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• Pickart CM
• MECHANISMS UNDERLYING UBIQUITINATION.
• Annu Rev Biochem. 2001; 70: 503-533
• Display abstract

• The conjugation of ubiquitin to other cellular proteins regulates a broad range of eukaryotic cell functions. The high efficiency and exquisite selectivity of ubiquitination reactions reflect the properties of enzymes known as ubiquitin-protein ligases or E3s. An E3 recognizes its substrates based on the presence of a specific ubiquitination signal, and catalyzes the formation of an isopeptide bond between a substrate (or ubiquitin) lysine residue and the C terminus of ubiquitin. Although a great deal is known about the molecular basis of E3 specificity, much less is known about molecular mechanisms of catalysis by E3s. Recent findings reveal that all known E3s utilize one of just two catalytic domains-a HECT domain or a RING finger-and crystal structures have provided the first detailed views of an active site of each type. The new findings shed light on many aspects of E3 structure, function, and mechanism, but also emphasize that key features of E3 catalysis remain to be elucidated.

• You J, Pickart CM
• A HECT domain E3 enzyme assembles novel polyubiquitin chains.
• J Biol Chem. 2001; 276: 19871-8
• Display abstract

• Although polyubiquitin chains linked through Lys(29) of ubiquitin have been implicated in the targeting of certain substrates to proteasomes, the signaling properties of these chains are poorly understood. We previously described a ubiquitin-protein isopeptide ligase (E3) from erythroid cells that assembles polyubiquitin chains through either Lys(29) or Lys(48) of ubiquitin (Mastrandrea, L. D., You, J., Niles, E. G., and Pickart, C. M. (1999) J. Biol. Chem. 274, 27299-27306). Here we describe the purification of this E3 based on its affinity for a linear fusion of ubiquitin to the ubiquitin-conjugating enzyme UbcH5A. Among five major polypeptides in the affinity column eluate, the activity of interest was assigned to the product of a previously cloned human cDNA known as KIAA10 (Nomura, N., Miyajima, N., Sazuka, T., Tanaka, A., Kawarabayasi, Y., Sato, S., Nagase, T., Seki, N., Ishikawa, K., and Tabata, S. (1994) DNA Res. 1, 27-35). The KIAA10 protein is a member of the HECT (homologous to E6-AP carboxyl terminus) domain family of E3s. These E3s share a conserved C-terminal (HECT) domain that functions in the catalysis of ubiquitination, while their divergent N-terminal domains function in cognate substrate binding (Huibregtse, J. M., Scheffner, M., Beaudenon, S., and Howley, P. M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 2563-2567). Recombinant KIAA10 catalyzed the assembly of both Lys(29)- and Lys(48)-linked polyubiquitin chains. Surprisingly, the C-terminal 428 residues of KIAA10 were both necessary and sufficient for this activity, suggesting that the ability to assemble polyubiquitin chains may be a general property of HECT domains. The N-terminal domain of KIAA10 interacted in vitro with purified 26 S proteasomes and with the isolated S2/Rpn1 subunit of the proteasome's 19 S regulatory complex, suggesting that the N-terminal domains of HECT E3s may function in proteasome binding as well as substrate binding.

• VerPlank L et al.
• Tsg101, a homologue of ubiquitin-conjugating (E2) enzymes, binds the L domain in HIV type 1 Pr55(Gag).
• Proc Natl Acad Sci U S A. 2001; 98: 7724-9
• Display abstract

• Ubiquitination appears to be involved in virus particle release from infected cells. Free ubiquitin (Ub), as well as Ub covalently bound to a small fraction of p6 Gag, is detected in mature HIV particles. Here we report that the p6 region in the Pr55(Gag) structural precursor polyprotein binds to Tsg101, a putative Ub regulator that is involved in trafficking of plasma membrane-associated proteins. Tsg101 was found to interact with Gag in (i) a yeast two-hybrid assay, (ii) in vitro coimmunoprecipitation by using purified Pr55(Gag) and rabbit reticulocyte lysate-synthesized Tsg101, and (iii) in vivo in the cytoplasm of COS cells transfected with gag. The PTAPP motif [or late (L) domain] within p6, which is required for release of mature virus from the plasma membrane, was the determinant for binding Pr55(Gag). The N-terminal region in Tsg101, which is homologous to the Ubc4 class of Ub-conjugating (E2) enzymes, was the determinant of interaction with p6. Mutation of Tyr-110 in Tsg101, present in place of the active-site Cys that binds Ub in E2 enzymes, and other residues unique to Tsg101, impaired p6 interaction, indicating that features that distinguish Tsg101 from active E2 enzymes were important for binding the viral protein. The results link L-domain function in HIV to the Ub machinery and a specific component of the cellular trafficking apparatus.

• Rankin CA, Joazeiro CA, Floor E, Hunter T
• E3 ubiquitin-protein ligase activity of parkin is dependent on cooperative interaction of ring finger (triad) elements.
• J Biomed Sci. 2001; 8: 421-9
• Display abstract

• The parkin gene codes for a 465-amino acid protein which, when mutated, results in autosomal recessive juvenile parkinsonism (AR-JP). Symptoms of AR-JP are similar to those of idiopathic Parkinson's disease, with the notable exception being the early onset of AR-JP. We have cloned and expressed human Parkin in Escherichia coli and have examined Parkin-mediated ubiquitination in an in vitro ubiquitination assay using purified recombinant proteins. We found that Parkin has E3 ubiquitin ligase activity in this system, demonstrating for the first time that the E3 activity is an intrinsic function of the Parkin protein and does not require posttranslational modification or association with cellular proteins other than an E2 (human Ubc4 E2 was utilized in this ubiquitination assay). Mutagenesis of individual elements of the conserved RING TRIAD domain indicated that at least two elements were required for ubiquitin ligase activity and suggested a functional cooperation between the RING finger elements. Since the activity assays were conducted with recombinant proteins purified from E. coli, this is the first time TRIAD element interaction has been demonstrated as an intrinsic feature of Parkin E3 activity. Copyright 2001 National Science Council, ROC and S. Karger AG, Basel

• Matsuda N, Suzuki T, Tanaka K, Nakano A
• Rma1, a novel type of RING finger protein conserved from Arabidopsis to human, is a membrane-bound ubiquitin ligase.
• J Cell Sci. 2001; 114: 1949-57
• Display abstract

• Rma1 is a protein with a RING finger motif and a C-terminal membrane-anchoring domain and is well conserved among higher eukaryotes. We show that fusion proteins between maltose binding protein (MBP) and human or Arabidopsis Rma1 are polyubiquitinated, when incubated with the rabbit reticulocyte or the wheat germ lysate, respectively. The polyubiquitination of MBP-Rma1 has been reconstituted by incubation with purified ubiquitin, the ubiquitin-activating enzyme E1, and one of the two ubiquitin-conjugating E2 enzymes (Ubc4 or UbcH5a). Other E2 enzymes tested, E2-20k, E2-25k, Ubc3 and Ubc8, are not able to confer this modification. Mutational analysis shows that the RING finger motif of Rma1 is necessary for the auto-ubiquitination of MBP-Rma1. Thus, Rma1 represents a novel, membrane-bound type of ubiquitin ligase E3, which probably functions with the Ubc4/5 subfamily of E2. The MBP moiety but not Rma1 itself is ubiquitinated in the auto-ubiquitination reaction of MBP-Rma1. Free MBP in solution is not a substrate of Rma1. These observations indicate that bringing the substrate into its physical vicinity is very important for the action of ubiquitin ligase.

• Pringa E, Martinez-Noel G, Muller U, Harbers K
• Interaction of the ring finger-related U-box motif of a nuclear dot protein with ubiquitin-conjugating enzymes.
• J Biol Chem. 2001; 276: 19617-23
• Display abstract

• The U-box domain has been suggested to be a modified RING finger motif where the metal-coordinating cysteines and histidines have been replaced with other amino acids. Known U-box-containing proteins have been implicated in the ubiquitin/proteasome system. In a search for proteins interacting with the ubiquitin-conjugating enzyme UbcM4/UbcH7, we have identified a novel U-box containing protein, termed UIP5, that is exclusively found in the nucleus as part of a nuclear dot-like structure. Interaction between UbcM4 and UIP5 was observed in vivo and in vitro with bacterially expressed proteins. In addition to UbcM4, several other ubiquitin-conjugating enzymes (E2s) that share the same sequence within the L1 loop bind to UIP5. Mutational analysis showed that the U-box, like the RING finger in other proteins, forms the physical basis for the interaction with E2 enzymes. Further support for the structural similarity between U-box and RING finger comes from the observation that, in both cases, the same regions within the UbcM4 molecule are required for interaction. Our results establish at the molecular level a link between the U-box and the ubiquitin conjugating system and strongly suggest that proteins containing U-box domains are functionally closely related to RING finger proteins.

• Dunn R, Hicke L
• Domains of the Rsp5 ubiquitin-protein ligase required for receptor-mediated and fluid-phase endocytosis.
• Mol Biol Cell. 2001; 12: 421-35
• Display abstract

• Yeast Rsp5p and its mammalian homologue, Nedd4, are hect domain ubiquitin-protein ligases (E3s) required for the ubiquitin-dependent endocytosis of plasma membrane proteins. Because ubiquitination is sufficient to induce internalization, E3-mediated ubiquitination is a key regulatory event in plasma membrane protein endocytosis. Rsp5p is an essential, multidomain protein containing an amino-terminal C2 domain, three WW protein-protein interaction domains, and a carboxy-terminal hect domain that carries E3 activity. In this study, we demonstrate that Rsp5p is peripherally associated with membranes and provide evidence that Rsp5p functions as part of a multimeric protein complex. We define the function of Rsp5p and its domains in the ubiquitin-dependent internalization of the yeast alpha-factor receptor, Ste2p. Temperature-sensitive rsp5 mutants were unable to ubiquitinate or to internalize Ste2p at the nonpermissive temperature. Deletion of the entire C2 domain had no effect on alpha-factor internalization; however, point mutations in any of the three WW domains impaired both receptor ubiquitination and internalization. These observations indicate that the WW domains play a role in the important regulatory event of selecting phosphorylated proteins as endocytic cargo. In addition, mutations in the C2 and WW1 domains had more severe defects on transport of fluid-phase markers to the vacuole than on receptor internalization, suggesting that Rsp5p functions at multiple steps in the endocytic pathway.

• Wang G, McCaffery JM, Wendland B, Dupre S, Haguenauer-Tsapis R, Huibregtse JM
• Localization of the Rsp5p ubiquitin-protein ligase at multiple sites within the endocytic pathway.
• Mol Cell Biol. 2001; 21: 3564-75
• Display abstract

• The Saccharomyces cerevisiae RSP5 gene encodes an essential HECT E3 ubiquitin-protein ligase. Rsp5p contains an N-terminal C2 domain, three WW domains in the central portion of the molecule, and a C-terminal catalytic HECT domain. A diverse group of substrates of Rsp5p and vertebrate C2 WW-domain-containing HECT E3s have been identified, including both nuclear and membrane-associated proteins. We determined the intracellular localization of Rsp5p and the determinants necessary for localization, in order to better understand how Rsp5p activities are coordinated. Using both green fluorescent protein fusions to Rsp5p and immunogold electron microscopy, we found that Rsp5p was distributed in a punctate pattern at the plasma membrane, corresponding to membrane invaginations that are likely sites of endosome formation, as well as at perivacuolar sites. The latter appeared to correspond to endocytic intermediates, as these structures were not seen in a sla2/end4-1 mutant, and double-immunogold labeling demonstrated colocalization of Rsp5p with the endosomal markers Pep12p and Vps32p. The C2 domain was an important determinant of localization; however, mutations that disrupted HECT domain function also caused mislocalization of Rsp5p, indicating that enzymatic activity is linked to localization. Deletion of the C2 domain partially stabilized Fur4p, a protein previously shown to undergo Rsp5p- and ubiquitin-mediated endocytosis; however, Fur4p was still ubiquitinated at the plasma membrane when the C2 domain was deleted from the protein. Together, these results indicate that Rsp5p is located at multiple sites within the endocytic pathway and suggest that Rsp5p may function at multiple steps in the ubiquitin-mediated endocytosis pathway.

• Ito K et al.
• N-Terminally extended human ubiquitin-conjugating enzymes (E2s) mediate the ubiquitination of RING-finger proteins, ARA54 and RNF8.
• Eur J Biochem. 2001; 268: 2725-32
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• We have previously cloned cDNAs encoding the N-terminally extended class III human ubiquitin-conjugating enzymes (E2s), UBE2E2 and UBE2E3, the biological functions of which are not known. In this study, we performed yeast two-hybrid screening for protein(s) interacting with UBE2E2, and two RING-finger proteins, ARA54 and RNF8, were identified. Both ARA54, a ligand-dependent androgen receptor coactivator, and RNF8 interacted with class III E2s (UBE2E2, UbcH6, and UBE2E3), but not with other E2s (UbcH5, UbcH7, UbcH10, hCdc34, and hBendless) in the yeast two-hybrid assay. The use of various deletion mutants of UBE2E2 and RING-finger proteins and two RING point mutants, ARA54 C(220)S and RNF8 C(403)S, in which the RING structure is disrupted, showed that the UBC domain of UBE2E2 and the RING domain of these RING-finger proteins were involved in this association. Wild-type ARA54 and RNF8, expressed in insect Sf9 cells, catalyzed E2-dependent autoubiquitination in vitro, whereas the point mutated proteins showed markedly reduced activity. Ubiquitination of wild-type ARA54 and RNF8, expressed in COS-7 cells, was also observed, and a proteasome inhibitor, MG132, prevented the degradation of these wild-type proteins, but was much less effective in protecting the RING mutants. Transfection of COS-7 cells with a green fluorescent protein chimera showed that RNF8 was localized in the nucleus, and ARA54 in both the cytoplasm and nucleus. Our results suggest that ARA54 and RNF8 possibly act as Ub-ligases (E3) in the ubiquitination of certain nuclear protein(s).

• Zheng N, Wang P, Jeffrey PD, Pavletich NP
• Structure of a c-Cbl-UbcH7 complex: RING domain function in ubiquitin-protein ligases.
• Cell. 2000; 102: 533-9
• Display abstract

• Ubiquitin-protein ligases (E3s) regulate diverse cellular processes by mediating protein ubiquitination. The c-Cbl proto-oncogene is a RING family E3 that recognizes activated receptor tyrosine kinases, promotes their ubiquitination by a ubiquitin-conjugating enzyme (E2) and terminates signaling. The crystal structure of c-Cbl bound to a cognate E2 and a kinase peptide shows how the RING domain recruits the E2. A comparison with a HECT family E3-E2 complex indicates that a common E2 motif is recognized by the two E3 families. The structure reveals a rigid coupling between the peptide binding and the E2 binding domains and a conserved surface channel leading from the peptide to the E2 active site, suggesting that RING E3s may function as scaffolds that position the substrate and the E2 optimally for ubiquitin transfer.

• Winkler AA, Korstanje R, Zonneveld BJ, Hooykaas PJ, Steensma HY
• Isolation and characterization of KIUBP2, a ubiquitin hydrolase gene of Kluyveromyces lactis that can suppress a ts-mutation in CBF2, a gene encoding a centromeric protein of Saccharomyces cerevisiae.
• Curr Genet. 2000; 38: 17-22
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• The Kluyveromyces lactis UBP2 gene was isolated as a suppressor of a temperature-sensitive mutation in CBF2, a gene coding for a centromere-binding protein of Saccharomyces cerevisiae. The UBP genes are hydrolases than can cleave a ubiquitin moiety from a protein substrate. KlUBP2 is not essential for growth since a disruption of the KlUBP2 gene had little effect, except for a slight decrease in the growth rate. The stability of centromere-containing plasmids was not influenced either. In addition to KlUBP2, five S. cerevisiae genes involved in the ubiquitination pathway could suppress the ts-mutation in the CBF2 gene, namely UBA1, UBA2, UBP1, UBP2 and YUH1, although YUH1 was the only one that could do this like KlUBP2 from a single-copy plasmid. Surprisingly, these genes encode proteins with antagonistic activity as two, UBA1 and UBA2, are ubiquitin-activating enzymes whereas the other three are de-ubiquitinating hydrolases.

• Duncan K, Umen JG, Guthrie C
• A putative ubiquitin ligase required for efficient mRNA export differentially affects hnRNP transport.
• Curr Biol. 2000; 10: 687-96
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• BACKGROUND: In the nucleus, mRNAs are bound by hnRNP proteins. A subset of hnRNP proteins shuttle between the nucleus and cytoplasm and are believed to promote mRNA export by acting as adaptors between mRNA and the transport machinery. The existence of multiple shuttling hnRNP proteins raises the question of whether differentially regulated, hnRNP-specific mRNA export pathways exist. RESULTS: We have determined that Tom1p, a conserved protein with a hect (homology to E6-AP carboxyl terminus) E3 ubiquitin ligase domain, is required for efficient mRNA export in S. cerevisiae, yet differentially affects hnRNP protein localization and export. Mutations in tom1 predicted to abolish ubiquitin ligase activity block efficient export of Nab2p and mRNA, causing Nab2p-mRNA complexes to accumulate in a punctate pattern coincident with the nuclear pore complex (NPC). Notably, the subcellular distribution of several other hnRNP proteins is not affected. In particular, Np13p remains mRNA-associated and continues to be efficiently exported in tom1 mutants. CONCLUSION: Our results demonstrate that mutations predicted to affect the enzymatic activity of the Tom1p ubiquitin ligase differentially affect export of hnRNP proteins in association with mRNA. We propose the existence of multiple mRNA export pathways, with export of Nab2p-associated mRNAs dependent on a branch of the ubiquitin protein modification pathway.

• Fang S, Jensen JP, Ludwig RL, Vousden KH, Weissman AM
• Mdm2 is a RING finger-dependent ubiquitin protein ligase for itself and p53.
• J Biol Chem. 2000; 275: 8945-51
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• Mdm2 has been shown to regulate p53 stability by targeting the p53 protein for proteasomal degradation. We now report that Mdm2 is a ubiquitin protein ligase (E3) for p53 and that its activity is dependent on its RING finger. Furthermore, we show that Mdm2 mediates its own ubiquitination in a RING finger-dependent manner, which requires no eukaryotic proteins other than ubiquitin-activating enzyme (E1) and an ubiquitin-conjugating enzyme (E2). It is apparent, therefore, that Mdm2 manifests an intrinsic capacity to mediate ubiquitination. Mutation of putative zinc coordination residues abrogated this activity, as did chelation of divalent cations. After cation chelation, the full activity could be restored by addition of zinc. We further demonstrate that the degradation of p53 and Mdm2 in cells requires additional potential zinc-coordinating residues beyond those required for the intrinsic activity of Mdm2 in vitro. Replacement of the Mdm2 RING with that of another protein (Praja1) reconstituted ubiquitination and proteasomal degradation of Mdm2. However, this RING was ineffective in ubiquitination and proteasomal targeting of p53, suggesting that there may be specificity at the level of the RING in the recognition of heterologous substrates.

• Furukawa K, Mizushima N, Noda T, Ohsumi Y
• A protein conjugation system in yeast with homology to biosynthetic enzyme reaction of prokaryotes.
• J Biol Chem. 2000; 275: 7462-5
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• Protein conjugation, such as ubiquitination, is the process by which the C-terminal glycine of a small modifier protein is covalently attached to target protein(s) through sequential reactions with an activating enzyme and conjugating enzymes. Here we report on a novel protein conjugation system in yeast. A newly identified ubiquitin related modifier, Urm1 is a 99-amino acid protein terminated with glycine-glycine. Urm1 is conjugated to target proteins, which requires the C-terminal glycine of Urm1. At the first step of this reaction, Urm1 forms a thioester with a novel E1-like protein, Uba4. Deltaurm1 and Deltauba4 cells showed a temperature-sensitive growth phenotype. Urm1 and Uba4 show similarity to prokaryotic proteins essential for molybdopterin and thiamin biosynthesis, although the Urm1 system is not involved in these pathways. This is the fifth conjugation system in yeast, following ubiquitin, Smt3, Rub1, and Apg12, but it is unique in respect to relation to prokaryotic enzyme systems. This fact may provide an important clue regarding evolution of protein conjugation systems in eukaryotic cells.

• Honda R, Yasuda H
• Activity of MDM2, a ubiquitin ligase, toward p53 or itself is dependent on the RING finger domain of the ligase.
• Oncogene. 2000; 19: 1473-6
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• We previously showed that oncoprotein MDM2 has ubiquitin ligase activity toward tumor suppressor p53. In that paper, we showed very weak homology in the carboxyl terminal portion between MDM2 and E6AP (HECT domain). We mutated the cysteine residue (C464) corresponding to the residue essential for the ubiquitin ligase activity of E6AP and this mutation diminished the ligase activity of MDM2. The cysteine residue described above is also one of the cysteine residues that form the RING finger domain of MDM2. We tried to find out whether the diminishing of the activity by the mutation is attributable to the disruption of the RING finger domain or not. When the ring finger domain of MDM2 was deleted, the truncation mutant did not have the ubiquitin ligase activity. When we mutated the seven cysteine residues of RING finger domain of MDM2 in the carboxyl terminus, the disruption of each residue in the RING finger completely diminished the ubiquitin ligase activity of MDM2 toward MDM2 itself and toward tumor suppressor p53. These data indicate that the RING finger domain in MDM2 is essential for its ubiquitin ligase activity toward p53 and itself.

• Huang Hk, Joazeiro CA, Bonfoco E, Kamada S, Leverson JD, Hunter T
• The inhibitor of apoptosis, cIAP2, functions as a ubiquitin-protein ligase and promotes in vitro monoubiquitination of caspases 3 and 7.
• J Biol Chem. 2000; 275: 26661-4
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• The inhibitor of apoptosis, cIAP2, contains a putative Ring finger motif at the C terminus. Using in vitro ubiquitination assays, we found that the Ring finger of cIAP2 alone possesses intrinsic ubiquitin ligase activity and promotes substrate-independent ubiquitination. It also promotes ubiquitination of caspases 3 and 7 but not caspase-1. The Ring fingers of c-Cbl and Apc11 failed to promote caspase-7 ubiquitination, suggesting that the Ring finger of cIAP2 itself is involved in substrate recognition.

• Qiu L et al.
• Recognition and ubiquitination of Notch by Itch, a hect-type E3 ubiquitin ligase.
• J Biol Chem. 2000; 275: 35734-7
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• Genetic studies identified Itch, which is a homologous to the E6-associated protein carboxyl terminus (Hect) domain-containing E3 ubiquitin-protein ligase that is disrupted in non-agouti lethal mice or Itchy mice. Itch-deficiency results in abnormal immune responses and constant itching in the skin. Here, Itch was shown to associate with Notch, a protein involved in cell fate decision in many mammalian cell types, including cells in the immune system. Itch binds to the N-terminal portion of the Notch intracellular domain via its WW domains and promotes ubiquitination of Notch through its Hect ubiquitin ligase domain. Thus, Itch may participate in the regulation of immune responses by modifying Notch-mediated signaling.

• Nakagawa S, Huibregtse JM
• Human scribble (Vartul) is targeted for ubiquitin-mediated degradation by the high-risk papillomavirus E6 proteins and the E6AP ubiquitin-protein ligase.
• Mol Cell Biol. 2000; 20: 8244-53
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• The high-risk human papillomavirus (HPV) E6 proteins stimulate the ubiquitination and degradation of p53, dependent on the E6AP ubiquitin-protein ligase. Other proteins have also been shown to be targeted for degradation by E6, including hDlg, the human homolog of the Drosophila melanogaster Discs large (Dlg) tumor suppressor. We show here that the human homolog of the Drosophila Scribble (Vartul) (hScrib) tumor suppressor protein is also targeted for ubiquitination by the E6-E6AP complex in vitro and that expression of E6 induces degradation of hScrib in vivo. Characterization of the E6AP-E6-hScrib complex indicated that hScrib binds directly to E6 and that the binding is mediated by the PDZ domains of hScrib and a carboxyl-terminal epitope conserved among the high-risk HPV E6 proteins. Green fluorescent protein-hScrib was localized to the periphery of MDCK cells, where it colocalized with ZO-1, a component of tight junctions. E6 expression resulted in loss of integrity of tight junctions, as measured by ZO-1 localization, and this effect was dependent on the PDZ binding epitope of E6. Thus, the high-risk HPV E6 proteins induce the degradation of the human homologs of two Drosophila PDZ domain-containing tumor suppressor proteins, hDlg and hScrib, both of which are associated with cell junction complexes. The fact that Scrib/Vart and Dlg appear to cooperate in a pathway that controls Drosophila epithelial cell growth suggests that the combined targeting of hScrib and hDlg is an important component of the biologic activity of high-risk HPV E6 proteins.

• Huang K et al.
• A HECT domain ubiquitin ligase closely related to the mammalian protein WWP1 is essential for Caenorhabditis elegans embryogenesis.
• Gene. 2000; 252: 137-45
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• The highly conserved ubiquitin/proteasome pathway controls the degradation of many critical regulatory proteins. Proteins are posttranslationally conjugated to ubiquitin through a concerted set of reactions involving activating (E1), conjugating (E2), and ligase (E3) enzymes. Ubiquitination targets proteins for proteolysis via the proteasome and may regulate protein function independent of proteolysis. We describe the cloning and functional analysis of new members of the HECT domain family of E3 ubiquitin ligases. Murine Wwp1 encoded a broadly expressed protein containing a C2 domain, four WW domains, and a catalytic HECT domain. A Caenorhabditis elegans gene was cloned encoding a HECT domain protein (CeWWP1), which was highly homologous to murine and human WWP1. Disruption of CeWwp1 via RNA interference yielded an embryonic lethal phenotype, despite the presence of at least six additional C. elegans genes encoding HECT domain proteins. The embryonic lethality was characterized by grossly abnormal morphogenesis during late embryogenesis, despite normal proliferation early in embryogenesis. CeWWP1 must therefore have unique and nonredundant functions critical for embryogenesis.

• Traidej M, Chen L, Yu D, Agrawal S, Chen J
• The roles of E6-AP and MDM2 in p53 regulation in human papillomavirus-positive cervical cancer cells.
• Antisense Nucleic Acid Drug Dev. 2000; 10: 17-27
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• The p53 tumor suppressor is regulated by the MDM2 oncoprotein through a negative feedback mechanism. MDM2 promotes the ubiquitination and proteasome-dependent degradation of p53, possibly by acting as a ubiquitin ligase. In cervical cancer cells containing high-risk human papillomaviruses (HPV), p53 is also targeted for degradation by the HPV E6 oncoprotein in combination with the cellular E6-AP ubiquitin ligase. In this report, we describe the identification of efficient antisense oligonucleotides against human E6-AP. The roles of MDM2 and E6-AP in p53 regulation were investigated using a novel E6-AP antisense oligonucleotide and a previously characterized MDM2 antisense oligonucleotide. In HPV16-positive and HPV-18 positive cervical cancer cells, inhibition of E6-AP, but not MDM2, expression results in significant induction of p53. In HPV-negative tumor cells, p53 is activated by inhibition of MDM2 but not E6-AP. Furthermore, treatment with both E6-AP and MDM2 antisense oligonucleotides in HPV-positive cells does not lead to further induction of p53 over inhibition of E6-AP alone. Therefore, E6-AP-mediated degradation is dominant over MDM2 in cervical cancer cells but does not have a significant role in HPV-negative cells.

• Gilon T, Chomsky O, Kulka RG
• Degradation signals recognized by the Ubc6p-Ubc7p ubiquitin-conjugating enzyme pair.
• Mol Cell Biol. 2000; 20: 7214-9
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• Proteolysis by the ubiquitin-proteasome system is highly selective. Specificity is achieved by the cooperation of diverse ubiquitin-conjugating enzymes (Ubcs or E2s) with a variety of ubiquitin ligases (E3s) and other ancillary factors. These recognize degradation signals characteristic of their target proteins. In a previous investigation, we identified signals directing the degradation of beta-galactosidase and Ura3p fusion proteins via a subsidiary pathway of the ubiquitin-proteasome system involving Ubc6p and Ubc7p. This pathway has recently been shown to be essential for the degradation of misfolded and regulated proteins in the endoplasmic reticulum (ER) lumen and membrane, which are transported to the cytoplasm via the Sec61p translocon. Mutant backgrounds which prevent retrograde transport of ER proteins (hrd1/der3Delta and sec61-2) did not inhibit the degradation of the beta-galactosidase and Ura3p fusions carrying Ubc6p/Ubc7p pathway signals. We therefore conclude that the ubiquitination of these fusion proteins takes place on the cytosolic face of the ER without prior transfer to the ER lumen. The contributions of different sequence elements to a 16-amino-acid-residue Ubc6p-Ubc7p-specific signal were analyzed by mutation. A patch of bulky hydrophobic residues was an essential element. In addition, positively charged residues were found to be essential. Unexpectedly, certain substitutions of bulky hydrophobic or positively charged residues with alanine created novel degradation signals, channeling the degradation of fusion proteins to an unidentified proteasomal pathway not involving Ubc6p and Ubc7p.

• Tokumoto M, Nagahama Y, Tokumoto T
• Molecular cloning of cDNA encoding a ubiquitin-activating enzyme (E1) from goldfish (Carassius auratus) and expression analysis of the cloned gene.
• Biochim Biophys Acta. 2000; 1492: 259-63
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• Destruction of cyclin B is required to the mitotic and meiotic cycles. A cyclin-specific ubiquitinating system, including ubiquitin-activating enzyme (E1), is thought to be responsible for cyclin B destruction. Here we present the cloning, sequencing and expression analysis of goldfish, Carassius auratus, E1 from goldfish ovary. The cloned cDNA is 4069 bp long and encodes 1059 amino acids. The deduced amino acid sequence is highly homologous to E1 from other species. Recombinant goldfish E1 could transfer ubiquitin to cyclin-selective ubiquitin-conjugating enzyme. Tissue distribution revealed a single 4.0-kb message ubiquitous among tissues.

• Kelley ML, Van Beneden RJ
• Identification of an E3 ubiquitin-protein ligase in the softshell clam (Mya arenaria).
• Mar Environ Res. 2000; 50: 289-93
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• Softshell clams (Mya arenaria) were exposed to dioxin in controlled laboratory experiments in order to study their molecular response to dioxin exposure. A complementary DNA (cDNA) fragment with sequence similarity to E3 ubiquitin-protein ligase appeared to be upregulated in dioxin-exposed clams compared to controls. E3 covalently ligates ubiquitin onto a protein, targeting it for degradation. Our findings suggest that the ubiquitin-mediated proteolytic pathway in the softshell clam may be activated by dioxin exposure. Because the clam E3-predicted amino acid sequence is most similar to a specific vertebrate E3 protein (E6-AP), we hypothesize that dioxin may stimulate ubiquitin-mediated degradation of cell-cycle regulatory proteins, such as the tumor suppressor p53, which promotes cell proliferation. This pathway has been observed in human cervical cancer. Partial cDNA sequence of the clam E3 has been identified using the differential display polymerase chain reaction (ddPCR) and RACE (Rapid Amplification of cDNA Ends) PCR; the full-length sequence is currently being determined. Discovering the molecular mechanism(s) stimulated by dioxin exposure in this invertebrate model may contribute to a better understanding of the effects of dioxin on marine organisms.

• Ulrich HD, Jentsch S
• Two RING finger proteins mediate cooperation between ubiquitin-conjugating enzymes in DNA repair.
• EMBO J. 2000; 19: 3388-97
• Display abstract

• Two ubiquitin-conjugating enzymes, RAD6 and the heteromeric UBC13-MMS2 complex, have been implicated in post-replicative DNA damage repair in yeast. Here we provide a mechanistic basis for cooperation between the two enzymes. We show that two chromatin-associated RING finger proteins, RAD18 and RAD5, play a central role in mediating physical contacts between the members of the RAD6 pathway. RAD5 recruits the UBC13-MMS2 complex to DNA by means of its RING finger domain. Moreover, RAD5 association with RAD18 brings UBC13-MMS2 into contact with the RAD6-RAD18 complex. Interaction between the two RING finger proteins thus promotes the formation of a heteromeric complex in which the two distinct ubiquitin-conjugating activities of RAD6 and UBC13-MMS2 can be closely coordinated. Surprisingly, UBC13 and MMS2 are largely cytosolic proteins, but DNA damage triggers their redistribution to the nucleus. These findings suggest a mechanism by which the activity of this DNA repair pathway could be regulated.

• Conklin D, Holderman S, Whitmore TE, Maurer M, Feldhaus AL
• Molecular cloning, chromosome mapping and characterization of UBQLN3 a testis-specific gene that contains an ubiquitin-like domain.
• Gene. 2000; 249: 91-8
• Display abstract

• The sequence of the ubiquitin protein is highly conserved between species and has facilitated the cloning of numerous ubiquitin-like proteins. In the present study, we report the cloning of the cDNA for human ubiquilin 3 (UBQLN3). The deduced amino acid sequence of UBQLN3 contains a UBQ domain (ubiquitin-like) in the amino terminus as well as two highly conserved domains found in several recently cloned ubiquitin-like proteins. One of these domains, termed the NP domain, is a highly conserved 93 amino acid region present in UBQLN3 and several ubiquitin-like proteins. The last conserved domain is the UBA domain (ubiquitin-associated) found in a variety of proteins of the ubiquination pathway. The human UBQLN3 gene was mapped to the 11p15 region of chromosome 11. Northern blot analysis of multiple human and mouse tissues demonstrated UBQLN3 mRNA expression specifically in testis.

• Schwienhorst I, Johnson ES, Dohmen RJ
• SUMO conjugation and deconjugation.
• Mol Gen Genet. 2000; 263: 771-86
• Display abstract

• Ligation of the ubiquitin-like protein SUMO (Smt3p) to other proteins is essential for viability of the yeast Saccharomyces cerevisiae. Like ubiquitin (Ub), SUMO undergoes ATP-dependent activation by a specific activating enzyme. SUMO-activating enzyme is a heterodimer composed of Uba2p and Aos1p, polypeptides with sequence similarities, respectively, to the C- and N-terminal parts of Ub-activating enzyme. To study the function of SUMO conjugation, we isolated uba2 mutants that were temperature-sensitive for growth. In these mutants conjugation of SUMO to other proteins was drastically reduced, even at the temperature permissive for growth. In a screen for spontaneous suppressors of the temperature-sensitive growth phenotype of the mutant uha2-ts9, we isolated a strain with a null mutation (sut9) in a gene of hitherto unknown function (SUT9/YIL031W/SMT4). This gene encodes a protein with similarities to Ulp1p, a dual-function protease that processes the SUMO precursor and deconjugates SUMO from its substrates. The novel protein was therefore termed Ulp2p. Inactivation of ULP2 in a strain expressing wild-type SUMO-activating enzyme resulted in slow and temperature-sensitive growth, and accumulation of SUMO conjugates. Thus, mutations in SUMO-activating enzyme and mutations in Ulp2p suppress each other, indicating that SUMO conjugation and deconjugation must be in balance for cells to grow normally. Other phenotypes of ulp2 mutants include a defect in cell cycle progression, hypersensitivity to DNA damage, and chromosome mis-segregation. Ulp2p is predominantly located within the nucleus, whereas Ulp1p colocalizes with nuclear pore complex proteins, indicating that the apparently distinct functions of the two SUMO deconjugating enzymes are spatially separated.

• Lisztwan J, Imbert G, Wirbelauer C, Gstaiger M, Krek W
• The von Hippel-Lindau tumor suppressor protein is a component of an E3 ubiquitin-protein ligase activity.
• Genes Dev. 1999; 13: 1822-33
• Display abstract

• pVHL, the product of the VHL tumor suppressor gene, plays an important role in the regulation of cell growth and differentiation of human kidney cells, and inactivation of the VHL gene is the most frequent genetic event in human kidney cancer. The biochemical function of pVHL is unknown. Here we report that pVHL exists in vivo in a complex that displays ubiquitination-promoting activity in conjunction with the universally required components E1, E2, and ubiquitin. pVHL-associated ubiquitination activity requires, at a minimum, pVHL to bind elongin C and Cul-2, relatives of core components of SCF (Skp1-Cdc53/Cul-1-F-box protein) E3 ligase complexes. Notably, certain tumor-derived mutants of pVHL demonstrate loss of associated ubiquitination promoting activity. These results identify pVHL as a component of a potential SCF-like E3 ubiquitin-protein ligase complex and suggest a direct link between pVHL tumor suppressor and the process of ubiquitination.

• Ito K, Kato S, Matsuda Y, Kimura M, Okano Y
• cDNA cloning, characterization, and chromosome mapping of UBE2E3 (alias UbcH9), encoding an N-terminally extended human ubiquitin-conjugating enzyme.
• Cytogenet Cell Genet. 1999; 84: 99-104
• Display abstract

• A cDNA encoding a third member of human class III ubiquitin-conjugating enzymes (E2s), UBE2E3, was cloned from a human gastric adenocarcinoma cDNA library. The deduced 207-amino acid protein shares over 94% amino acid identity with the UBC domains of class III E2s, UbcH6, UBE2E2, UbcM2, UbcM3, and UbcD2. But the N-terminal extension exhibited little homology among these, except for UbcM2, which showed 100% identity, and which is thought to be a mouse counterpart. Northern hybridization analysis exhibited a strong 1.9-kb band of UBE2E3 in skeletal muscle. Recombinant fusion protein of GST-UBE2E3 was found to form a thioester bond with ubiquitin (Ub) in an E1-dependent manner, demonstrating that the cDNA encodes a functional E2. In addition, a UBE2E3 mutant of cysteine-145 to serine failed in UBE2E3-Ub complex formation, indicating that the cysteine is essential for E2 function. Using FISH and PCR analysis of radiation hybrid and somatic cell hybrid panels the UBE2E3 gene was mapped to human chromosome 2q32.1 and showed strong linkage to SHGC-8506 (LOD = 11.52) between D2S1302 and D2S364.

• Ohsako S, Takamatsu Y
• Identification and characterization of a Drosophila homologue of the yeast UBC9 and hus5 genes.
• J Biochem (Tokyo). 1999; 125: 230-5
• Display abstract

• The yeast UBC9 and hus5 gene products have been identified as putative E2 members of the ubiquitin-conjugating enzyme (UBC) family and have been shown to play an essential role in cell cycle progression. We have identified a Drosophila Ubc9/Hus5 homologue (termed dUBC9) in an attempt to identify proteins that interact with the amino-terminal transcriptional repression domain of the Groucho corepressor by use of the yeast two-hybrid system. The predicted dUBC9 protein consists of 159 amino acids and shows 85, 68, and 54% amino acid sequence identities with human UBC9 homologue, Schizosaccharomyces pombe Hus5, and Saccharomyces cerevisiae Ubc9 proteins, respectively. Expression of dUBC9 cDNA complements a temperature-sensitive ubc9-1 mutation of S. cerevisiae to fully restore normal growth, indicating that the dUBC9 protein can act as a substitute for the yeast Ubc9 protein. The dUBC9 transcripts were about 1.2 kb and were detected at all stages of Drosophila development and in ovaries and Schneider cells. However, an increased level was observed in early embryos and ovaries. The dUBC9 gene is present as a single copy in the genome and localized in segment 21C-D on the left arm of the second chromosome.

• Moynihan TP et al.
• The ubiquitin-conjugating enzymes UbcH7 and UbcH8 interact with RING finger/IBR motif-containing domains of HHARI and H7-AP1.
• J Biol Chem. 1999; 274: 30963-8
• Display abstract

• Ubiquitinylation of proteins appears to be mediated by the specific interplay between ubiquitin-conjugating enzymes (E2s) and ubiquitin-protein ligases (E3s). However, cognate E3s and/or substrate proteins have been identified for only a few E2s. To identify proteins that can interact with the human E2 UbcH7, a yeast two-hybrid screen was performed. Two proteins were identified and termed human homologue of Drosophila ariadne (HHARI) and UbcH7-associated protein (H7-AP1). Both proteins, which are widely expressed, are characterized by the presence of RING finger and in between RING fingers (IBR) domains. No other overt structural similarity was observed between the two proteins. In vitro binding studies revealed that an N-terminal RING finger motif (HHARI) and the IBR domain (HHARI and H7-AP1) are involved in the interaction of these proteins with UbcH7. Furthermore, binding of these two proteins to UbcH7 is specific insofar that both HHARI and H7-AP1 can bind to the closely related E2, UbcH8, but not to the unrelated E2s UbcH5 and UbcH1. Although it is not clear at present whether HHARI and H7-AP1 serve, for instance, as substrates for UbcH7 or represent proteins with E3 activity, our data suggests that a subset of RING finger/IBR proteins are functionally linked to the ubiquitin/proteasome pathway.

• Nuber U, Scheffner M
• Identification of determinants in E2 ubiquitin-conjugating enzymes required for hect E3 ubiquitin-protein ligase interaction.
• J Biol Chem. 1999; 274: 7576-82
• Display abstract

• Members of the hect domain protein family are characterized by sequence similarity of their C-terminal regions to the C terminus of E6-AP, an E3 ubiquitin-protein ligase. An essential intermediate step in E6-AP-dependent ubiquitination is the formation of a thioester complex between E6-AP and ubiquitin in the presence of distinct E2 ubiquitin-conjugating enzymes including human UbcH5, a member of the UBC4/UBC5 subfamily of E2s. Similarly, several hect domain proteins, including Saccharomyces cerevisiae RSP5, form ubiquitin thioester complexes, indicating that hect domain proteins in general have E3 activity. We show here, by the use of chimeric E2s generated between UbcH5 and other E2s, that a region of UbcH5 encompassing the catalytic site cysteine residue is critical for its ability to interact with E6-AP and RSP5. Of particular importance is a phenylalanine residue at position 62 of UbcH5 that is conserved among the members of the UBC4/UBC5 subfamily but is not present in any of the other known E2s, whereas the N-terminal 60 amino acids do not contribute significantly to the specificity of these interactions. The conservation of this phenylalanine residue throughout evolution underlines the importance of the ability to interact with hect domain proteins for the cellular function of UBC4/UBC5 subfamily members.

• Gong L, Yeh ET
• Identification of the activating and conjugating enzymes of the NEDD8 conjugation pathway.
• J Biol Chem. 1999; 274: 12036-42
• Display abstract

• NEDD8 is a ubiquitin-like molecule that can be covalently conjugated to a limited number of cellular proteins, such as Cdc53/cullin. We have previously reported that the C terminus of NEDD8 is efficiently processed to expose Gly-76, which is required for conjugation to target proteins. A combination of data base searches and polymerase chain reaction cloning was used to identify a cDNA encoding human UBA3, which is 38% identical to the yeast homologue, 22% identical to human UBA2, and 19% identical to the C-terminal region of human UBE1. The human UBA3 gene is located on chromosome 3p13 and gave rise to a 2.2-kilobase pair transcript that was detected in all tissues. Human UBA3 could be precipitated with glutathione S-transferase (GST)-NEDD8, but not with GST-ubiquitin or GST-sentrin-1. Moreover, human UBA3 could form a beta-mercaptoethanol-sensitive conjugate with NEDD8 in the presence of APP-BP1, a protein with sequence homology to the N-terminal half of ubiquitin-activating enzyme. We have also cloned human UBC12 and demonstrated that it could form a thiol ester linkage with NEDD8 in the presence of the activating enzyme complex. Identification of the activating and conjugating enzymes of the NEDD8 conjugation pathway should allow for a more detailed study of the role of NEDD8 modification in health and disease.

• Lin H, Wing SS
• Identification of rabbit reticulocyte E217K as a UBC7 homologue and functional characterization of its core domain loop.
• J Biol Chem. 1999; 274: 14685-91
• Display abstract

• The structural basis by which ubiquitin (Ub)-conjugating enzymes (E2s) determine substrate specificity remains unclear. We cloned rabbit reticulocyte E217K because unlike the similarly sized class I E2s, E214K and UBC4, it is unable to support ubiquitin-protein ligase (E3)-dependent conjugation to endogenous proteins. RNA analysis revealed that this E2 was expressed in all tissues tested, with higher levels in the testis. Analysis of testis RNA from rats of different ages showed that E217K mRNA was induced from days 15 to 30. The predicted amino acid sequence indicates that E217K is a 19. 5-kDa class I E2 but differs from other class I enzymes in possessing an insertion of 13 amino acids distal to the active site cysteine. E217K shows 74% amino acid identity with Saccharomyces cerevisiae UBC7, and therefore, we rename it mammalian UBC7. Yeast UBC7 crystal structure indicates that this insertion forms a loop out of the otherwise conserved folding structure. Sequence analysis of E2s had previously suggested that this loop is a hypervariable region and may play a role in substrate specificity. We created mutant UBC7 lacking the loop (ubc7Deltaloop) and a mutant E214k with an inserted loop (E214k+loop) and characterized their biochemical functions. Ubc7Deltaloop had higher affinity for the E1-Ub thiol ester than native UBC7 and permitted conjugation of Ub to selected proteins in the testis but did not permit the broad spectrum E3-dependent conjugation to endogenous reticulocyte proteins. Surprisingly, E214k+loop was unable to accept Ub from ubiquitin-activating enzyme (E1) but was able to accept NEDD8 from E1. E214k+loop was able to support conjugation of NEDD8 to endogenous reticulocyte proteins but with much lower efficiency than E214k. Thus, the loop can influence interactions of the E2 with charged E1 as well as with E3s or substrates, but the exact nature of these interactions depends on divergent sequences in the remaining conserved core domain.

• Fischle W et al.
• A new family of human histone deacetylases related to Saccharomyces cerevisiae HDA1p.
• J Biol Chem. 1999; 274: 11713-20
• Display abstract

• Histone deacetylases are the catalytic subunits of multiprotein complexes that are targeted to specific promoters through their interaction with sequence-specific DNA-binding factors. We have cloned and characterized a new human cDNA, HDAC-A, with homology to the yeast HDA1 family of histone deacetylases. Analysis of the predicted amino acid sequence of HDAC-A revealed an open reading frame of 967 amino acids containing two domains: a NH2-terminal domain with no homology to known proteins and a COOH-terminal domain with homology to known histone deacetylases (42% similarity to RPD3, 60% similarity to HDA1). Three additional human cDNAs with high homology to HDAC-A were identified in sequence data bases, indicating that HDAC-A itself is a member of a new family of human histone deacetylases. The mRNA encoding HDAC-A was differentially expressed in a variety of human tissues. The expressed protein, HDAC-Ap, exhibited histone deacetylase activity and this activity mapped to the COOH-terminal region (amino acids 495-967) with homology to HDA1p. In immunoprecipitation experiments, HDAC-A interacted specifically with several cellular proteins, indicating that it might be part of a larger multiprotein complex.

• Martinez-Noel G, Niedenthal R, Tamura T, Harbers K
• A family of structurally related RING finger proteins interacts specifically with the ubiquitin-conjugating enzyme UbcM4.
• FEBS Lett. 1999; 454: 257-61
• Display abstract

• The ubiquitin-conjugating enzyme UbcM4 was previously shown to be necessary for normal mouse development. As a first step in identifying target proteins or proteins involved in the specificity of UbcM4-mediated ubiquitylation, we have isolated seven cDNAs encoding proteins that specifically interact with UbcM4 but with none of the other Ubcs tested. This interaction was observed in yeast as well as in mammalian cells. With one exception, all UbcM4-interacting proteins (UIPs) belong to a family of proteins that contain a RING finger motif. As they are structurally related to RING finger proteins that have recently been shown to play an essential role in protein ubiquitylation and degradation, the possibility is discussed that UIPs are involved in the specific recognition of substrate proteins of UbcM4.

• Desterro JM, Rodriguez MS, Kemp GD, Hay RT
• Identification of the enzyme required for activation of the small ubiquitin-like protein SUMO-1.
• J Biol Chem. 1999; 274: 10618-24
• Display abstract

• The ubiquitin-like protein SUMO-1 is conjugated to a variety of proteins including Ran GTPase-activating protein 1 (RanGAP1), IkappaBalpha, and PML. SUMO-1-modified proteins display altered subcellular targeting and/or stability. We have purified the SUMO-1-activating enzyme from human cells and shown that it contains two subunits of 38 and 72 kDa. Isolation of cDNAs for each subunit indicates that they are homologous to ubiquitin-activating enzymes and to the Saccharomyces cerevisiae enzymes responsible for conjugation of Smt3p and Rub-1p. In vitro, recombinant SAE1/SAE2 (SUMO-1-activating enzyme) was capable of catalyzing the ATP-dependent formation of a thioester linkage between SUMO-1 and SAE2. The addition of the SUMO-1-conjugating enzyme Ubch9 resulted in efficient transfer of the thioester-linked SUMO-1 from SAE2 to Ubch9. In the presence of SAE1/SAE2, Ubch9, and ATP, SUMO-1 was efficiently conjugated to the protein substrate IkappaBalpha. As SAE1/SAE2, Ubch9, SUMO-1, and IkappaBalpha are all homogeneous, recombinant proteins, it appears that SUMO-1 conjugation of IkappaBalpha in vitro does not require the equivalent of an E3 ubiquitin protein ligase activity.

• del Pozo JC, Estelle M
• The Arabidopsis cullin AtCUL1 is modified by the ubiquitin-related protein RUB1.
• Proc Natl Acad Sci U S A. 1999; 96: 15342-7
• Display abstract

• The ubiquitin-like protein RUB1 is conjugated to target proteins by a mechanism similar to that of ubiquitin conjugation. Genetic studies in Arabidopsis thaliana have implicated the RUB-conjugation pathway in auxin response. The first step in the pathway is RUB activation by a bipartite enzyme composed of the AXR1 and ECR1 proteins. Ubiquitin activation is an ATP-dependent process that involves the formation of an AMP-ubiquitin intermediate. Here we show that RUB activation by AXR1-ECR1 also involves formation of an AMP-RUB intermediate and that this reaction is catalyzed by the ECR1 subunit alone. In addition, we identified an Arabidopsis protein called RCE1 that is a likely RUB-conjugating enzyme. RCE1 works together with AXR1-ECR1 to promote formation of a stable RUB conjugate with the Arabidopsis cullin AtCUL1 in vitro. Using a tagged version of RUB1, we show that this modification occurs in vivo. Because AtCUL1 is a component of the ubiquitin protein ligase SCF(TIR1), a complex that also functions in auxin response, we propose that RUB modification of AtCUL1 is important for auxin response.

• Wang G, Yang J, Huibregtse JM
• Functional domains of the Rsp5 ubiquitin-protein ligase.
• Mol Cell Biol. 1999; 19: 342-52
• Display abstract

• RSP5, an essential gene of Saccharomyces cerevisiae, encodes a hect domain E3 ubiquitin-protein ligase. Hect E3 proteins have been proposed to consist of two broad functional domains: a conserved catalytic carboxyl-terminal domain of approximately 350 amino acids (the hect domain) and a large, nonconserved amino-terminal domain containing determinants of substrate specificity. We report here the mapping of the minimal region of Rsp5 necessary for its essential in vivo function, the minimal region necessary to stably interact with a substrate of Rsp5 (Rpb1, the large subunit of RNA polymerase II), and the finding that the hect domain, by itself, is sufficient for formation of the ubiquitin-thioester intermediate. Mutations within the hect domain that affect either the ability to form a ubiquitin-thioester or to catalyze substrate ubiquitination abrogate in vivo function, strongly suggesting that the ubiquitin-protein ligase activity of Rsp5 is intrinsically linked to its essential function. The amino-terminal region of Rsp5 contains three WW domains and a C2 calcium-binding domain. Two of the three WW domains are required for the essential in vivo function, while the C2 domain is not, and requirements for Rpb1 binding and ubiquitination lie within the region required for in vivo function. Together, these results support the two-domain model for hect E3 function and indicate that the WW domains play a role in the recognition of at least some of the substrates of Rsp5, including those related to its essential function. In addition, we show that haploid yeast strains bearing complete disruptions of either of two other hect E3 genes of yeast, designated HUL4 (YJR036C) and HUL5 (YGL141W), are viable.

• Bates PW, Vierstra RD
• UPL1 and 2, two 405 kDa ubiquitin-protein ligases from Arabidopsis thaliana related to the HECT-domain protein family.
• Plant J. 1999; 20: 183-95
• Display abstract

• The ubiquitin/26S proteasome pathway is a major route for degrading abnormal and important short-lived regulatory proteins in eukaryotes. Covalent attachment of ubiquitin, which triggers entry of target proteins into the pathway, is accomplished by an ATP-dependent reaction cascade involving the sequential action of three enzymes, E1s, E2s and E3s. Although much of the substrate specificity of the pathway is determined by E3s (or ubiquitin-protein ligases, UPLs), little is known about these enzymes in plants and how they choose appropriate targets for ubiquitination. Here, we describe two 405 kDa E3s (UPL1 and 2) from Arabidopsis thaliana related to the HECT-E3 family that is essential in yeast and animals. UPL1 and 2 are encoded by 13 kbp genes 26 cM apart on chromosome I, that are over 95% identical within both the introns and exons, suggesting that the two loci arose from a recent gene duplication. The C-terminal HECT domain of UPL1 is necessary and sufficient to conjugate ubiquitin in vitro in a reaction that requires the positionally conserved cysteine within the HECT domain, E1, and an E2 of the UBC8 family. Given that HECT E3s help define target specificity of the ubiquitin conjugation, a continued characterization of UPL1 and 2 should be instrumental in understanding the functions of ubiquitin-dependent protein turnover in plants and for identifying pathway substrates.

• Utsugi T, Hirata A, Sekiguchi Y, Sasaki T, Toh-e A, Kikuchi Y
• Yeast tom1 mutant exhibits pleiotropic defects in nuclear division, maintenance of nuclear structure and nucleocytoplasmic transport at high temperatures.
• Gene. 1999; 234: 285-95
• Display abstract

• A tom1-1 mutant was isolated from Saccharomyces cerevisiae. At high temperatures, 60% of the cells were arrested as dumbbell forms with a single large nucleus containing duplicated DNA and a short spindle. Electron-microscopy showed electron-dense structures scattered within the nucleus. Indirect immunofluorescent microscopy revealed these structures to be fragmented nucleoli since the dotted structures were stained with anti-Nop1(fibrillarin) antibody in large regions of the nuclei. Fluorescent in situ hybridization analysis using oligo(dT) revealed nuclear accumulation of poly(A)+RNA. We cloned TOM1 which encodes a large protein (380kDa) with a hect (homologous to E6-AP C terminus)-domain at its C terminus. Deletions of either this hect-region or the entire gene made cellular growth temperature-sensitive. Site-directed mutagenesis of the conserved cysteine residue (tom1C3235A) in the hect-domain, supposed to be necessary for thioester-bond formation with ubiquitin, abolished the gene function. When a functional glutathione S-transferase (GST)-tagged hect protein was overproduced, it facilitated the protein conjugation with a myc-tagged ubiquitinRA, while this was not seen when GST-hectC3235A was overproduced. The protein conjugation with a hemagglutinin-tagged Smt3 was not affected by the overproduction of GST-hect. Taken together, we suggest that Tom1 is a ubiquitin ligase. As a multi-copy suppressor of tom1, we isolated STM3/NPI46/FPR3 which encodes a nucleolar nucleolin-like protein. We discuss possible functions of Tom1 with respect to the pleiotropic defects of nuclear division, maintenance of nuclear structure, and nucleocytoplasmic transport.

• Gong L, Li B, Millas S, Yeh ET
• Molecular cloning and characterization of human AOS1 and UBA2, components of the sentrin-activating enzyme complex.
• FEBS Lett. 1999; 448: 185-9
• Display abstract

• Sentrin-1/SUMO-1 is a novel ubiquitin-like protein, which can covalently modify a limited number of cellular proteins. Here we report the identification of the sentrin-activating enzyme complex, which consists of two proteins AOS1 and UBA2. Human AOS1 is homologous to the N-terminal half of E1, whereas human UBA2 is homologous to the C-terminal half of E1. The human UBA2 gene is located on chromosome 19q12. Human UBA2 could form a beta-mercaptoethanol-sensitive conjugate with members of the sentrin family, but not with ubiquitin of NEDD8, in the presence of AOS1. Identification of human UBA2 and AOS1 should allow a more detailed analysis of the enzymology of the activation of ubiquitin-like proteins.

• Moynihan TP et al.
• Characterization of the mouse ubiquitin-conjugating enzyme gene UbcM4.
• Mamm Genome. 1999; 10: 977-82
• Display abstract

• The ubiquitination pathway targets not only normal (short-lived) intracellular eukaryotic proteins for degradation when appropriate, but also serves to eliminate mutant/misfolded proteins from the cell. An understanding of the molecular basis of the interaction between the ubiquitin-conjugating enzymes (E2s), ubiquitin protein ligases (E3s), and target proteins is essential to explain the process in normal cellular function and in disease. UbcM4 is the mouse ortholog of the human E2, UbcH7, which can participate in the in vitro degradation of many proteins including p53. We describe the characterization of the mouse UbcM4 gene and the identification of a UbcM4 pseudogene. Four UbcM4 transcripts of approximately 0.7, 1.5, 2.1, and 2.6 kb, observed on Northern blots, are differentiated by their utilization of alternative UbcM4 polyadenylation sites. A single alternative splice variant cDNA, termed UbcM4Deltaex2, was also identified. The polypeptide encoded by UbcM4Deltaex2 is incapable of forming an ubiquitin-thioester in contrast to UbcM4, despite retaining the key cysteine residue essential for ubiquitin thioester formation and the active site consensus sequence that defines the ubiquitin-conjugating enzyme class. These observations are of particular relevance for analysis of UbcM4 function in vivo as our studies indicate that the targeted deletion of the coding exon absent in UbcM4Deltaex2 would produce an inactive UbcM4 protein and presents an alternative to disruption of its transcriptional initiation site/promoter region. Furthermore, it suggests that a similar strategy may be applicable to disrupt the function of other ubiquitin-conjugating enzymes in vivo.

• Anan T et al.
• Human ubiquitin-protein ligase Nedd4: expression, subcellular localization and selective interaction with ubiquitin-conjugating enzymes.
• Genes Cells. 1998; 3: 751-63
• Display abstract

• BACKGROUND: Nedd4 is a ubiquitin-protein ligase containing a calcium/lipid-binding domain, multiple WW domains and a C-terminal Hect domain, which is required for both the ubiquitin transfer and the association with E2 ubiquitin-conjugating enzymes. Nedd4 has been reported to be involved in the selective ubiquitination of some regulatory proteins in transcription and membrane transport. RESULTS: Three mRNA species for human Nedd4 were found to be 6.4-, 7.8- and 9.5-kb in size, and their expression patterns varied among normal tissues and cancer cell lines, indicating the tissue- and cell-specificities of Nedd4 expression. The Nedd4 protein, approximately 120 kDa in weight, was found in the cytoplasm, mainly in the perinuclear region and cytoplasmic periphery, of human cultured cells. Neural differentiation induced not only the down-regulation of Nedd4 but also the localization of the protein to both the cytoplasm and neurites. To identify the ubiquitination pathway that is linked to Nedd4, we demonstrated that specific E2 enzymes, including human Ubc4, UbcH5B, UbcH5C, UbcH6 and UbcH7, could transfer ubiquitin molecules to Nedd4 at the active cysteine residue, whereas E6AP accepted ubiquitins from Ubc4, UbcH5B, UbcH5C and UbcH7. Furthermore, nuclear localization of N-terminal deletion mutant Nedd4 enabled us to investigate the interaction between Nedd4 and E2 enzyme (Ubc4 or UbcH7) in the cell. The simultaneous expression of the full-length Nedd4 and E2 enzyme revealed the both proteins mostly colocalized in the cytoplasmic periphery, while the N-terminal deleted Nedd4 induced the nuclear and perinuclear colocalization with E2 enzyme. CONCLUSION: Our findings suggested that Nedd4 plays an important role in the cell regulation, including neural differentiation through cooperation with specific E2 ubiquitination pathways.

• Hauser HP, Bardroff M, Pyrowolakis G, Jentsch S
• A giant ubiquitin-conjugating enzyme related to IAP apoptosis inhibitors.
• J Cell Biol. 1998; 141: 1415-22
• Display abstract

• 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.

• Talis AL, Huibregtse JM, Howley PM
• The role of E6AP in the regulation of p53 protein levels in human papillomavirus (HPV)-positive and HPV-negative cells.
• J Biol Chem. 1998; 273: 6439-45
• Display abstract

• The E6 protein encoded by the oncogenic human papillomaviruses (HPVs) targets p53 for ubiquitin-dependent proteolysis. E6-mediated p53 degradation requires the 100-kDa cellular protein E6-associated protein (E6AP). E6AP and E6 together provide the E3-ubiquitin protein ligase activity in the transfer of ubiquitin to p53. In vitro studies have shown that E6AP can form a high energy thiolester bond with ubiquitin and, in the presence of E6, transfer ubiquitin to p53. In this study we have addressed the role of E6AP in vivo in the degradation of p53. Overexpression of wild-type E6AP in HeLa cells, which are HPV18-positive and express E6, resulted in a decreased steady state level of p53 and a decrease in the half-life of p53. Mutant forms of E6AP proteins were identified that were catalytically incapable of participating in E6-dependent ubiquitination of p53 and functioned in a dominant-negative manner in that they inhibited the E6-mediated ubiquitination of p53 by the wild-type E6AP in vitro. Transient transfection of one of these dominant negative (dn) mutants resulted in an increase in both the steady state level and half-life of p53 in vivo in HeLa cells. Consistent with this observation, overexpression of the dn E6AP resulted in a marked G1 shift in the cell cycle profile. In contrast, dn E6AP had no effect on p53 levels in U2OS cells, an HPV-negative cell line that contains wild-type p53. These studies provide evidence for the involvement of E6AP in E6-mediated p53 degradation in vivo and also indicate that E6AP may not be involved in the regulation of p53 ubiquitination in the absence of E6.

• Gilon T, Chomsky O, Kulka RG
• Degradation signals for ubiquitin system proteolysis in Saccharomyces cerevisiae.
• EMBO J. 1998; 17: 2759-66
• Display abstract

• Combinations of different ubiquitin-conjugating (Ubc) enzymes and other factors constitute subsidiary pathways of the ubiquitin system, each of which ubiquitinates a specific subset of proteins. There is evidence that certain sequence elements or structural motifs of target proteins are degradation signals which mark them for ubiquitination by a particular branch of the ubiquitin system and for subsequent degradation. Our aim was to devise a way of searching systematically for degradation signals and to determine to which ubiquitin system subpathways they direct the proteins. We have constructed two reporter gene libraries based on the lacZ or URA3 genes which, in Saccharomyces cerevisiae, express fusion proteins with a wide variety of C-terminal extensions. From these, we have isolated clones producing unstable fusion proteins which are stabilized in various ubc mutants. Among these are 10 clones whose products are stabilized in ubc6, ubc7 or ubc6ubc7 double mutants. The C-terminal extensions of these clones, which vary in length from 16 to 50 amino acid residues, are presumed to contain degradation signals channeling proteins for degradation via the UBC6 and/or UBC7 subpathways of the ubiquitin system. Some of these C-terminal tails share similar sequence motifs, and a feature common to almost all of these sequences is a highly hydrophobic region such as is usually located inside globular proteins or inserted into membranes.

• Aravind L, Koonin EV
• The HORMA domain: a common structural denominator in mitotic checkpoints, chromosome synapsis and DNA repair.
• Trends Biochem Sci. 1998; 23: 284-6

• Callaghan MJ, Russell AJ, Woollatt E, Sutherland GR, Sutherland RL, Watts CK
• Identification of a human HECT family protein with homology to the Drosophila tumor suppressor gene hyperplastic discs.
• Oncogene. 1998; 17: 3479-91
• Display abstract

• Use of the differential display technique to isolate progestin-regulated genes in T-47D human breast cancer cells led to identification of a novel gene, EDD. The cDNA sequence contains a 2799 amino acid open reading frame sharing 40% identity with the predicted 2894 amino acid product of the Drosophila melanogaster tumor suppressor gene hyperplastic discs, while the carboxy-terminal 889 amino acids show 96% identity to a rat 100 kDa HECT domain protein. EDD mRNA was progestin-induced in T-47D cells and was highly abundant in testes and expressed at moderately high levels in other tissues, suggesting a broad role for EDD. Anti-EDD antibodies immunoprecipitated an approximately 300 kDa protein from T-47D cell lysates. HECT family proteins function as E3 ubiquitin-protein ligases, targeting specific proteins for ubiquitin-mediated proteolysis. EDD is likely to function as an E3 as in vitro translated protein bound ubiquitin reversibly through a conserved HECT domain cysteine residue. EDD was localized by FISH to chromosome 8q22, a locus disrupted in a variety of cancers. Given the homology between EDD and the hyperplastic discs protein, which is required for control of imaginal disc growth in Drosophila, EDD potentially has a role in regulation of cell proliferation or differentiation.

• Schwarz SE, Matuschewski K, Liakopoulos D, Scheffner M, Jentsch S
• The ubiquitin-like proteins SMT3 and SUMO-1 are conjugated by the UBC9 E2 enzyme.
• Proc Natl Acad Sci U S A. 1998; 95: 560-4
• Display abstract

• The ubiquitin-like protein SMT3 from Saccharomyces cerevisiae and SUMO-1, its mammalian homolog, can be covalently attached to other proteins posttranslationally. Conjugation of ubiquitin requires the activities of ubiquitin-activating (E1) and -conjugating (E2) enzymes and proceeds via thioester-linked enzyme-ubiquitin intermediates. Herein we show that UBC9, one of the 13 different E2 enzymes from yeast, is required for SMT3 conjugation in vivo. Moreover, recombinant yeast and mammalian UBC9 enzymes were found to form thioester complexes with SMT3 and SUMO-1, respectively. This suggests that UBC9 functions as an E2 in a SMT3/SUMO-1 conjugation pathway analogous to ubiquitin-conjugating enzymes. The role of yeast UBC9 in cell cycle progression may thus be mediated through its SMT3 conjugation activity.

• Chen JJ, Hong Y, Rustamzadeh E, Baleja JD, Androphy EJ
• Identification of an alpha helical motif sufficient for association with papillomavirus E6.
• J Biol Chem. 1998; 273: 13537-44
• Display abstract

• We recently identified a cellular protein named E6BP or ERC-55 that binds cancer-related papillomavirus E6 proteins (Chen, J. J., Reid, C. E., Band, V., and Androphy, E. J. (1995) Science 269, 529-531). By construction of a series of deletion mutants, the region of E6BP that is necessary and sufficient for complex formation with human papillomavirus type 16 E6 has been mapped to a 25-amino acid domain. The corresponding peptide was synthesized and found by nuclear magnetic resonance spectroscopy to bind calcium and fold into a classical helix-loop-helix EF-hand conformation. Additional deletion mutagenesis showed that 13 amino acids that form the second alpha helix mediated E6 association. Alanine replacement mutagenesis indicated that amino acids of this helix were most important for E6 binding. Alignment of this alpha helical E6 binding peptide with the 18-amino acid E6 binding region of E6AP (Huibregtse, J. M., Scheffner, M., and Howley, P. M. (1993) Mol. Cell. Biol. 13, 4918-4927) and the first LD repeat of another E6-binding protein, paxillin (Tong, X., and Howley, P. M. (1997) J. Biol. Chem. 272, 33373-33376), revealed substantial similarities among these E6 binding domains. The extent of homology and the mutational data define the peptide as an E6 binding motif.

• Nuber U, Schwarz SE, Scheffner M
• The ubiquitin-protein ligase E6-associated protein (E6-AP) serves as its own substrate.
• Eur J Biochem. 1998; 254: 643-9
• Display abstract

• Recognition of substrate proteins by the ubiquitin-conjugation system is a highly specific and regulated event and involves the action of ubiquitin-conjugating enzymes (E2) and ubiquitin-protein ligases (E3). However, the E2 and E3 involved in the recognition of particular substrates have been identified in only a few cases. The ubiquitin-protein ligase E6-associated protein (E6-AP) was originally identified as a protein involved in the human papillomavirus E6-oncoprotein-induced degradation of p53. The substrate proteins of E6-AP in the absence of the E6 oncoprotein, however, have not been identified. We show here that E6-AP can target itself for ubiquitination in vitro and provide evidence that, under conditions of overexpression, E6-AP efficiently promotes its own degradation in vivo. Autoubiquitination of E6-AP is mediated mainly by intermolecular transfer of ubiquitin. In addition, highly ubiquitinated forms of E6-AP cannot bind to p53 in the presence of the E6 oncoprotein and, conversely, binding of E6-AP to p53 interferes with ubiquitination of E6-AP. These results suggest that autoubiquitination and subsequent degradation of E6-AP represents a mechanism to control intracellular E6-AP levels by inactivating E6-AP molecules that are not bound to substrate proteins.

• Lehman AL et al.
• A very large protein with diverse functional motifs is deficient in rjs (runty, jerky, sterile) mice.
• Proc Natl Acad Sci U S A. 1998; 95: 9436-41
• Display abstract

• Three radiation-induced alleles of the mouse p locus, p6H, p25H, and pbs, cause defects in growth, coordination, fertility, and maternal behavior in addition to p gene-related hypopigmentation. These alleles are associated with disruption of the p gene plus an adjacent gene involved in the disorders listed. We have identified this adjacent gene, previously named rjs (runty jerky sterile), by positional cloning. The rjs cDNA is very large, covering 15,264 nucleotides. The predicted rjs-encoded protein (4,836 amino acids) contains several sequence motifs, including three RCC1 repeats, a structural motif in common with cytochrome b5, and a HECT domain in common with E6-AP ubiquitin ligase. On the basis of sequence homology and conserved synteny, the rjs gene is the single mouse homolog of a previously described five- or six-member human gene family. This family is represented by at least two genes, HSC7541 and KIAA0393, from human chromosome 15q11-q13. HSC7541 and KIAA0393 lie close to, or within, a region commonly deleted in most Prader-Willi syndrome patients. Previous work has suggested that the multiple phenotypes in rjs mice might be due to a common neuroendocrine defect. In addition to this proposed mode of action, alternative functions of the rjs gene are evaluated in light of its known protein homologies.

• Honda R, Tanaka H, Yasuda H
• Oncoprotein MDM2 is a ubiquitin ligase E3 for tumor suppressor p53.
• FEBS Lett. 1997; 420: 25-7
• Display abstract

• The tumor suppressor p53 is degraded by the ubiquitin-proteasome system. p53 was polyubiquitinated in the presence of E1, UbcH5 as E2 and MDM2 oncoprotein. A ubiquitin molecule bound MDM2 through sulfhydroxy bond which is characteristic of ubiquitin ligase (E3)-ubiquitin binding. The cysteine residue in the carboxyl terminus of MDM2 was essential for the activity. These data suggest that the MDM2 protein, which is induced by p53, functions as a ubiquitin ligase, E3, in human papillomavirus-uninfected cells which do not have E6 protein.

• Zoladek T, Tobiasz A, Vaduva G, Boguta M, Martin NC, Hopper AK
• MDP1, a Saccharomyces cerevisiae gene involved in mitochondrial/cytoplasmic protein distribution, is identical to the ubiquitin-protein ligase gene RSP5.
• Genetics. 1997; 145: 595-603
• Display abstract

• Alteration of the subcellular distribution of Mod5p-I, a tRNA modification enzyme, member of the sorting isozyme family, affects tRNA-mediated nonsense suppression. Altered suppression efficiency was used to identify MDP genes, which, when mutant, change the mitochondrial/cytosolic distribution of Mod5p-I,KR6. MDP2 is the previously identified VRP1, which encodes verprolin, required for proper organization of the actin cytoskeleton. MDP3 is identical to PAN1, which encodes a protein involved in initiation of translation and actin cytoskeleton organization. We report here the cloning and characterization of wild-type and mutant MDP1 alleles and the isolation and characterization of a multicopy suppressor of mdp1 mutations. MDP1 is identical to RSP5, which encodes ubiquitin-protein ligase, and mdp1 mutations are suppressed by high copy expression of ubiquitin. All four characterized mdp1 mutations cause missense changes located in the hect domain of Rsp5p that is highly conserved among ubiquitin-protein ligases. In addition to its well-known function in protein turnover, ubiquitination has been proposed to play roles in subcellular sorting of proteins via endocytosis and in delivery of proteins to peroxisomes, the endoplasmic reticulum and mitochondria. mdp1, as well as mdp2/vrp1 and mdp3/pan1 mutations, affect endocytosis. Further, mdp1 mutations show synthetic interactions with mdp2/vrp1 and mdp3/pan1. Identification of MDP1 as RSP5, along with our previous identification of MDP2/VRP1 and MDP3/PAN1, implicate interactions of the ubiquitin system, the actin cytoskeleton and protein synthesis in the subcellular distribution of proteins.

• Sun B, Jeyaseelan K, Chung MC, Teo TS
• Rabbit ubiquitin-activating enzyme E1: cDNA cloning, sequence and expression.
• Gene. 1997; 196: 19-23
• Display abstract

• A cDNA clone encoding ubiquitin-activating enzyme E1 has been isolated from a rabbit heart cDNA library and sequenced. The 3.485 kb cDNA contains an open reading frame of 1058 amino acid residues which predicts a protein of approx. 118 kDa. The deduced protein sequence exhibits a very high homology to other ubiquitin-activating enzymes identified in a variety of organisms. Northern blot analysis reveals a single transcript of approx. 3.5 kb in all the rabbit tissues examined. The entire coding region of the rabbit E1 cDNA has been expressed as a his-tagged protein. The recombinant protein has been verified by its ability to cross-react with anti-human E1 antibodies. Ubiquitin thiolester assay shows that the recombinant rabbit E1 protein is functional.

• Wu X, Lieber MR
• Interaction between DNA-dependent protein kinase and a novel protein, KIP.
• Mutat Res. 1997; 385: 13-20
• Display abstract

• DNA-dependent protein kinase (DNA-PKcs) is the only eukaryotic kinase activated by DNA ends. Mutation of DNA-PKcs results in murine severe combined immune deficiency in mice and radiation sensitivity. Both the immune and the radiation defects are due to a failure in double-strand break repair. Biochemical studies indicate that DNA-PKcs kinase activity is stimulated by the presence of the DNA end binding protein. Ku. Autophosphorylation of DNA-PKcs results in its inactivation. Based on these studies, DNA-PKcs is presumed to play a direct and important role in the repair of double-strand breaks, but the details of its role are quite unclear. We have done two-hybrid analysis of this entire protein to identify other proteins with which it interacts. Thus far, extensive analysis has only revealed one strong interaction that satisfies both high genetic and biochemical stringency. The interaction is with a novel human protein that has 26% amino acid identity with the phosphatase component, calcineurin B. We discuss the interaction of DNA-PKcs with this novel calcium-binding protein family member in the context of possible kinase-phosphatase regulation of DNA end joining.

• Huibregtse JM, Yang JC, Beaudenon SL
• The large subunit of RNA polymerase II is a substrate of the Rsp5 ubiquitin-protein ligase.
• Proc Natl Acad Sci U S A. 1997; 94: 3656-61
• Display abstract

• The E3 ubiquitin-protein ligases play an important role in controlling substrate specificity of the ubiquitin proteolysis system. A biochemical approach was taken to identify substrates of Rsp5, an essential hect (homologous to E6-AP carboxyl terminus) E3 of Saccharomyces cerevisiae. We show here that Rsp5 binds and ubiquitinates the largest subunit of RNA polymerase II (Rpb1) in vitro. Stable complex formation between Rsp5 and Rpb1 was also detected in yeast cell extracts, and repression of RSP5 expression in vivo led to an elevated steady-state level of Rpb1. The amino-terminal domain of Rsp5 mediates binding to Rpb1, while the carboxyl-terminal domain of Rpb1, containing the heptapeptide repeats characteristic of polymerase II, is necessary and sufficient for binding to Rsp5. Fusion of the Rpb1 carboxyl-terminal domain to another protein also causes that protein to be ubiquitinated by Rsp5. These findings indicate that Rsp5 targets at least a subset of cellular Rpb1 molecules for ubiquitin-dependent degradation and may therefore play a role in regulating polymerase II activities. In addition, the results support a model for hect E3 function in which the amino-terminal domain mediates substrate binding, while the carboxyl-terminal hect domain catalyzes ubiquitination of bound substrates.

• Masson M, Menissier-de Murcia J, Mattei MG, de Murcia G, Niedergang CP
• Poly(ADP-ribose) polymerase interacts with a novel human ubiquitin conjugating enzyme: hUbc9.
• Gene. 1997; 190: 287-96
• Display abstract

• Poly(ADP-ribose) polymerase (PARP) has been suggested to play a regulatory role in vivo, in DNA replication and/or DNA repair based mainly on its capacity to bind to DNA strand breaks. This interaction is modulated through auto poly(ADP-ribosylation). However, the biological function of PARP may also involve interactions with proteins such as topoisomerase I or DNA polymerase alpha, which may or may not be themselves ADP-ribosylated. Using the yeast two-hybrid method search for other proteins interacting with PARP, we have isolated a full-length cDNA clone coding for a protein of 158 amino acid residues. This amino acid sequence is 66 and 56% identical to yeast ubiquitin-conjugating enzymes Hus5 and Ubc9 of Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively. Moreover, we have demonstrated that the expressed protein complements a S. cerevisiae yeast strain deficient for Ubc9. The protein encoded by the isolated cDNA is thus a new human counterpart of the ubiquitin-conjugating enzyme family and has been called hUbc9. The hubc9 gene locus has been assigned to the chromosomal location 16p13.2-p13.3. By means of two-hybrid analysis it was discovered that hUbc9 interacts with the automodification domain of PARP. This interaction was further confirmed using GST (glutathione-S-transferase) tagged fusion proteins: (i) in vivo, by transfecting cos7 cells with hUbc9 cloned in an eukaryotic expression vector, and (ii) in vitro, by mixing purified PARP with hUbc9 purified and expressed in bacteria. The possible significance and function of this interaction is discussed while taking into account the possible intracellular role of hUbc9.

• Yamamoto Y, Huibregtse JM, Howley PM
• The human E6-AP gene (UBE3A) encodes three potential protein isoforms generated by differential splicing.
• Genomics. 1997; 41: 263-6
• Display abstract

• The E6-AP gene (UBE3A) encodes an E3 ubiquitin-protein ligase that binds the human papillomavirus E6 oncoprotein and catalyzes the ubiquitination of p53. Recent studies have also established that mutations in E6-AP are the genetic basis of the Angelman syndrome in humans. In this study we present the genomic structure of the coding region of E6-AP and an analysis of a set of five E6-AP mRNAs with the potential to encode three protein isoforms of the E6-AP protein (isoforms I, II, and III) that differ at their extreme amino-termini. These transcripts were expressed in a variety of different cell lines examined.

• Kimura M et al.
• cDNA cloning, characterization, and chromosome mapping of UBE2E2 encoding a human ubiquitin-conjugating E2 enzyme.
• Cytogenet Cell Genet. 1997; 78: 107-11
• Display abstract

• A cDNA encoding a human ubiquitin-conjugating enzyme (E2) with N-terminal extension (UBE2E2/UbcH8) was isolated. Amino acid sequence within the UBC domain of UBE2E2 shares over 90% identity with human UbcH6, mouse UbcM2, and Drosophila UbcD2, whereas the N-terminal region shows little amino acid sequence similarity with known proteins. The UBE2E2 gene is transcribed in various tissues as a 1.9-kb transcript. The UBE2E2 protein formed a thioester bond with ubiquitin in an E1-dependent manner, indicating that the gene product is a functional E2 enzyme. The UBE2E2 gene was assigned to human chromosome 3p24.2 by fluorescence in situ hybridization.

• Yu H, King RW, Peters JM, Kirschner MW
• Identification of a novel ubiquitin-conjugating enzyme involved in mitotic cyclin degradation.
• Curr Biol. 1996; 6: 455-66
• Display abstract

• BACKGROUND: The destruction of cyclin B is required for exit from mitosis, and is mediated by the ubiquitin pathway. Recently, a 20S complex, termed the anaphase-promoting complex (APC) or the cyclosome, has been genetically and biochemically identified as the cyclin-specific ubiquitin ligase (E3). In addition, a ubiquitin-conjugating enzyme (E2), UBC4, was shown to be involved in cyclin ubiquitination in Xenopus egg extracts. Another E2 activity, designated UBCx, can independently support cyclin ubiquitination in Xenopus. A similar activity (E2-C) has also been observed in clams. However, the molecular identity of Xenopus UBCx or clam E2-C has not been established. RESULTS: We have purified and cloned Xenopus UBCx. Sequence comparisons with known E2s reveal that UBCx is a novel ubiquitin-conjugating enzyme. Purified recombinant UBCx is sufficient to complement purified APC and E1 in destruction box-dependent cyclin ubiquitination. UBCx and UBC4 are active in a similar concentration range and with similar kinetics. At saturating enzyme concentrations, UBCx converts twice as much substrate into ubiquitin conjugates, but generates conjugates of lower molecular mass than UBC4. CONCLUSIONS: UBCx is a novel ubiquitin-conjugating enzyme involved in cyclin ubiquitination in Xenopus. Like UBC4, ubiquitination catalyzed by UBCx is dependent on both the destruction box and the APC, suggesting that these E2s function through a similar mechanism. However, as the patterns of conjugates generated by these E2s are distinct, these enzymes may play different roles in promoting cyclin proteolysis in mitosis.

• Nefsky B, Beach D
• Pub1 acts as an E6-AP-like protein ubiquitiin ligase in the degradation of cdc25.
• EMBO J. 1996; 15: 1301-12
• Display abstract

• The level of the mitotic activating tyrosine phosphatase cdc25 is regulated by both transcriptional and post-transcriptional mechanisms in the fission yeast Schizosaccharomyces pombe. We have found that cdc25 is ubiquitinated and have cloned pub1, a gene which regulates this event. Pub1 contains a region highly homologous to the putative catalytic domain of the human protein ubiquitin ligase E6-AP. Disruption of pub1 elevates the level of cdc25 protein in vivo rendering cells relatively resistant to the cdc25-opposing tyrosine kinases wee1 and mik1. In addition, loss of wee1 activity in a pub1-disruption background results in a lethal premature entry into mitosis which can be rescued by loss of cdc25 function. A ubiquitin-thioester adduct of pub1 was isolated from fission yeast and disruption of pub1 dramatically reduced ubiquitination of cdc25 in vivo. These results suggest that pub1 directly ubiquitinates cdc25 in vivo.

• Kanda T
• A ubiquitin-protein ligase (E3) mutation of Saccharomyces cerevisiae suppressed by co-overexpression of two ubiquitin-specific processing proteases.
• Genes Genet Syst. 1996; 71: 75-83
• Display abstract

• To isolate mutations related to the ubiquitin system, I constructed a plasmid carrying the YUH1 and UBP1 genes (genes of ubiquitin-specific processing proteases) whose expressions were under the control of the galactose-inducible GAL1-GAL10 promoter. Cells of a strain carrying the plasmid were mutagenized with ethyl methanesulfonate. One mutant, which showed galactose-dependent growth at a high temperature (37 degrees C), was isolated from about 380,000 mutagenized colonies. The mutation responsible for galactose-dependent growth at 37 degrees C was a single nuclear recessive mutation designated as uby1-1. UBP1 and YUH1 as well as the GAL1-GAL10 promoter are required to suppress uby1-1. At the restrictive temperature, a uby1-1 mutant did not arrest at a specific phase of the cell cycle, but still lost viability. Even at the permissive temperature (30 degrees C), the uby1-1 mutant grew somewhat slowly and showed pleiotropic phenotypes including hypersensitivity to stresses such as cadmium and canavanine, and sporulation defects. The genomic DNA fragments in a single-copy plasmid which complemented uby1-1 were isolated. Chromosomal mapping, sequencing and subcloning analyses indicated that the gene complementing uby1-1 is RSP5, which encodes a ubiquitin-protein ligase (E3) homologous to E6-AP (E6 associated protein). Deletion, complementation and linkage analyses revealed that UBY1 and RSP5 are the same gene. Therefore, the E3 protein encoded by RSP5 (UBY1) is required for vegetative growth, sporulation and stress response. The present procedure using suppression by co-overexpression of two cloned genes will be useful to isolate mutations of related genes and to analyze biochemical pathways and gene-interactions.

• Prendergast JA et al.
• Identification of a positive regulator of the cell cycle ubiquitin-conjugating enzyme Cdc34 (Ubc3).
• Mol Cell Biol. 1996; 16: 677-84
• Display abstract

• The Cdc34 (Ubc3) ubiquitin-conjugating enzyme from Saccharomyces cerevisiae plays an essential role in the progression of cells from the G1 to S phase of the cell division cycle. Using a high-copy suppression strategy, we have identified a yeast gene (UBS1) whose elevated expression suppresses the conditional cell cycle defects associated with cdc34 mutations. The UBS1 gene encodes a 32.2-kDa protein of previously unknown function and is identical in sequence to a genomic open reading frame on chromosome II (GenBank accession number Z36034). Several lines of evidence described here indicate that Ubs1 functions as a general positive regulator of Cdc34 activity. First, overexpression of UBS1 suppresses not only the cell proliferation and morphological defects associated with cdc34 mutants but also the inability of cdc34 mutant cells to degrade the general amino acid biosynthesis transcriptional regulator, Gcn4. Second, deletion of the UBS1 gene profoundly accentuates the cell cycle defect when placed in combination with a cdc34 temperature-sensitive allele. Finally, a comparison of the Ubs1 and Cdc34 polypeptide sequences reveals two noncontiguous regions of similarity, which, when projected onto the three-dimensional structure of a ubiquitin-conjugating enzyme, define a single region situated on its surface. While cdc34 mutations corresponding to substitutions outside this region are suppressed by UBS1 overexpression, Ubs1 fails to suppress amino acid substitutions made within this region. Taken together with other findings, the allele specificity exhibited by UBS1 expression suggests that Ubs1 regulates Cdc34 by interaction or modification.

• Gonen H et al.
• Isolation, characterization, and partial purification of a novel ubiquitin-protein ligase, E3. Targeting of protein substrates via multiple and distinct recognition signals and conjugating enzymes.
• J Biol Chem. 1996; 271: 302-10
• Display abstract

• Degradation of a protein via the ubiquitin system involves two discrete steps, conjugation of ubiquitin to the substrate and degradation of the adduct. Conjugation follows a three-step mechanism. First, ubiquitin is activated by the ubiquitin-activating enzyme, E1. Following activation, one of several E2 enzymes (ubiquitin-carrier proteins or ubiquitin-conjugating enzymes, UBCs) transfers ubiquitin from E1 to the protein substrate that is bound to one of several ubiquitin-protein ligases, E3s. These enzymes catalyze the last step in the process, covalent attachment of ubiquitin to the protein substrate. The binding of the substrate to E3 is specific and implies that E3s play a major role in recognition and selection of proteins for conjugation and subsequent degradation. So far, only a few ligases have been identified, and it is clear that many more have not been discovered yet. Here, we describe a novel ligase that is involved in the conjugation and degradation of non "N-end rule" protein substrates such as actin, troponin T, and MyoD. This substrate specificity suggests that the enzyme may be involved in degradation of muscle proteins. The ligase acts in concert with E2-F1, a previously described non N-end rule UBC. Interestingly, it is also involved in targeting lysozyme, a bona fide N-end substrate that is recognized by E3 alpha and E2-14 kDa. The novel ligase recognizes lysozyme via a signal(s) that is distinct from the N-terminal residue of the protein. Thus, it appears that certain proteins can be targeted via multiple recognition motifs and distinct pairs of conjugating enzymes. We have purified the ligase approximately 200-fold and demonstrated that it is different from other known E3s, including E3 alpha/UBR1, E3 beta, and E6-AP. The native enzyme has an apparent molecular mass of approximately 550 kDa and appears to be a homodimer. Because of its unusual size, we designated this novel ligase E3L (large). E3L contains an -SH group that is essential for its activity. Like several recently described E3 enzymes, including E6-AP and the ligase involved in the processing of p105, the NF-kappa B precursor, the novel ligase is found in mammalian tissues but not in wheat germ.

• Keeling PJ, Doherty-Kirby AL, Teh EM, Doolittle WF
• Linked genes for calmodulin and E2 ubiquitin-conjugating enzyme in Trichomonas vaginalis.
• J Eukaryot Microbiol. 1996; 43: 468-74
• Display abstract

• In searching the genomes of early-diverging protists to study whether the possession of calmodulin is ancestral to all eukaryotes, the gene for calmodulin was identified in Trichomonas vaginalis. This flagellate is a member of the Parabasalia, one of the earliest lineages of recognized eukaryotes to have diverged. This sequence was used to isolate a homologous 1.250-kb fragment from the T. vaginalis genome by inverse polymerase chain reaction. This fragment was also completely sequenced and shown to contain the 3' end of the single-copy calmodulin gene and the 3' end of a gene encoding a protein with high similarity to E2 ubiquitin-conjugating enzymes, a family which has previously only been identified in animals, plants, and fungi. Phylogenetic analysis of 50 members of the E2 family distinguishes at least nine separate subfamilies one of which includes the T. vaginalis E2-homologue and an uncharacterized gene from yeast chromosome XII.

• Yashiroda H, Oguchi T, Yasuda Y, Toh-E A, Kikuchi Y
• Bul1, a new protein that binds to the Rsp5 ubiquitin ligase in Saccharomyces cerevisiae.
• Mol Cell Biol. 1996; 16: 3255-63
• Display abstract

• We characterized a temperature-sensitive mutant of Saccharomyces cerevisiae in which a mini-chromosome was unstable at a high temperature and cloned a new gene which encodes a basic and hydrophilic protein (110 kDa). The disruption of this gene caused the same temperature-sensitive growth as the original mutation. By using the two-hybrid system, we further isolated RSP5 (reverses Spt- phenotype), which encodes a hect (homologous to E6-AP C terminus) domain, as a gene encoding a ubiquitin ligase. Thus, we named our gene BUL1 (for a protein that binds to the ubiquitin ligase). BUL1 seems to be involved in the ubiquitination pathway, since a high dose of UBI1, encoding a ubiquitin, partially suppressed the temperature sensitivity of the bul1 disruptant as well as that of a rsp5 mutant. Coexpression of RSP5 and BUL1 on a multicopy plasmid was toxic for mitotic growth of the wild-type cells. Pulse-chase experiments revealed that Bul1 in the wild-type cells remained stable, while the bands of Bul1 in the rsp5 cells were hardly detected. Since the steady-state levels of the protein were the same in the two strains as determined by immunoblotting analysis, Bul1 might be easily degraded during immunoprecipitation in the absence of intact Rsp5. Furthermore, both Bul1 and Rsp5 appeared to be associated with large complexes which were separated through a sucrose gradient centrifugation, and Rsp5 was coimmunoprecipitated with Bul1. We discuss the possibility that Bul1 functions together with Rsp5 in protein ubiquitination.

• Jiang W, Koltin Y
• Two-hybrid interaction of a human UBC9 homolog with centromere proteins of Saccharomyces cerevisiae.
• Mol Gen Genet. 1996; 251: 153-60
• Display abstract

• Using a two-hybrid system, we cloned a human cDNA encoding a ubiquitin-conjugating enzyme (UBC), hUBC9, which interacts specifically with all three subunits of the Saccharomyces cerevisiae centromere DNA-binding core complex, CBF3. The hUBC9 protein shows highest homology to a new member of the UBC family: 54% identity to S. cerevisiae Ubc9p and 64% identity to Schizosaccharomyces pombe (Sp) hus5. Overexpression of hUBC9 partially suppresses a S. cerevisiae ubc9 temperature-sensitive mutation, indicating that the UBC9 gene family is also functionally conserved. Like hUBC9, Sphus5 also interacts specifically with all three subunits of the CBF3 complex. However, S. cerevisiae Ubc9p interacts only with the Cbf3p subunit (64 kDa) of the CBF3 complex, indicating the specificity of the interaction between S. cerevisiae Ubc9 and Cbf3p proteins. The function of Ubc9p in the G2/M phase of S. cerevisiae could be related to regulation of centromere proteins in chromosome segregation in mitosis. Therefore, the ubiquitination process and centromere function may be linked to chromosome segregation. We also provide further in vivo evidence that Mck1p, a protein kinase, is specifically associated with the centromere proteins Cbf2p and Cbf5p, which were previously shown to interact in vitro.

• Harbers K, Muller U, Grams A, Li E, Jaenisch R, Franz T
• Provirus integration into a gene encoding a ubiquitin-conjugating enzyme results in a placental defect and embryonic lethality.
• Proc Natl Acad Sci U S A. 1996; 93: 12412-7
• Display abstract

• Ubiquitin-conjugating enzymes (E2 or Ubc) constitute a family of conserved proteins that play a key role in ubiquitin-dependent degradation of proteins in eukaryotes. We describe here a transgenic mouse strain where retrovirus integration into an Ubc gene, designated UbcM4, results in a recessive-lethal mutation. UbcM4 is the mouse homologue of the previously described human UbcH7 that is involved in the in vitro ubiquitination of several proteins including the tumor suppressor protein p53. The provirus is located in the first intron of the gene. When both alleles are mutated the level of steady-state mRNA is reduced by about 70%. About a third of homozygous mutant embryos die around day 11.5 of gestation. Embryos that survive that stage are growth retarded and die perinatally. The lethal phenotype is most likely caused by impairment of placenta development as this is the only organ that consistently showed pathological defects. The placental labyrinth is drastically reduced in size and vascularization is disturbed. The UbcM4 mouse mutant represents the first example in mammals of a mutation in a gene involved in ubiquitin conjugation. Its recessive-lethal phenotype demonstrates that the ubiquitin system plays an essential role during mouse development.

• McDonough M, Sangan P, Gonda DK
• Characterization of novel yeast RAD6 (UBC2) ubiquitin-conjugating enzyme mutants constructed by charge-to-alanine scanning mutagenesis.
• J Bacteriol. 1995; 177: 580-5
• Display abstract

• Ubiquitination of intracellular proteins by the yeast RAD6 (UBC2) ubiquitin-conjugating (E2) enzyme is required for cellular processes as diverse as DNA repair, selective proteolysis, and normal growth. For most RAD6-dependent functions, the relevant in vivo targets, as well as the mechanisms and cofactors that govern RAD6 substrate selectivity, are unknown. We have explored the utility of "charge-to-alanine" scanning mutagenesis to generate novel RAD6 mutants that are enzymatically competent with respect to unfacilitated (E3-independent) ubiquitination but that are nevertheless severely handicapped with respect to several in vivo functions. Five of the nine mutants we generated show defects in their in vivo functions, but almost all of the most severely affected mutants displayed unfacilitated ubiquitin-conjugating activity in vitro. We suggest that E2 mutants obtained by this approach are likely to be defective with respect to interaction with other, trans-acting factors required for their intracellular activity or substrate selectivity and therefore will be useful for further genetic and biochemical studies of ubiquitin-conjugating enzyme function.

• Hein C, Springael JY, Volland C, Haguenauer-Tsapis R, Andre B
• NPl1, an essential yeast gene involved in induced degradation of Gap1 and Fur4 permeases, encodes the Rsp5 ubiquitin-protein ligase.
• Mol Microbiol. 1995; 18: 77-87
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• When yeast cells growing on a poor nitrogen source are supplied with NH4+ ions, several nitrogen permeases including the general amino acid permease (Gap1p) are rapidly and completely inactivated. This report shows that inactivation by NH4+ of the Gap1 permease is accompanied by its degradation. A functional NPl1 gene product is required for both inactivation and degradation of Gap1p. Molecular analysis of the NPl1 gene showed that it is identical to RSP5. The RSP5 product is a ubiquitin-protein ligase (E3 enzyme) whose physiological function was, however, unknown. Its C-terminal region is very similar to that of other members of the E6-AP-like family of ubiquitin-protein ligases. Its N-terminal region contains a single C2 domain that may be a Ca(2+)-dependent phospholipid interaction motif, followed by several copies of a recently identified domain called WW(P). The Npi1/Rsp5 protein has a homologue both in humans and in mice, the latter being involved in brain development. Stress-induced degradation of the uracil permease (Fur4p), a process in which ubiquitin is probably involved, was also found to require a functional NPl1/RSP5 product. Chromosomal deletion of NPl1/RSP5 showed that this gene is essential for cell viability. In the viable npi1/rsp5 strain, expression of NPl1/RSP5 is reduced as a result of insertion of a Ty1 element in its 5' region. Our results show that the Npi1/Rsp5 ubiquitin-protein ligase participates in induced degradation of at least two permeases, Gap1p and Fur4p, and probably also other proteins.

• Wefes I, Kaiser P, Schneider R, Pickart CM, Finley D
• Characterization of a cDNA clone encoding E2-20K, a murine ubiquitin-conjugating enzyme.
• Gene. 1995; 163: 321-2
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• The 20-kDa ubiquitin-conjugating enzyme E2-20K is induced specifically during a late stage of erythroid differentiation. Here we report the sequence of a murine cDNA encoding E2-20K. Northern blot analysis identified polyadenylated transcripts of 3.5 and 6.5 kb which are present at comparable levels in many nonerythroid tissues.

• Ciechanover A, Shkedy D, Oren M, Bercovich B
• Degradation of the tumor suppressor protein p53 by the ubiquitin-mediated proteolytic system requires a novel species of ubiquitin-carrier protein, E2.
• J Biol Chem. 1994; 269: 9582-9
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• The tumor suppressor protein p53 is extremely unstable in most cell lines. In contrast, many mutant and oncogenic species of the protein are stable. The degradation of p53 in vivo requires metabolic energy; however, the proteolytic system(s) involved have not been identified. The ubiquitin system has been implicated in the degradation of p53 in vitro. The degradation is stimulated significantly by the human papillomavirus (HPV) oncoprotein E6 that associates with p53 and facilitates conjugate formation and subsequent degradation. Complex formation between E6 and p53 is promoted by a cellular protein designated E6-associated protein (E6-AP). Initial dissection of the conjugation process have demonstrated a role for the ubiquitin-activating enzyme, E1, but the ubiquitin-carrier protein (E2, UBC) and the ubiquitin protein ligase, E3, have not been identified. In this study, we report that a novel species of ubiquitin-carrier protein designated E2-F1 (Blumenfeld, N., Gonen, H., Mayer, A., Smith, C., Siegel, N.R., Schwartz, A.L., and Ciechanover, A. (1994) J. Biol. Chem. 269, 9574-9581) is involved in the conjugation and degradation of p53. This E2 enzyme recognizes non-"N-end rule" protein substrates and appears to mediate their conjugation via a novel species of E3. The process of recognition appears to be selective; E2-F1 is not required for the conjugation and degradation of human N-myc. The involvement of E2-F1 in the in vitro process appears to be physiologically meaningful and to reproduce the in vivo process; mutant species of p53 that do not interact with E6 and are stable in vivo are not recognized by the cell free system.

• Blumenfeld N et al.
• Purification and characterization of a novel species of ubiquitin-carrier protein, E2, that is involved in degradation of non-"N-end rule" protein substrates.
• J Biol Chem. 1994; 269: 9574-81
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• Ubiquitin-carrier proteins (E2s, ubiquitin-conjugating enzymes, UBCs) participate in proteolysis by catalyzing transfer of activated ubiquitin to the protein substrates, which are bound to specific ubiquitin-protein ligases (E3s). Yeast UBC2 (RAD6) and the mammalian E2(14kDa) bind to the ligase that recognizes and is involved in the degradation of certain free amino-terminal substrates ("N-end rule" substrates). As such proteins are rather scarce, the role of these E2s in general proteolysis is probably limited. Here, we report the purification and characterization of a novel 18-kDa species of E2 from rabbit reticulocytes. Unlike most members of the E2 family, this enzyme does not adsorb to anion exchange resin in neutral pH, and it is purified from the unadsorbed material (Fraction 1). Thus, it is designated E2-F1. Like all members of the E2 family, it generates a thiol ester with ubiquitin that serves as an intermediate in the conjugation reaction. Sequence analysis revealed a significant homology to many known species of E2s. The enzyme generates multiply ubiquitinated proteins in the presence of an E3 that has not been characterized yet. Most importantly, the ubiquitination via this E2 leads to the degradation of certain non-"N-end rule" substrates such as glyceraldehyde-3-phosphate dehydrogenase (Val at the NH2 terminus) and to the ubiquitination and degradation of certain N-alpha-acetylated proteins such as histone H2A, actin, and alpha-crystallin. The enzyme is also involved in the conjugation and degradation of the tumor suppressor protein p53.

• Scheffner M, Huibregtse JM, Howley PM
• Identification of a human ubiquitin-conjugating enzyme that mediates the E6-AP-dependent ubiquitination of p53.
• Proc Natl Acad Sci U S A. 1994; 91: 8797-801
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• The E6 protein of the oncogenic human papillomavirus types 16 and 18 facilitates the rapid degradation of the tumor-suppressor protein p53 via the ubiquitin-dependent proteolytic pathway. The E6 protein binds to a cellular protein of 100 kDa termed E6-AP. The complex of E6 and E6-AP specifically interacts with p53 and induces the ubiquitination of p53 in a reaction which requires the ubiquitin-activating enzyme (E1) and a cellular fraction thought to contain a mammalian ubiquitin-conjugating enzyme (E2). This mammalian E2 activity could be replaced with bacterially expressed UBC8 from Arabidopsis thaliana, which belongs to a subfamily of E2s including yeast UBC4 and UBC5 which are highly conserved at the amino acid level. In this paper we describe the cloning of a human cDNA encoding a human E2 that we have designated UbcH5 and that is related to Arabidopsis UBC8 and the other members of this subfamily. We demonstrate that UbcH5 can function in the E6/E6-AP-induced ubiquitination of p53.

• Keen N, Elston R, Crawford L
• Interaction of the E6 protein of human papillomavirus with cellular proteins.
• Oncogene. 1994; 9: 1493-9
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• The E6 proteins of the oncogenic Human Papillomavirus (HPV) types 16 and 18 are known to bind two cellular proteins, the tumor suppressor protein p53 and a 100 kDa protein named E6-AP. In this paper we describe the expression and purification of biologically active E6 fusion proteins and their specific association with additional cellular proteins. HPV16E6 specifically associated with at least seven cellular proteins which have been designated pp212, pp182, p100, p81, p75, p53 and p33 respectively, on the basis of molecular mass and phosphorylation. We have also shown that the complex of cellular proteins associated with HPV16, 18, 6 and 11 E6 proteins contains a protein kinase. This protein kinase phosphorylated exogenous histone H1 and the E6 associated protein pp182.

• Shkedy D, Gonen H, Bercovich B, Ciechanover A
• Complete reconstitution of conjugation and subsequent degradation of the tumor suppressor protein p53 by purified components of the ubiquitin proteolytic system.
• FEBS Lett. 1994; 348: 126-30
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• The wild-type tumor suppressor protein p53 is a short-lived protein that plays important roles in regulation of cell cycle, differentiation, and survival. Mutations that inactivate or alter the tumor suppressor activity of the protein seem to be the most common genetic change in human cancer and are frequently associated with changes in its stability. The ubiquitin system has been implicated in the degradation of p53 both in vivo and in vitro. A mutant cell line that harbors a thermolabile ubiquitin-activating enzyme, E1, fails to degrade p53 at the nonpermissive temperature. Studies in cell-free extracts have shown that covalent attachment of ubiquitin to the protein requires the three conjugating enzymes: E1, a novel species of ubiquitin-carrier protein (ubiquitin-conjugating enzyme; UBC),E2-F1, and an ubiquitin-protein ligase, E3. Recognition of p53 by the ligase is facilitated by formation of a complex between the protein and the human papillomavirus (HPV) oncoprotein E6. Therefore, the ligase has been designated E6-associated protein (E6-AP). However, these in vitro studies have not demonstrated that the conjugates serve as essential intermediates in the proteolytic process. In fact, in many cases, conjugation of ubiquitin to the target protein does not signal its degradation. Thus, it is essential to demonstrate that p53-ubiquitin adducts serve as essential proteolytic intermediates and are recognized and degraded by the 26S protease complex, the proteolytic arm of the ubiquitin pathway. In this study, we demonstrate that conjugates of p53 generated in the presence of purified, E1, E2, E6-AP, E6, ubiquitin and ATP, are specifically recognized by the 26S protease complex and degraded. In contrast, unconjugated p53 remains stable. The ability to reconstitute the system from purified components will enable detailed analysis of the recognition process and the structural motifs involved in targeting the protein for degradation.

• Huibregtse JM, Scheffner M, Howley PM
• Localization of the E6-AP regions that direct human papillomavirus E6 binding, association with p53, and ubiquitination of associated proteins.
• Mol Cell Biol. 1993; 13: 4918-27
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• E6-AP is a 100-kDa cellular protein that mediates the interaction of the human papillomavirus type 16 and 18 E6 proteins with p53. The association of p53 with E6 and E6-AP promotes the specific ubiquitination and subsequent proteolytic degradation of p53 in vitro. We recently isolated a cDNA encoding E6-AP and have now mapped functional domains of E6-AP involved in binding E6, association with p53, and ubiquitination of p53. The E6 binding domain consists of an 18-amino-acid region within the central portion of the molecule. Deletion of these 18 amino acids from E6-AP results in loss of both E6 and p53 binding activities. The region that directs p53 binding spans the E6 binding domain and consists of approximately 500 amino acids. E6-AP sequences in addition to those required for formation of a stable ternary complex with E6 and p53 are necessary to stimulate the ubiquitination of p53. These sequences lie within the C-terminal 84 amino acids of E6-AP. The entire region required for E6-dependent ubiquitination of p53 is also required for the ubiquitination of an artificial E6 fusion protein.

• Picton S, Gray JE, Lowe A, Barton SL, Grierson D
• Sequence of a cloned tomato ubiquitin conjugating enzyme.
• Plant Physiol. 1993; 103: 1471-2

• Huibregtse JM, Scheffner M, Howley PM
• Cloning and expression of the cDNA for E6-AP, a protein that mediates the interaction of the human papillomavirus E6 oncoprotein with p53.
• Mol Cell Biol. 1993; 13: 775-84
• Display abstract

• The E6 oncoproteins of the cancer-associated or high-risk human papillomaviruses (HPVs) target the cellular p53 protein. The association of E6 with p53 leads to the specific ubiquitination and degradation of p53 in vitro, suggesting a model by which E6 deregulates cell growth control by the elimination of the p53 tumor suppressor protein. Complex formation between E6 and p53 requires an additional cellular factor, designated E6-AP (E6-associated protein), which has a native and subunit molecular mass of approximately 100 kDa. Here we report the purification of E6-AP and the cloning of its corresponding cDNA, which contains a novel open reading frame encoding 865 amino acids. E6-AP, translated in vitro, has the following properties: (i) it associates with wild-type p53 in the presence of the HPV16 E6 protein and simultaneously stimulates the association of E6 with p53, (ii) it associates with the high-risk HPV16 and HPV18 E6 proteins in the absence of p53, and (iii) it induces the E6- and ubiquitin-dependent degradation of p53 in vitro.

• Scheffner M, Huibregtse JM, Vierstra RD, Howley PM
• The HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53.
• Cell. 1993; 75: 495-505
• Display abstract

• The ubiquitin-dependent proteolytic pathway plays a major role in selective protein degradation. Ubiquitination of proteins requires the sequential action of the ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzymes (E2), and in some cases ubiquitin-protein ligases (E3s). The oncogenic human papillomavirus (HPV) types 16 and 18 utilize this cellular proteolytic system to target the tumor suppressor protein p53. The HPV E6 oncoprotein binds to a cellular protein of 100 kd, termed E6-associated protein (E6-AP). The E6-E6-AP complex specifically interacts with p53, resulting in the rapid ubiquitin-dependent degradation of p53. Here we report the purification and identification of the factors necessary for the E6-E6-AP-mediated ubiquitination of p53. The ubiquitination of p53 requires the E1 enzyme and a novel E2 in mammalian cells, while E3 activity is conferred by the E6-E6-AP complex. Furthermore, E6-AP appears to have ubiquitin-protein ligase activity in the absence of E6.

• Berleth ES, Kasperek EM, Grill SP, Braunscheidel JA, Graziani LA, Pickart CM
• Inhibition of ubiquitin-protein ligase (E3) by mono- and bifunctional phenylarsenoxides. Evidence for essential vicinal thiols and a proximal nucleophile.
• J Biol Chem. 1992; 267: 16403-11
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• Trivalent arsenoxides bind to vicinal thiol groups of proteins. We showed previously that the simplest trivalent arsenoxide, inorganic arsenite, inhibits ubiquitin-dependent protein degradation in rabbit reticulocyte lysate (Klemperer, N.S., and Pickart, C.M. (1989) J. Biol. Chem. 264, 19245-19242). We now show that, relative to arsenite, phenylarsenoxides are 10-165-fold more potent inhibitors of protein degradation in the same system (K0.5 for inhibition by p-aminophenylarsenoxide was 3.5-20 microM, depending on the substrate). In the ubiquitin-dependent proteolytic pathway, covalent ligation of ubiquitin to protein substrates targets the latter for degradation. In certain cases, specificity in ubiquitin-substrate conjugation depends critically upon the properties of ubiquitin-protein ligase or E3. Among other effects, p-aminophenylarsenoxide decreased the steady-state level of ubiquitinated human alpha-lactalbumin; this is a substrate which is acted upon directly by ubiquitin-protein ligase-alpha (E3-alpha). This finding suggests that phenylarsenoxides (unlike arsenite) inhibit E3. Several other lines of evidence confirm this conclusion. 1) A complex of E3-alpha and the 14-kDa ubiquitin-conjugating (E2) isozyme binds to phenylarsenoxide-Sepharose resin, with the E3 component of the complex mediating binding. 2) p-Aminophenylarsenoxide inhibited isolated E3 (K0.5 approximately 50 microM); inhibition was readily reversed by addition of dithiothreitol (which contains a competing vicinal thiol group), but not by beta-mercaptoethylamine (a monothiol). 3) A bifunctional phenylarsenoxide (bromoacetylaminophenylarsenoxide) rapidly and irreversibly inactivated E3; bromoacetyl aniline, which lacks an arsenoxide moiety, did not inhibit E3. These results suggest that E3 possesses essential vicinal thiol groups and that there is a reactive nucleophile proximal to the vicinal thiol site. The bifunctional phenylarsenoxide should be a useful tool for probing the relationship between structure and function in E3. As expected from prior results with arsenite, p-aminophenylarsenoxide was also a potent inhibitor of the turnover of ubiquitin-(human) alpha-lactalbumin conjugates.

• Reiss Y, Heller H, Hershko A
• Binding sites of ubiquitin-protein ligase. Binding of ubiquitin-protein conjugates and of ubiquitin-carrier protein.
• J Biol Chem. 1989; 264: 10378-83
• Display abstract

• It was found previously that the enzyme ubiquitin-protein ligase (E3) contains specific protein substrate binding sites that are responsible for the selection of proteins for degradation by the ubiquitin system. In the present study, we have tried to gain more insight into the mode of action of E3 by the characterization of other binding sites of this enzyme. Following the ligation of ubiquitin to 125I-lysozyme, the conjugates produced are very tightly bound to E3, as indicated by size analysis on glycerol density gradient centrifugation. The strong binding of ubiquitin-protein conjugates to the enzyme may account for the apparently processive addition of multiple molecules of ubiquitin to the protein substrate. Both the protein substrate moiety and the ubiquitin moiety participate in the interaction of ubiquitin-protein conjugates with E3, as indicated by competition with specific agents and by the comparison of the binding of ubiquitin-conjugated protein to that of free protein. In addition to the binding of its substrates and products, E3 also appears to interact with some of the enzymes with which it acts in concert. When E3 is incubated with the ubiquitin-carrier protein E2, a complex is formed between the two enzymes as analyzed on glycerol gradients. The formation of an E2.E3 complex may facilitate the transfer of activated ubiquitin from E2 to the protein substrate bound to the ligase.

• Hershko A, Heller H, Eytan E, Reiss Y
• The protein substrate binding site of the ubiquitin-protein ligase system.
• J Biol Chem. 1986; 261: 11992-9
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• In order to gain insight into the mechanisms that determine the selectivity of the ubiquitin proteolytic pathway, the protein substrate binding site of the ubiquitin-protein ligase system was identified and examined. Previous studies had shown that the ligase system consists of three components: a ubiquitin-activating enzyme (E1), ubiquitin-carrier protein (E2), and a third enzyme, E3, the mode of action of which has not been defined. E3 from rabbit reticulocytes was further purified by a combination of affinity chromatography, hydrophobic chromatography, and gel filtration procedures. A 180-kDa protein was identified as the subunit of E3. Two independent methods indicate that E3 has the protein binding site of the ubiquitin ligase system. These are the chemical cross-linking of 125I-labeled proteins to the E3 subunit and the functional conversion of enzyme-bound labeled proteins to ubiquitin conjugates in pulse-chase experiments. The trapping of E3-bound protein for labeled product formation was allowed by the slow dissociation of E3 X protein complex. The specificity of binding of different proteins to E3, examined by both methods, showed a direct correlation with their susceptibility to degradation by the ubiquitin system. Proteins with free alpha-NH2 groups, which are good substrates, bind better to E3 than corresponding proteins with blocked NH2 termini, which are not substrates. Oxidation of methionine residues to sulfoxide derivatives greatly increases the susceptibility of some proteins to ligation with ubiquitin, with a corresponding increase in their binding to E3. However, a protein derivative which was subjected to both amino group modification and oxidation binds strongly to the enzyme, even though it cannot be ligated to ubiquitin. It thus seems that the substrate binding site of E3 participates in determining the specificity of proteins that enter the ubiquitin pathway of protein degradation.

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