4.613-625.2002 ◽

2002 ◽

Vol 1 (4) ◽

pp. 613-625 ◽

Cited By ~ 14

Author(s):

Inga Sig Nielsen ◽

Olaf Nielsen ◽

Johanne M. Murray ◽

Geneviève Thon

Keyword(s):

Fission Yeast ◽

Rna Polymerase Ii ◽

Mating Type ◽

26S Proteasome ◽

Obvious Effect ◽

Type Region ◽

Ubiquitin Conjugating Enzyme ◽

Conjugating Enzymes ◽

Ubiquitin Conjugating Enzymes ◽

Active Site Mutants

ABSTRACT Genes transcribed by RNA polymerase II are silenced when introduced near the mat2 or mat3 mating-type loci of the fission yeast Schizosaccharomyces pombe. Silencing is mediated by a number of gene products and cis-acting elements. We report here the finding of novel trans-acting factors identified in a screen for high-copy-number disruptors of silencing. Expression of cDNAs encoding the putative E2 ubiquitin-conjugating enzymes UbcP3, Ubc15 (ubiquitin-conjugating enzyme), or Rhp6 (Rad homolog pombe) from the strong nmt1 promoter derepressed the silent mating-type loci mat2 and mat3 and reporter genes inserted nearby. Deletion of rhp6 slightly derepressed an ade6 reporter gene placed in the mating-type region, whereas disruption of ubcP3 or ubc15 had no obvious effect on silencing. Rhp18 is the S. pombe homolog of Saccharomyces cerevisiae Rad18p, a DNA-binding protein that physically interacts with Rad6p. Rhp18 was not required for the derepression observed when UbcP3, Ubc15, or Rhp6 was overproduced. Overexpressing Rhp6 active-site mutants showed that the ubiquitin-conjugating activity of Rhp6 is essential for disruption of silencing. However, high dosage of UbcP3, Ubc15, or Rhp6 was not suppressed by a mutation in the 26S proteasome, suggesting that loss of silencing is not due to an increased degradation of silencing factors but rather to the posttranslational modification of proteins by ubiquitination. We discuss the implications of these results for the possible modes of action of UbcP3, Ubc15, and Rhp6.

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Evolution and expression analysis of the sorghum ubiquitin-conjugating enzyme family

Functional Plant Biology ◽

10.1071/fp18184 ◽

2019 ◽

Vol 46 (3) ◽

pp. 236

Author(s):

Liqiang Jia ◽

QiuFang Zhao ◽

Shu Chen

Keyword(s):

Current Knowledge ◽

Functional Divergence ◽

Signalling Pathways ◽

E3 Ligases ◽

Ubiquitin Conjugating Enzyme ◽

Multiple Treatments ◽

Ubiquitin Conjugating Enzymes ◽

Encoding Genes ◽

Sorghum Bicolor L ◽

Conjugating Enzyme

Ubiquitin-conjugating enzymes (UBCs), which catalyse the transfer of ubiquitin to substrate or E3 ligases, are key enzymes in ubiquitination modifications of target proteins. Current knowledge regarding the sorghum (Sorghum bicolor (L.) Moench) ubiquitin-conjugating enzyme (SbUBC) family remains very limited. We identified 53 UBC-encoding genes in the sorghum genome and divided these into 18 groups according to their phylogenetic relationship with Arabidopsis thaliana (L.) Heynh., which was further supported by conserved motif and gene structure analyses. Different expression levels under a variety of abiotic stresses suggested that these might participate in distinct signalling pathways and that they underwent functional divergence during evolution. Furthermore, several SbUBC genes responded to single treatments, and individual SbUBC genes responded to multiple treatments, suggesting that sorghum UBCs may mediate crosstalk among different signalling pathways. Overall, the results provide valuable information for better understanding the classification and putative functions of sorghum UBC-encoding genes.

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Interaction with a Host Ubiquitin-Conjugating Enzyme Is Required for the Pathogenicity of a Geminiviral DNA β Satellite

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-22-6-0737 ◽

2009 ◽

Vol 22 (6) ◽

pp. 737-746 ◽

Cited By ~ 50

Author(s):

Omid Eini ◽

Satish Dogra ◽

Luke A. Selth ◽

Ian B. Dry ◽

John W. Randles ◽

...

Keyword(s):

Single Gene ◽

Bimolecular Fluorescence Complementation ◽

Cotton Leaf ◽

Specific Symptom ◽

Ubiquitin Conjugating Enzyme ◽

Ubiquitin Proteasome ◽

Ubiquitin Conjugating Enzymes ◽

Conjugating Enzyme

DNA β is a single-stranded satellite DNA which encodes a single gene, βC1. To better understand the role of βC1 in the pathogenicity of DNA β, a yeast two-hybrid screen of a tomato cDNA library was carried out using βC1 from Cotton leaf curl Multan virus (CLCuMV) DNA β as the bait. A ubiquitin-conjugating enzyme, designated SlUBC3, which functionally complemented a yeast mutant deficient in ubiquitin-conjugating enzymes was identified. The authenticity and specificity of the interaction between βC1 and SlUBC3 was confirmed both in vivo, using a bimolecular fluorescence complementation assay, and in vitro, using a protein-binding assay. Analysis of deletion mutants of the βC1 protein showed that a myristoylation-like motif is required both for its interaction with SlUBC3 and the induction of DNA-β-specific symptoms in host plants. The level of polyubiquitinated proteins in transgenic tobacco plants expressing βC1 was found to be reduced compared with wild-type plants. These results are consistent with the hypothesis that interaction of βC1 with SlUBC3 is required for DNA-β-specific symptom induction, and that this is possibly due to downregulation of the host ubiquitin proteasome pathway.

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Molecular Basis for Lysine Specificity in the Yeast Ubiquitin-Conjugating Enzyme Cdc34

Molecular and Cellular Biology ◽

10.1128/mcb.01094-09 ◽

2010 ◽

Vol 30 (10) ◽

pp. 2316-2329 ◽

Cited By ~ 35

Author(s):

Martin Sadowski ◽

Randy Suryadinata ◽

Xianning Lai ◽

Jörg Heierhorst ◽

Boris Sarcevic

Keyword(s):

Amino Acids ◽

Catalytic Core ◽

Ubiquitination Site ◽

New Findings ◽

Lysine Residues ◽

Ubiquitin Conjugating Enzyme ◽

Conjugating Enzymes ◽

Ubiquitin Conjugating Enzymes ◽

Key Residues ◽

Ring E3

ABSTRACT Ubiquitin (Ub)-conjugating enzymes (E2s) and ubiquitin ligases (E3s) catalyze the attachment of Ub to lysine residues in substrates and Ub during monoubiquitination and polyubiquitination. Lysine selection is important for the generation of diverse substrate-Ub structures, which provides versatility to this pathway in the targeting of proteins to different fates. The mechanisms of lysine selection remain poorly understood, with previous studies suggesting that the ubiquitination site(s) is selected by the E2/E3-mediated positioning of a lysine(s) toward the E2/E3 active site. By studying the polyubiquitination of Sic1 by the E2 protein Cdc34 and the RING E3 Skp1/Cul1/F-box (SCF) protein, we now demonstrate that in addition to E2/E3-mediated positioning, proximal amino acids surrounding the lysine residues in Sic1 and Ub are critical for ubiquitination. This mechanism is linked to key residues composing the catalytic core of Cdc34 and independent of SCF. Changes to these core residues altered the lysine preference of Cdc34 and specified whether this enzyme monoubiquitinated or polyubiquitinated Sic1. These new findings indicate that compatibility between amino acids surrounding acceptor lysine residues and key amino acids in the catalytic core of ubiquitin-conjugating enzymes is an important mechanism for lysine selection during ubiquitination.

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Two Ubiquitin-Conjugating Enzymes, Rhp6 and UbcX, Regulate Heterochromatin Silencing in Schizosaccharomyces pombe

Molecular and Cellular Biology ◽

10.1128/mcb.22.23.8366-8374.2002 ◽

2002 ◽

Vol 22 (23) ◽

pp. 8366-8374 ◽

Cited By ~ 8

Author(s):

Eun Shik Choi ◽

Hyun Soo Kim ◽

Yeun Kyu Jang ◽

Seung Hwan Hong ◽

Sang Dai Park

Keyword(s):

Schizosaccharomyces Pombe ◽

Chromatin Immunoprecipitation ◽

Histone H3 ◽

Silent Chromatin ◽

Type Region ◽

Inactive X Chromosome ◽

Ubiquitin Conjugating Enzyme ◽

Conjugating Enzymes ◽

Heterochromatin Silencing ◽

Ubiquitin Conjugating Enzymes

ABSTRACT Methylation of histone H3 has been linked to the assembly of higher-order chromatin structures. Very recently, several examples, including the Schizosaccharomyces pombe mating-type region, chicken β-globin locus, and inactive X-chromosome, revealed that H3-Lys9-methyl (Me) is associated with silent chromatin while H3-Lys4-Me is prominent in active chromatin. Surprisingly, it was shown that homologs of Drosophila Su(var)3-9 specifically methylate the Lys9 residue of histone H3. Here, to identify putative enzymes responsible for destabilization of heterochromatin, we screened genes whose overexpressions disrupt silencing at the silent mat3 locus in fission yeast. Interestingly, we identified two genes, rhp6+ and ubcX+ (ubiquitin-conjugating enzyme participating in silencing), both of which encode ubiquitin-conjugating enzymes. Their overexpression disrupted silencing at centromeres and telomeres as well as at mat3. Additionally, the overexpression interfered with centromeric function, as confirmed by elevated minichromosome loss and antimicrotubule drug sensitivity. On the contrary, deletion of rhp6+ or ubcX+ enhanced silencing at all heterochromatic regions tested, indicating that they are negative regulators of silencing. More importantly, chromatin immunoprecipitation showed that their overexpression alleviated the level of H3-Lys9-Me while enhancing the level of H3-Lys4-Me at the silent regions. On the contrary, their deletions enhanced the level of H3-Lys9-Me while alleviating that of H3-Lys4-Me. Taken together, the data suggest that two ubiquitin-conjugating enzymes, Rhp6 and UbcX, affect methylation of histone H3 at silent chromatin, which then reconfigures silencing.

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Crystal Structure of a Fragment of Mouse Ubiquitin-activating Enzyme

Journal of Biological Chemistry ◽

10.1074/jbc.m502583200 ◽

2005 ◽

Vol 280 (23) ◽

pp. 22006-22011 ◽

Cited By ~ 31

Author(s):

Roman H. Szczepanowski ◽

Renata Filipek ◽

Matthias Bochtler

Keyword(s):

Crystal Structure ◽

Sequence Similarity ◽

Adenylation Domain ◽

Protein Ubiquitination ◽

Catalytic Cysteine ◽

Ubiquitin Ligase E3 ◽

Ubiquitin Conjugating Enzyme ◽

Conjugating Enzymes ◽

Neuronal Precursor Cell ◽

Ubiquitin Conjugating Enzymes

Protein ubiquitination requires the sequential activity of three enzymes: a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin-ligase (E3). The ubiquitin-transfer machinery is hierarchically organized; for every ubiquitin-activating enzyme, there are several ubiquitin-conjugating enzymes, and most ubiquitin-conjugating enzymes can in turn interact with multiple ubiquitin ligases. Despite the central role of ubiquitin-activating enzyme in this cascade, a crystal structure of a ubiquitin-activating enzyme is not available. The enzyme is thought to consist of an adenylation domain, a catalytic cysteine domain, a four-helix bundle, and possibly, a ubiquitin-like domain. Its adenylation domain can be modeled because it is clearly homologous to the structurally known adenylation domains of the activating enzymes for the small ubiquitin-like modifier (SUMO) and for the protein encoded by the neuronal precursor cell-expressed, developmentally down-regulated gene 8 (NEDD8). Low sequence similarity and vastly different domain lengths make modeling difficult for the catalytic cysteine domain that results from the juxtaposition of two catalytic cysteine half-domains. Here, we present a biochemical and crystallographic characterization of the two half-domains and the crystal structure of the larger, second catalytic cysteine half-domain of mouse ubiquitin-activating enzyme. We show that the domain is organized around a conserved folding motif that is also present in the NEDD8- and SUMO-activating enzymes, and we propose a tentative model for full-length ubiquitin-activating enzyme.

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