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

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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OTUB1 non-catalytically stabilizes the E2 ubiquitin-conjugating enzyme UBE2E1 by preventing its autoubiquitination

Journal of Biological Chemistry ◽

10.1074/jbc.ra118.004677 ◽

2018 ◽

Vol 293 (47) ◽

pp. 18285-18295 ◽

Cited By ~ 11

Author(s):

Nagesh Pasupala ◽

Marie E. Morrow ◽

Lauren T. Que ◽

Barbara A. Malynn ◽

Averil Ma ◽

...

Keyword(s):

Polyubiquitin Chains ◽

Protein Ubiquitination ◽

Ubiquitin Conjugating Enzyme ◽

Conjugating Enzymes ◽

E2 Enzymes ◽

Ubiquitin Conjugating Enzymes ◽

In Cells ◽

Conjugating Enzyme

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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A ubiquitin-conjugating enzyme in fission yeast that is essential for the onset of anaphase in mitosis.

Molecular and Cellular Biology ◽

10.1128/mcb.17.6.3388 ◽

1997 ◽

Vol 17 (6) ◽

pp. 3388-3397 ◽

Cited By ~ 35

Author(s):

F Osaka ◽

H Seino ◽

T Seno ◽

F Yamao

Keyword(s):

Fission Yeast ◽

Cell Cycle Progression ◽

Protein S ◽

Growth Defect ◽

Restrictive Temperature ◽

Checkpoint Pathway ◽

Mitotic Cyclin ◽

Ubiquitin Conjugating Enzyme ◽

Conjugating Enzyme

A cDNA encoding a ubiquitin-conjugating enzyme designated UbcP4 in fission yeast was isolated. Disruption of its genomic gene revealed that it was essential for cell viability. In vivo depletion of the UbcP4 protein demonstrated that it was necessary for cell cycle progression at two phases, G2/M and metaphase/anaphase transitions. The G2 arrest of UbcP4-depleted cells was dependent upon chk1, which mediates checkpoint pathway. UbcP4-depleted cells arrested at metaphase had condensed chromosomes but were defective in separation. However, septum formation and cytokinesis were not restrained during the metaphase arrest. Overexpression of UbcP4 specifically rescued the growth defect of cut9ts cells at a restrictive temperature. cut9 encodes a component of the anaphase-promoting complex (APC) which is required for chromosome segregation at anaphase and moreover is defined as cyclin-specific ubiquitin ligase. Cdc13, a mitotic cyclin in fission yeast, was accumulated in the UbcP4-depleted cells. These results strongly suggested that UbcP4 is a ubiquitin-conjugating enzyme working in conjunction with APC and mediates the ubiquitin pathway for degradation of "sister chromatid holding protein(s)" at the onset of anaphase and possibly of mitotic cyclin at the exit of mitosis.

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

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s0907444998002480 ◽

1998 ◽

Vol 54 (5) ◽

pp. 891-898 ◽

Cited By ~ 41

Author(s):

M. F. Giraud ◽

J. M. P. Desterro ◽

J. H. Naismith

Keyword(s):

Ubiquitin Conjugating Enzyme ◽

Conjugating Enzymes ◽

Ubiquitin Conjugating Enzymes ◽

Conjugating Enzyme

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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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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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Regulation of canonical Wnt signalling by the ciliopathy protein MKS1 and the E2 ubiquitin-conjugating enzyme UBE2E1

10.1101/2020.01.08.897959 ◽

2020 ◽

Author(s):

Katarzyna Szymanska ◽

Karsten Boldt ◽

Clare V. Logan ◽

Matthew Adams ◽

Philip A. Robinson ◽

...

Keyword(s):

Primary Cilium ◽

Ubiquitin Proteasome System ◽

Wnt Signalling ◽

Abnormal Accumulation ◽

Ubiquitin Conjugating Enzyme ◽

Ubiquitin Proteasome ◽

Developmental Conditions ◽

Canonical Wnt ◽

Conjugating Enzyme

AbstractA functional primary cilium is essential for normal and regulated signalling. Primary ciliary defects cause a group of developmental conditions known as ciliopathies, but the precise mechanisms of signal regulation by the cilium remain unclear. Previous studies have implicated the ubiquitin proteasome system (UPS) in regulation of Wnt signalling at the ciliary basal body. Here, we provide mechanistic insight into ciliary ubiquitin processing in cells and for a ciliopathy mouse model lacking the ciliary protein Mks1. In vivo loss of Mks1 sensitizes cells to proteasomal disruption, leading to abnormal accumulation of ubiquitinated proteins. To substantiate a direct link between MKS1 and the UPS, we identified UBE2E1, an E2 ubiquitin-conjugating enzyme that polyubiquitinates β-catenin, and RNF34, an E3 ligase, as novel interactants of MKS1. UBE2E1 and MKS1 colocalized, particularly during conditions of ciliary resorption, and loss of UBE2E1 recapitulates the ciliary and Wnt signalling phenotypes observed during loss of MKS1. Levels of UBE2E1 and MKS1 are co-dependent and UBE2E1 mediates both regulatory and degradative ubiquitination of MKS1. Furthermore, we demonstrate that processing of phosphorylated β-catenin occurs at the ciliary base through the functional interaction between UBE2E1 and MKS1. These observations suggest that correct β-catenin levels are tightly regulated at the primary cilium by a ciliary-specific E2 (UBE2E1) and a regulatory substrate-adaptor (MKS1), confirming the fundamental role of UPS defects in the molecular pathogenesis of ciliopathies.

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