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

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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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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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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Functional Interaction between Human Herpesvirus 6 Immediate-Early 2 Protein and Ubiquitin-Conjugating Enzyme 9 in the Absence of Sumoylation

Journal of Virology ◽

10.1128/jvi.00375-06 ◽

2006 ◽

Vol 80 (20) ◽

pp. 10218-10228 ◽

Cited By ~ 21

Author(s):

Andru Tomoiu ◽

Annie Gravel ◽

Robert M. Tanguay ◽

Louis Flamand

Keyword(s):

Human Herpesvirus ◽

Immediate Early ◽

Human Herpesvirus 6 ◽

Interacting Protein ◽

Ubiquitin Conjugating Enzyme ◽

Herpesvirus 6 ◽

Transcriptional Complex ◽

Conjugating Enzyme

ABSTRACT The immediate-early 2 (IE2) protein of human herpesvirus 6 is a potent transactivator of cellular and viral promoters. To better understand the biology of IE2, we generated a LexA-IE2 fusion protein and screened, using the yeast two-hybrid system, a Jurkat T-cell cDNA library for proteins that could interact with IE2. The most frequently isolated IE2-interacting protein was the human ubiquitin-conjugating enzyme 9 (Ubc9), a protein involved in the small ubiquitin-like modifier (SUMO) conjugation pathway. Using deletion mutants of IE2, we mapped the IE2-Ubc9-interacting region to residues 989 to 1037 of IE2. The interaction was found to be of functional significance to IE2, as Ubc9 overexpression significantly repressed promoter activation by IE2. The C93S Ubc9 mutant exhibited a similar effect on IE2, indicating that the E2 SUMO-conjugating function of Ubc9 is not required for its repressive action on IE2. No consensus sumoylation sites or evidence of IE2 conjugation to SUMO could be demonstrated under in vivo or in vitro conditions. Moreover, expression levels and nuclear localization of IE2 were not altered by Ubc9 overexpression, suggesting that Ubc9's repressive function likely occurs at the transcriptional complex level. Overall, our results indicate that Ubc9 influences IE2's function and provide new information on the complex interactions that occur between herpesviruses and the sumoylation pathway.

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The APC11 RING-H2 Finger Mediates E2-Dependent Ubiquitination

Molecular Biology of the Cell ◽

10.1091/mbc.11.7.2315 ◽

2000 ◽

Vol 11 (7) ◽

pp. 2315-2325 ◽

Cited By ~ 114

Author(s):

Joel D. Leverson ◽

Claudio A.P. Joazeiro ◽

Andrew M. Page ◽

Han-kuei Huang ◽

Philip Hieter ◽

...

Keyword(s):

Ubiquitin Ligase ◽

26S Proteasome ◽

Anaphase Promoting Complex ◽

Mitotic Progression ◽

Protein Substrates ◽

Ubiquitin Ligase Activity ◽

Ubiquitin Conjugating Enzyme ◽

Ubiquitin Conjugating Enzymes

Polyubiquitination marks proteins for degradation by the 26S proteasome and is carried out by a cascade of enzymes that includes ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and ubiquitin ligases (E3s). The anaphase-promoting complex or cyclosome (APC/C) comprises a multisubunit ubiquitin ligase that mediates mitotic progression. Here, we provide evidence that theSaccharomyces cerevisiae RING-H2 finger protein Apc11 defines the minimal ubiquitin ligase activity of the APC. We found that the integrity of the Apc11p RING-H2 finger was essential for budding yeast cell viability, Using purified, recombinant proteins we showed that Apc11p interacted directly with the Ubc4 ubiquitin conjugating enzyme (E2). Furthermore, purified Apc11p was capable of mediating E1- and E2-dependent ubiquitination of protein substrates, including Clb2p, in vitro. The ability of Apc11p to act as an E3 was dependent on the integrity of the RING-H2 finger, but did not require the presence of the cullin-like APC subunit Apc2p. We suggest that Apc11p is responsible for recruiting E2s to the APC and for mediating the subsequent transfer of ubiquitin to APC substrates in vivo.

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Genetic and biochemical interactions between an essential kinetochore protein, Cbf2p/Ndc10p, and the CDC34 ubiquitin-conjugating enzyme.

Molecular and Cellular Biology ◽

10.1128/mcb.15.9.4835 ◽

1995 ◽

Vol 15 (9) ◽

pp. 4835-4842 ◽

Cited By ~ 20

Author(s):

H J Yoon ◽

J Carbon

Keyword(s):

Yeast Cells ◽

Temperature Sensitive ◽

Kinetochore Protein ◽

Ubiquitin Conjugating Enzyme ◽

Endogenous Substrate ◽

Growth Phenotype ◽

Kinetochore Function ◽

Conjugating Enzyme

CBF2/NDC10/CTF14 encodes the 110-kDa subunit of CBF3, a key component of the yeast centromere/kinetochore. Overexpression of yeast CDC34 specifically suppresses the temperature-sensitive growth phenotype of the ndc10-1 mutation. Mutations in CDC34, which specifies a ubiquitin-conjugating enzyme, arrest yeast cells in the G1 phase of the cell cycle, with no intact spindles formed (M. G. Goebl, J. Yochem, S. Jentsch, J. P. McGrath, A. Varshavsky, and B. Byers, Science 241:1331-1335, 1988). The cdc34-2 mutation drastically alters the pattern of Cbf2p modification. Results of experiments using antibodies against Cbf2p and ubiquitin indicate that Cbf2p is ubiquitinated in vivo. Purified Cdc34p catalyzes the formation of Cbf2p-monoubiquitin conjugate in vitro. These data suggest that Cbf2p is an endogenous substrate of the CDC34 ubiquitin-conjugating enzyme and imply that ubiquitination of a kinetochore protein plays a regulatory role in kinetochore function.

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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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OTUB1 non-catalytically regulates the stability of the E2 ubiquitin conjugating enzyme UBE2E1

10.1101/359414 ◽

2018 ◽

Author(s):

Nagesh Pasupala ◽

Marie E. Morrow ◽

Lauren T. Que ◽

Barbara A. Malynn ◽

Averil Ma ◽

...

Keyword(s):

Catalytic Mechanism ◽

Polyubiquitin Chains ◽

Protein Ubiquitination ◽

Ubiquitin Conjugating Enzyme ◽

E2 Enzymes ◽

The Stability ◽

In Cells ◽

Conjugating Enzyme

AbstractOTUB1 is a deubiquitinating enzyme that cleaves K48-linked polyubiquitin chains and also regulates ubiquitin signaling through a unique, non-catalytic 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 members of the UBE2D (UBCH5) and UBE2E families, as well as to UBC13 (UBE2N). Cellular roles have been identified for the interaction of OTUB1 with UBC13 and members of the UBE2D family, but not for UBE2E E2 enzymes. We report here a novel role for OTUB1-E2 interactions in modulating E2 protein ubiquitination. We find that depletion of OTUB1 dramatically destabilizes the E2 conjugating enzyme UBE2E1 (UBE2E1) in cells and that this effect is independent of the catalytic activity of OTUB1 but depends on the ability of OTUB1 to bind to UBE2E1. We show that OTUB1 suppresses UBE2E1 autoubiquitinationin vitroand in cells, thereby preventing UBE2E1 from being targeted to the proteasome for degradation. Taken together, we have found a new role for OTUB1 in rescuing specific E2s from degradationin vivo.

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Effect of Arabidopsis COP10 ubiquitin E2 enhancement activity across E2 families and functional conservation among its canonical homologues

Biochemical Journal ◽

10.1042/bj20081943 ◽

2009 ◽

Vol 418 (3) ◽

pp. 683-690 ◽

Cited By ~ 18

Author(s):

On Sun Lau ◽

Xing Wang Deng

Keyword(s):

Dna Damage ◽

Arabidopsis Thaliana ◽

Plant Development ◽

Functional Conservation ◽

Human Ubiquitin ◽

Ubiquitin Conjugating Enzyme ◽

Variant Protein ◽

Conjugating Enzyme

Arabidopsis thaliana COP10 (constitutive photomorphogenic 10) is a UEV [Ub (ubiquitin)-conjugating enzyme (E2) variant protein] that is required for repression of seedling photomorphogenesis in darkness. COP10 forms a complex {the CDD complex [COP10–DET1 (de-etiolated 1)–DDB1 (DNA damage binding protein 1) complex]} with DET1 and DDB1a in vivo and can enhance the activity of Ub-conjugating enzyme (E2) in vitro. To investigate whether COP10 might act as a general regulator of E2s, we tested the specificity of COP10 E2 enhancement activity across E2 families of Arabidopsis. We found that COP10 is capable of enhancing members of four E2 subgroups significantly, while having a milder effect on another. Surprisingly, we found that close canonical E2 homologues of COP10, such as UbcH5a (human ubiquitin-conjugating enzyme 5), are also capable of enhancing E2s. Furthermore, we detected direct interactions between COP10 and three of the enhanced E2s, hinting at a possible mechanism for the enhancements. The present study suggests that some E2s, including the generic Ubc4/5p families involved in many processes, might possess dual activities: the formation of the classic E2–Ub thiol ester and the previously unknown E2 enhancement activity. Therefore COP10, despite being a catalytically inactive E2, might still enhance a variety of E2s and regulate numerous aspects of plant development.

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Cdc34p Ubiquitin-Conjugating Enzyme Is a Component of the Tombusvirus Replicase Complex and Ubiquitinates p33 Replication Protein

Journal of Virology ◽

10.1128/jvi.00702-08 ◽

2008 ◽

Vol 82 (14) ◽

pp. 6911-6926 ◽

Cited By ~ 102

Author(s):

Zhenghe Li ◽

Daniel Barajas ◽

Tadas Panavas ◽

David A. Herbst ◽

Peter D. Nagy

Keyword(s):

Replication Protein ◽

Host Proteins ◽

A Genome ◽

Associated Proteins ◽

Ubiquitin Conjugating Enzyme ◽

Wide Scale ◽

Split Ubiquitin ◽

Conjugating Enzyme

ABSTRACT To identify host proteins interacting with Tomato bushy stunt virus (TBSV) replication proteins in a genome-wide scale, we have used a yeast (Saccharomyces cerevisiae) proteome microarray carrying 4,088 purified proteins. This approach led to the identification of 58 yeast proteins that interacted with p33 replication protein. The identified host proteins included protein chaperones, ubiquitin-associated proteins, translation factors, RNA-modifying enzymes, and other proteins with yet-unknown functions. We confirmed that 19 of the identified host proteins bound to p33 in vitro or in a split-ubiquitin-based two-hybrid assay. Further analysis of Cdc34p E2 ubiquitin-conjugating enzyme, which is one of the host proteins interacting with p33, revealed that Cdc34p is a novel component of the purified viral replicase. Downregulation of Cdc34p expression in yeast, which supports replication of a TBSV replicon RNA (repRNA), reduced repRNA accumulation and the activity of the tombusvirus replicase by up to fivefold. Overexpression of wild-type Cdc34p, but not that of an E2-defective mutant of Cdc34p, increased repRNA accumulation, suggesting a significant role for the ubiquitin-conjugating enzyme function of Cdc34p in TBSV replication. Also, Cdc34p was able to ubiquitinate p33 in vitro. In addition, we have shown that p33 becomes ubiquitinated in vivo. We propose that ubiquitination of p33 likely alters its function or affects the recruitment of host factors during TBSV replication.

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Distinct activation of an E2 ubiquitin-conjugating enzyme by its cognate E3 ligases

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1415621112 ◽

2015 ◽

Vol 112 (7) ◽

pp. E625-E632 ◽

Cited By ~ 11

Author(s):

Itamar Cohen ◽

Reuven Wiener ◽

Yuval Reiss ◽

Tommer Ravid

Keyword(s):

Transition State ◽

Protein Quality ◽

Noncovalent Interactions ◽

Cellular Protein ◽

Ring Domain ◽

E3 Ligases ◽

Ubiquitin Conjugating Enzyme ◽

Conjugating Enzyme

A significant portion of ubiquitin (Ub)-dependent cellular protein quality control takes place at the endoplasmic reticulum (ER) in a process termed “ER-associated degradation” (ERAD). Yeast ERAD employs two integral ER membrane E3 Ub ligases: Hrd1 (also termed “Der3”) and Doa10, which recognize a distinct set of substrates. However, both E3s bind to and activate a common E2-conjugating enzyme, Ubc7. Here we describe a novel feature of the ERAD system that entails differential activation of Ubc7 by its cognate E3s. We found that residues within helix α2 of Ubc7 that interact with donor Ub were essential for polyUb conjugation. Mutagenesis of these residues inhibited the in vitro activity of Ubc7 by preventing the conjugation of donor Ub to the acceptor. Unexpectedly, Ub chain formation by mutant Ubc7 was restored selectively by the Hrd1 RING domain but not by the Doa10 RING domain. In agreement with the in vitro data, Ubc7 α2 helix mutations selectively impaired the in vivo degradation of Doa10 substrates but had no apparent effect on the degradation of Hrd1 substrates. To our knowledge, this is the first example of distinct activation requirements of a single E2 by two E3s. We propose a model in which the RING domain activates Ub transfer by stabilizing a transition state determined by noncovalent interactions between the α2 helix of Ubc7 and Ub and that this transition state may be stabilized further by some E3 ligases, such as Hrd1, through additional interactions outside the RING domain.

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