Spectrin ubiquitination and oxidative stress: potential roles in blood and neurological disorders

This review covers the observations that erythrocyte spectrin has a E2 ubiquitin conjugating enzymatic activity that allows it to transfer ubiquitin to a target site in the alpha-spectrin repeats 20/21. The position of this ubiquitination site suggests that ubiquitination may regulate alpha beta spectrin heterodimer nucleation, spectrin-4.1-actin ternary complex formation, and adducin stimulated spectrin-actin attachment in the mature erythrocyte. In sickle cells, which contain altered redox status (high GSSG/GSH ratio), ubiquitin attachment to the E2 and target sites in alpha-spectrin is greatly diminished. We propose that this attenuated ubiquitination of spectrin may be due to glutathiolation of the E2 active site cysteine leading to diminished ubiquitin-spectrin adduct and conjugate formation. Furthermore we propose that lack of ubiquitin-spectrin complex formation leads to dysregulation of the membrane skeleton in mature SS erythrocytes and may diminish spectrin turnover in SS erythropoietic cells via the ubiquitin proteasome machinery. In hippocampal neurons, spectrin is the major ubiquitinated protein and a component of the cytoplasmic ubiquitinated inclusions observed in Alzheimer's and Parkinson's diseases. The two primary neuronal spectrin isoforms: alpha SpI Sigma*/beta SpI Sigma 2 and alpha SpII Sigma 1/beta SpII Sigma 1 are both ubiquitinated. Future work will resolve whether neuronal spectrins also contain E2-ubiquitin conjugating activity and the molecular basis for formation of ubiquitinated inclusions in neurological disorders.     

Structural insights into the conformation and oligomerization of E2~ubiquitin conjugates

Post-translational modification of proteins by ubiquitin (Ub) regulates a host of cellular processes, including protein quality control, DNA repair, endocytosis, and cellular signaling. In the ubiquitination cascade, a thioester-linked conjugate between the C-terminus of Ub and the active site cysteine of a ubiquitin-conjugating enzyme (E2) is formed. The E2~Ub conjugate interacts with a ubiquitin ligase (E3) to transfer Ub to a lysine residue on a target protein. The flexibly linked E2~Ub conjugates have been shown to form a range of structures in solution. In addition, select E2~Ub conjugates oligomerize through a noncovalent "backside" interaction between Ub and E2 components of different conjugates. Additional studies are needed to bridge the gap between the dynamic monomeric conjugates, E2~Ub oligomers, and the mechanisms of ubiquitination. We present a new 2.35 ? crystal structure of an oligomeric UbcH5c~Ub conjugate. The conjugate forms a staggered linear oligomer that differs substantially from the "infinite spiral" helical arrangement of the only previously reported structure of an oligomeric conjugate. Our structure also differs in intraconjugate conformation from other structurally characterized conjugates. Despite these differences, we find that the backside interaction mode is conserved in different conjugate oligomers and is independent of intraconjugate relative E2-Ub orientations. We delineate a common intraconjugate E2-binding surface on Ub. In addition, we demonstrate that an E3 CHIP (carboxyl terminus of Hsp70 interacting protein) interacts directly with UbcH5c~Ub oligomers, not only with conjugate monomers. These results provide insights into the conformational diversity of E2~Ub conjugates and conjugate oligomers, and into their compatibility and interactions with E3s, which have important consequences for the ubiquitination process.     

Noncovalent interaction between ubiquitin and the human DNA repair protein Mms2 is required for Ubc13-mediated polyubiquitination

Ubiquitin-conjugating enzyme variants share significant sequence similarity with typical E2 (ubiquitin-conjugating) enzymes of the protein ubiquitination pathway but lack their characteristic active site cysteine residue. The MMS2 gene of Saccharomyces cerevisiae encodes one such ubiquitin-conjugating enzyme variant that is involved in the error-free DNA postreplicative repair pathway through its association with Ubc13, an E2. The Mms2-Ubc13 heterodimer is capable of linking ubiquitin molecules to one another through an isopeptide bond between the C terminus and Lys-63. Using highly purified components, we show here that the human forms of Mms2 and Ubc13 associate into a heterodimer that is stable over a range of conditions. The ubiquitin-thiol ester form of the heterodimer can be produced by the direct activation of its Ubc13 subunit with E1 (ubiquitin-activating enzyme) or by the association of Mms2 with the Ubc13-ubiquitin thiol ester. The activated heterodimer is capable of transferring its covalently bound ubiquitin to Lys-63 of an untethered ubiquitin molecule, resulting in diubiquitin as the predominant species. In (1)H (15)N HSQC ((1)H (15)N heteronuclear single quantum coherence) NMR experiments, we have mapped the surface determinants of tethered and untethered ubiquitin that interact with Mms2 and Ubc13 in both their monomeric and dimeric forms. These results have identified a surface of untethered ubiquitin that interacts with Mms2 in the monomeric and heterodimeric form. Furthermore, the C-terminal tail of ubiquitin does not participate in this interaction. These results suggest that the role of Mms2 is to correctly orient either a target-bound or untethered ubiquitin molecule such that its Lys-63 is placed proximally to the C terminus of the ubiquitin molecule that is linked to the active site of Ubc13.     

Rbx1 Flexible Linker Facilitates Cullin-RING Ligase Function Before Neddylation and After Deneddylation

In ubiquitination, cullin-RING E3 ubiquitin ligases (CRLs) assist in ubiquitin transfer from ubiquitin-conjugating enzyme E2 to the substrate. Neddylation, which involves NEDD8 transfer from E2 to E3-cullin, stimulates ubiquitination by inducing conformational change in CRLs. However, deneddylation, which removes NEDD8 from cullin, does not suppress ubiquitination in vivo, raising the question of how neddylation/deneddylation exerts its effects. Using molecular-dynamics simulations, we demonstrate that before neddylation occurs, the linker flexibility of Rbx1, a CRL component, leads to conformational changes in CRLs that allow neddylation and initiation of ubiquitination. These large NEDD8-induced conformational changes are retained after deneddylation, allowing both initiation of the ubiquitination process and ubiquitin chain elongation after deneddylation. Furthermore, mutation of lysine, the cullin residue to which NEDD8 covalently attaches, dramatically reduces CRL conformational changes, suggesting that the acceptor lysine allosterically regulates CRLs. Thus, our results imply that neddylation stimulates ubiquitination by CRL conformational control via lysine modification.     

Essential role for ubiquitin-ubiquitin-conjugating enzyme interaction in ubiquitin discharge from Cdc34 to substrate

During ubiquitin conjugation, the thioester bond that links "donor" ubiquitin to ubiquitin-conjugating enzyme (E2) undergoes nucleophilic attack by the ?-amino group of an acceptor lysine, resulting in formation of an isopeptide bond. Models of ubiquitination have envisioned the donor ubiquitin to be a passive participant in this process. However, we show here that the I44A mutation in ubiquitin profoundly inhibits its ability to serve as a donor for ubiquitin chain initiation or elongation, but can be rescued by computationally predicted compensatory mutations in the E2 Cdc34. The donor defect of ubiquitin-I44A can be partially suppressed either by using a low pKa amine (hydroxylamine) as the acceptor or by performing reactions at higher pH, suggesting that the discharge defect arises in part due to inefficient deprotonation of the acceptor lysine. We propose that interaction between Cdc34 and the donor ubiquitin organizes the active site to promote efficient ubiquitination of substrate.     

In vivo action of the HRD ubiquitin ligase complex: mechanisms of endoplasmic reticulum quality control and sterol regulation

Ubiquitination is used to target both normal proteins for specific regulated degradation and misfolded proteins for purposes of quality control destruction. Ubiquitin ligases, or E3 proteins, promote ubiquitination by effecting the specific transfer of ubiquitin from the correct ubiquitin-conjugating enzyme, or E2 protein, to the target substrate. Substrate specificity is usually determined by specific sequence determinants, or degrons, in the target substrate that are recognized by the ubiquitin ligase. In quality control, however, a potentially vast collection of proteins with characteristic hallmarks of misfolding or misassembly are targeted with high specificity despite the lack of any sequence similarity between substrates. In order to understand the mechanisms of quality control ubiquitination, we have focused our attention on the first characterized quality control ubiquitin ligase, the HRD complex, which is responsible for the endoplasmic reticulum (ER)-associated degradation (ERAD) of numerous ER-resident proteins. Using an in vivo cross-linking assay, we directly examined the association of the separate HRD complex components with various ERAD substrates. We have discovered that the HRD ubiquitin ligase complex associates with both ERAD substrates and stable proteins, but only mediates ubiquitin-conjugating enzyme association with ERAD substrates. Our studies with the sterol pathway-regulated ERAD substrate Hmg2p, an isozyme of the yeast cholesterol biosynthetic enzyme HMG-coenzyme A reductase (HMGR), indicated that the HRD complex discerns between a degradation-competent "misfolded" state and a stable, tightly folded state. Thus, it appears that the physiologically regulated, HRD-dependent degradation of HMGR is effected by a programmed structural transition from a stable protein to a quality control substrate.     

Molecular characterization of the thermosensitive E1 ubiquitin-activating enzyme cell mutant A31N-ts20. Requirements upon different levels of E1 for the ubiquitination/degradation of the various protein substrates in vivo.

According to our current knowledge, protein ubiquitination involves three steps: activation of ubiquitin through formation of an energy-rich bond with an E1 ubiquitin-activating enzyme; and transfer of activated ubiquitin onto E2 ubiquitin-conjugating enzymes, which, in turn, alone, or in combination with E3 ubiquitin-protein ligase enzymes, transfer ubiquitin onto target proteins. A31N-ts20 cells are mouse embryo fibroblasts, thermosensitive for E1. We show here that: (a) the enzymatic activity of the enzyme is heat-inactivatable in vitro; and (b) a major mechanism responsible for E1 inactivation in vivo consists of accelerated destruction. Surprisingly, a >90% reduction in E1 abundance little alters the formation of the bulk of protein-ubiquitin conjugates when A31N-ts20 cells are grown at the nonpermissive temperature, indicating that cautious interpretation of results is required when studying ubiquitination of specific substrates using this cell line. Surprisingly, our data also indicate that, in vivo, ubiquitination of the various protein substrates in A31N-ts20 cells requires different amounts of E1, indicating that this mutant cell line can be used for unveiling the existence of differences in the intimate mechanisms responsible for the ubiquitination of the various cell proteins in vivo, and for providing criteria of reliability when developing in vitro ubiquitination assays for specific proteins.     

Structure of a c-Cbl-UbcH7 complex: RING domain function in ubiquitin-protein ligases

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.     

Solution structure of the ubiquitin-conjugating enzyme UbcH5B

The ubiquitination pathway is the main pathway for protein degradation in eukaryotic cells. The attachment of ubiquitin to a substrate protein is catalyzed by three types of enzymes, namely a ubiquitin activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3). Here, the structure of the human ubiquitin-conjugating enzyme (E2) UbcH5B has been solved by a combination of homology modeling, NMR relaxation data and automated NOE assignments. Comparison to E2 structures solved previously by X-ray crystallography or NMR shows in all cases the same compact fold, but differences are observed in the orientation of both N and C-terminal alpha-helices. The N-terminal helix that is involved in binding to ubiquitin ligases (E3) displays a different position, which could have consequences for precise E2-E3 recognition. In addition, multiple conformations of the side-chain of Asn77 are found in solution, which contrasts the single hydrogen-bonded conformation in the crystal structures of E2 enzymes. The possible implication of this conformational freedom of Asn77 for its catalytic function is discussed.     

A simple and high-sensitivity method for analysis of ubiquitination and polyubiquitination based on wheat cell-free protein synthesis

Background  

Ubiquitination is mediated by the sequential action of at least three enzymes: the E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme) and E3 (ubiquitin ligase) proteins. Polyubiquitination of target proteins is also implicated in several critical cellular processes. Although Arabidopsis genome research has estimated more than 1,300 proteins involved in ubiquitination, little is known about the biochemical functions of these proteins. Here we demonstrate a novel, simple and high-sensitive method for in vitro analysis of ubiquitination and polyubiquitination based on wheat cell-free protein synthesis and luminescent detection.     

Control of ubiquitination of proteins in rat tissues by ubiquitin conjugating enzymes and isopeptidases

The activity of the ubiquitin-dependent proteolytic system in differentiated tissues under basal conditions remains poorly explored. We measured rates of ubiquitination in rat tissue extracts. Accumulation of ubiquitinated proteins increased in the presence of ubiquitin aldehyde, indicating that deubiquitinating enzymes can regulate ubiquitination. Rates of ubiquitination varied fourfold, with the highest rate in the testis. We tested whether ubiquitin-activating enzyme (E1) or ubiquitin-conjugating enzymes (E2s) could be limiting for conjugation. Immunodepletion of the E2s UBC2 or UBC4 lowered rates of conjugation similarly. Supplementation of extracts with excess UBC2 or UBC4, but not E1, stimulated conjugation. However, UBC2-stimulated rates of ubiquitination still differed among tissues, indicating that tissue differences in E3s or substrate availability may also be rate controlling. UBC2 and UBC4 stimulated conjugation half-maximally at concentrations of 10-50 and 28-44 nM, respectively. Endogenous tissue levels of UBC2, but not UBC4, appeared saturating for conjugation, suggesting that in vivo modulation of UBC4 levels can likely control ubiquitin conjugation. Thus the pool of ubiquitin conjugates and therefore the rate of degradation of proteins by this system may be controlled by E2s, E3s, and isopeptidases. The regulation of the ubiquitin pathway appears complex, but precise.     

The APC11 RING-H2 finger mediates E2-dependent ubiquitination

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

Mdm2 is a RING finger-dependent ubiquitin protein ligase for itself and p53

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.     

Evidence for a particulate location of ubiquitin conjugates and ubiquitin-conjugating enzymes in rabbit brain.

Conjugate ubiquitin was previously found in the nucleus, cytoplasm, and membranes of eukaryotic cells while the enzymes of the ubiquitin-conjugating system appear to be cytoplasmic. We have prepared the mitochondrial fraction from rabbit brain by discontinuous density gradient ultracentrifugation and by Western blotting, using a specific antibody against conjugate ubiquitin, showing that it contains ubiquitin conjugates in a very wide molecular weight range. Electron microscopy and measurement of specific enzyme markers show that this fraction not only contains mitochondria but also some endoplasmic reticulum vesicles. Immunostaining with anti-ubiquitin IgG followed by immunodecoration with colloidal gold particles provides evidence for the presence of conjugate ubiquitin both in mitochondria and in the endoplasmic reticulum. Furthermore, this "mitochondrial fraction" shows a pronounced ATP-dependent ability to conjugate 125I-ubiquitin into a number of endogenous proteins as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Addition of E1, E2, and E3, the enzymes of the ubiquitin conjugating system purified from rabbit reticulocytes, does not further increase this ubiquitination nor incorporate 125I-ubiquitin into additional protein bands. The same mitochondrial fraction is not able to carry out any ATP-dependent degradation of 125I-albumin; however, it contains an isopeptidase activity able to release the covalently incorporated 125I-ubiquitin and is also able to conjugate 125I-ubiquitin to exogenous proteins as oxidized RNase. By affinity chromatography on ubiquitin-agarose of fraction II of a crude Triton X-100 extract of the mitochondrial fraction, several proteins corresponding in Mr to the E1 and E2s enzymes were obtained. These proteins were also able to form specific ubiquitin-thiol ester bounds on sodium dodecyl sulfate-polyacrylamide gels and to support 125I-ubiquitin conjugation to oxidized RNase. Detergent fractionation of the mitochondrial fraction provided evidence for a possible localization of the ubiquitin conjugating activity in the mitochondrial external membrane and endoplasmic reticulum. The presence of an active ubiquitin protein conjugating system in mitochondria and endoplasmic reticulum may be related to the turnover of organelle proteins as well as to specific cell functions such as import of proteins into mitochondria and ubiquitination of externally oriented membrane-bound proteins.     

Activation of UBC5 ubiquitin-conjugating enzyme by the RING finger of ROC1 and assembly of active ubiquitin ligases by all cullins

Protein ubiquitination plays an important role in regulating the abundance and conformation of a broad range of eukaryotic proteins. This process involves a cascade of enzymes including ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3). E1 and E2 represent two families of structurally related proteins and are relatively well characterized. In contrast, the nature and mechanism of E3, proposed to contain activities in catalyzing isopeptide bond formation (ubiquitin ligation) and substrate targeting, remains inadequately understood. Two major families of E3 ubiquitin ligases, the HECT (for homologous to E6-AP C terminus) family and the RING family, have been identified that utilize distinct mechanisms in promoting isopeptide bond formation. Here, we showed that purified RING finger domain of ROC1, an essential subunit of SKP1-cullin/CDC53-F box protein ubiquitin ligases, was sufficient to activate UBCH5c to synthesize polyubiquitin chains. The sequence flanking the RING finger in ROC1 did not contribute to UBCH5c activation, but was required for binding with CUL1. We demonstrated that all cullins, through their binding with ROC proteins, constituted active ubiquitin ligases, suggesting the existence in vivo of a large number of cullin-RING ubiquitin ligases. These results are consistent with the notion that the RING finger domains allosterically activate E2. We suggest that RING-E2, rather than cullin-RING, constitutes the catalytic core of the ubiquitin ligase and that one major function of the cullin subunit is to assemble the RING-E2 catalytic core and substrates together.     

ASB2 is an Elongin BC-interacting protein that can assemble with Cullin 5 and Rbx1 to reconstitute an E3 ubiquitin ligase complex

The ankyrin repeat-containing protein with a suppressor of cytokine signaling box-2 (ASB2) gene was identified as a retinoic acid-response gene and a target of the promyelocytic leukemia-retinoic acid receptor-alpha oncogenic protein characteristic of acute promyelocytic leukemia. Expression of ASB2 in myeloid leukemia cells inhibits growth and promotes commitment, recapitulating an early step known to be critical for differentiation. Here we show that ASB2, by interacting with the Elongin BC complex, can assemble with Cullin5.Rbx1 to form an E3 ubiquitin ligase complex that stimulates polyubiquitination by the E2 ubiquitin-conjugating enzyme Ubc5. This is a first indication that a member of the ASB protein family, ASB2, is a subunit of an ECS (Elongin C-Cullin-SOCS box)-type E3 ubiquitin ligase complex. Altogether, our results strongly suggest that ASB2 targets specific proteins to destruction by the proteasome in leukemia cells that have been induced to differentiate.     

Expression,purification and characterization of human ubiquitin-activating enzyme,UBE1

UBE1 plays an important role in the first step of ubiquitin–proteasome pathway to activate ubiquitin. Both the structure and biochemical property research of human UBE1 protein, and the activity analysis of those enzymes which are related with ubiquitination pathway, are based on high purity of UBE1 protein. To obtain human UBE1 protein, the full length of human UBE1 was expressed in E. coli and purified by Ni-NTA superflow sepharose and strep-tactin sepharose which based on UB–UBE1 high-energy thioester bonded intermediate complex. It was demonstrated that purified UBE1 could activate and conjugate UB to ubiquitin-conjugating enzyme E2s. The purified large amount of UBE1 could be used for in vitro studies of ubiquitin pathway and structural studies.     

The Protein Stability of Axin,a Negative Regulator of Wnt Signaling,Is Regulated by Smad Ubiquitination Regulatory Factor 2 (Smurf2)

Axin is a negative regulator of Wnt/β-catenin signaling via regulating the level of β-catenin, which is a key effector molecule. Therefore, controlling the level of Axin is a critical step for the regulation of Wnt/β-catenin signaling. It has been shown that ubiquitination-mediated proteasomal degradation may play a critical role in the regulation of Axin; however, the E3 ubiquitin ligase(s), which attaches ubiquitin to a target protein in combination with an E2 ubiquitin-conjugating enzyme, for Axin has not yet been identified. Here, we show that Smurf2 is an E3 ubiquitin ligase for Axin. Transient expression of Smurf2 down-regulated the level of Axin and increased the ubiquitination of Axin. Conversely, shRNA specific to Smurf2 blocked Axin ubiquitination. Essential domains of Axin responsible for Smurf2 interaction as well as Smurf2-mediated down-regulation and ubiquitination were identified. In vitro ubiquitination assays followed by analysis using mass spectroscopy revealed that Smurf2 specifically ubiquitinylated Lys⁵⁰⁵ of Axin and that the Axin(K505R) mutant resisted degradation. Knockdown of endogenous Smurf2 increased the level of endogenous Axin and resulted in reduced β-catenin/Tcf reporter activity. Overall, our data strongly suggest that Smurf2 is a genuine E3 ligase for Axin.     

Characterization of E3Histone, a novel testis ubiquitin protein ligase which ubiquitinates histones

During spermatogenesis, a large fraction of cellular proteins is degraded as the spermatids evolve to their elongated mature forms. In particular, histones must be degraded in early elongating spermatids to permit chromatin condensation. Our laboratory previously demonstrated the activation of ubiquitin conjugation during spermatogenesis. This activation is dependent on the ubiquitin-conjugating enzyme (E2) UBC4, and a testis-particular isoform, UBC4-testis, is induced when histones are degraded. Therefore, we tested whether there are UBC4-dependent ubiquitin protein ligases (E3s) that can ubiquitinate histones. Indeed, a novel enzyme, E3Histone, which could conjugate ubiquitin to histones H1, H2A, H2B, H3, and H4 in vitro, was found. Only the UBC4/UBC5 family of E2s supported E3Histone-dependent ubiquitination of histone H2A, and of this family, UBC4-1 and UBC4-testis are the preferred E2s. We purified this ligase activity 3,600-fold to near homogeneity. Mass spectrometry of the final material revealed the presence of a 482-kDa HECT domain-containing protein, which was previously named LASU1. Anti-LASU1 antibodies immunodepleted E3Histone activity. Mass spectrometry and size analysis by gel filtration and glycerol gradient centrifugation suggested that E3Histone is a monomer of LASU1. Our assays also show that this enzyme is the major UBC4-1-dependent histone-ubiquitinating E3. E3Histone is therefore a HECT domain E3 that likely plays an important role in the chromatin condensation that occurs during spermatid maturation.