Novel CDC34 (UBC3) ubiquitin-conjugating enzyme mutants obtained by charge-to-alanine scanning mutagenesis.

CDC34 (UBC3) encodes a ubiquitin-conjugating (E2) enzyme required for transition from the G1 phase to the S phase of the budding yeast cell cycle. CDC34 consists of a 170-residue catalytic N-terminal domain onto which is appended an acidic C-terminal domain. A portable determinant of cell cycle function resides in the C-terminal domain, but determinants for specific function must reside in the N-terminal domain as well. We have explored the utility of "charge-to-alanine" scanning mutagenesis to identify novel N-terminal domain mutants of CDC34 that are enzymatically competent with respect to unfacilitated (E3-independent) ubiquitination but that nevertheless are defective with respect to its cell cycle function. Such mutants may reveal determinants of specific in vivo function, such as those required for interaction with substrates or trans-acting regulators of activity and substrate selectivity. Three of 18 "single-scan" mutants (in which small clusters of charged residues were mutated to alanine) were compromised with respect to in vivo function. One mutant (cdc34-109, 111, 113A) targeted a 12-residue segment of the Cdc34 protein not found in most other E2s and was unable to complement a cdc34 null mutant at low copy numbers but could complement a null mutant when overexpressed from an induced GAL1 promoter. Combining adjacent pairs of single-scan mutants to produce "double-scan" mutants yielded four additional mutants, two of which showed heat and cold sensitivity conditional defects. Most of the mutant proteins expressed in Escheria coli displayed unfacilitated (E3-independent) ubiquitin-conjugating activity, but two mutants differed from wild-type and other mutant Cdc34 proteins in the extent of multiubiquitination they catalyzed during an autoubiquitination reation-conjugating enzyme function and have identified additional mutant alleles of CDC34 that will be valuable in further genetic and biochemical studies of Cdc34-dependent ubiquitination.     

The structure and function of RAD6 and RAD18 DNA repair genes of Saccharomyces cerevisiae

The RAD6 and RAD18 genes of Saccharomyces cerevisiae are required for postreplication repair of discontinuities occurring in newly synthesized DNA following exposure to uv light. In addition, rad6 mutants are highly defective in mutagenesis induced by uv and other DNA damaging agents and in sporulation. RAD6 encodes a protein of 172 amino acids with a highly acidic carboxyl terminus. Deletion of the carboxyl terminal 23 residues, 20 of which are acidic, has little or no effect on uv sensitivity or uv mutagenesis, but sporulation is greatly reduced. Addition of the first four residues of the polyacidic tail restores sporulation to 50% the level observed in RAD+/RAD+ diploids. RAD6 protein has been previously shown to be a ubiquitin-conjugating (E2) enzyme that attaches ubiquitin to histones H2A and H2B in vitro. Our experiments show that deletion of varying lengths of the polyacidic tail of RAD6 protein greatly reduces its ubiquitin-conjugating activity. The RAD18 encoded protein contains features which suggest that it binds DNA and nucleotides. Ten of the 12 cysteine residues occur in regions that could form zinc finger domains for nucleic acid binding. The other interesting feature in RAD18 protein is the presence of a putative nucleotide binding sequence. The possible in vivo functions of the RAD6 and RAD18 proteins are discussed.     

A site-directed approach for constructing temperature-sensitive ubiquitin-conjugating enzymes reveals a cell cycle function and growth function for RAD6.

We have determined the gene sequence of a temperature-sensitive allele of the cell cycle-related ubiquitin-conjugating enzyme CDC34 (UBC 3) from Saccharomyces cerevisiae. The basis of temperature sensitivity is a missense mutation resulting in a proline to serine substitution at a residue that is conserved in all ubiquitin-conjugating enzymes identified thus far. This observation raised the possibility that other temperature-sensitive ubiquitin-conjugating enzymes could be generated in the same way. We therefore created the corresponding substitution in the DNA repair-related ubiquitin-conjugating enzyme, RAD6 (UBC2), and examined the effect of temperature on the cell proliferation and DNA repair-related functions of this altered polypeptide. Yeast strains carrying this mutation proved to be temperature-sensitive with respect to cell proliferation but not with respect to the DNA damage-processing phenotypes exhibited by other rad6 mutants. Upon further investigation of the proliferation defect exhibited by this mutant, we discovered that other rad6 gene mutants deleted for the gene undergo cell cycle arrest at the nonpermissive temperature, whereas the engineered temperature-sensitive allele showed no evidence of a cell cycle defect. From these findings, we conclude that the proliferation function of RAD6 can be subdivided into a growth component and a cell division cycle component and that the growth component is unrelated to the DNA repair functions of RAD6. A reasonable interpretation of these results is that different proteins are targeted for ubiquitination in each case. The conserved proline residue of RAD6 and CDC34 is part of a turn motif common to all ubiquitin-conjugating enzymes. It is therefore likely that site-directed substitution of prolines located in turns can be generally applied for the creation of other temperature-sensitive ubiquitin-conjugating enzymes and possibly other proteins as well.     

RAD6 gene product of Saccharomyces cerevisiae requires a putative ubiquitin protein ligase (E3) for the ubiquitination of certain proteins.

The RAD6 (UBC2) gene of Saccharomyces cerevisiae which is involved in DNA repair, induced mutagenesis, and sporulation, encodes a ubiquitin-conjugating enzyme (E2). Since the RAD6 gene product can transfer ubiquitin directly to histones in vitro without the participation of a ubiquitin protein ligase (E3), it has been suggested that in vivo it also acts by the unassisted conjugation of ubiquitin to histones or to other target proteins. Here we show that the RAD6 protein can ligate ubiquitin in vitro to a hitherto unknown set of exogenous target proteins (alpha-, beta-, and kappa-casein and beta-lactoglobulin) when supplemented by a putative ubiquitin protein ligase (E3-R) from S. cerevisiae. RAD6 supplemented with E3-R ligates 1 or, sometimes, 2 ubiquitin molecules to the target protein molecule. UBC3 (CDC34) protein in the presence of E3-R has barely detectable activity on the non-histone substrates. Other ubiquitin-conjugating enzymes tested (products of the UBC1 and UBC4 genes) do not cooperate with E3-R in conjugating ubiquitin to the same substrates. Thus, E3-R apparently interacts selectively with RAD6 protein. These findings suggest that some of the in vivo activities of the RAD6 gene may involve E3-R.     

Identification of a portable determinant of cell cycle function within the carboxyl-terminal domain of the yeast CDC34 (UBC3) ubiquitin conjugating (E2) enzyme.

The ubiquitin conjugating (E2) enzyme encoded by CDC34 (UBC3) in Saccharomyces cerevisiae is required for the G1 to S transition of the cell cycle. CDC34 consists of a 170 residue amino-terminal domain that is homologous to that found in other E2s, followed by a 125 residue carboxyl-terminal domain that is specific to CDC34. We found that a truncation mutant of CDC34 which lacked the CDC34 carboxyl-terminal domain could not support the essential function of CDC34 in the cell cycle in vivo. To explore further the role of the carboxyl-terminal domain in determining the cell cycle function of CDC34, we constructed and characterized genes encoding chimeric E2s incorporating sequences from CDC34 and the related but functionally distinct E2 RAD6 (UBC2). We found that a construct encoding a chimeric RAD6-CDC34 ubiquitin conjugating enzyme, in which the 21 residue acidic carboxyl-terminal domain of RAD6 has been replaced with the 125 residue carboxyl-terminal domain of CDC34, performed the essential functions of CDC34 in vivo. This chimeric E2 also complemented the growth deficiency, UV sensitivity and sporulation deficiency of rad6 mutant strains. Deletion analysis of the CDC34 carboxyl-terminal domain in both CDC34 and the RAD6-CDC34 chimeric E2 identified a region comprising residues 171-244 of CDC34 that was sufficient to confer CDC34 function on the amino-terminal domains of CDC34 and RAD6. We suggest that this region interacts with substrates of CDC34 or with trans-acting factors (such as CDC34-specific ubiquitin protein ligases) that govern the substrate selectivity of CDC34. Congruent results demonstrating a positive role for the carboxyl-terminal domain of CDC34 in the essential function of CDC34 have also been obtained by Silver et al. (1992) and are reported in the accompanying paper.     

Creation of a pluripotent ubiquitin-conjugating enzyme

We describe the creation of a pluripotent ubiquitin-conjugating enzyme (E2) generated through a single amino acid substitution within the catalytic domain of RAD6 (UBC2). This RAD6 derivative carries out the stress-related function of UBC4 and the cell cycle function of CDC34 while maintaining its own DNA repair function. Furthermore, it carries out CDC34's function in the absence of the CDC34 carboxy-terminal extension. By using sequence and structural comparisons, the residues that define the unique functions of these three E2s were found on the E2 catalytic face partitioned to either side by a conserved divide. One of these patches corresponds to a binding site for both HECT and RING domain proteins, suggesting that a single substitution in the catalytic domain of RAD6 confers upon it the ability to interact with multiple ubiquitin protein ligases (E3s). Other amino acid substitutions made within the catalytic domain of RAD6 either caused loss of its DNA repair function or modified its ability to carry out multiple E2 functions. These observations suggest that while HECT and RING domain binding may generally be localized to a specific patch on the E2 surface, other regions of the functional E2 face also play a role in specificity. Finally, these data also indicate that RAD6 uses a different functional region than either UBC4 or CDC34, allowing it to acquire the functions of these E2s while maintaining its own. The pluripotent RAD6 derivative, coupled with sequence, structural, and phylogenetic data, suggests that E2s have diverged from a common multifunctional progenitor.     

Protein-protein interactions within an E2-RING finger complex. Implications for ubiquitin-dependent DNA damage repair

The RING finger protein RAD5 interacts and cooperates with the UBC13-MMS2 ubiquitin-conjugating enzyme in postreplication DNA damage repair in yeast. Previous observations implied that the function of UBC13 and MMS2 is dependent on the presence of RAD5, suggesting that the RING finger protein might act as a ubiquitin-protein ligase specific for the UBC13-MMS2 complex. In support of this notion it is shown here that the contact surfaces between the RAD5 RING domain and UBC13 correspond to those found in other pairs of ubiquitin-conjugating enzymes and ubiquitin-protein ligases. Mutations that compromise the protein-protein interactions either between the RING domain and UBC13 or within the UBC13-MMS2 dimer were found to have variable effects on repair activity in vivo that strongly depended on the expression levels of the corresponding mutants. Quantitative analysis of the affinity and kinetics of the UBC13-MMS2 interaction suggests a highly dynamic association model in which compromised mutual interactions result in phenotypic effects only under conditions where protein levels become limiting. Finally, this study demonstrates that beyond its cooperation with the UBC13-MMS2 dimer, RAD5 must have an additional role in DNA damage repair independent of its RING finger domain.     

Stable ester conjugate between the Saccharomyces cerevisiae RAD6 protein and ubiquitin has no biological activity.

The RAD6 gene of Saccharomyces cerevisiae, which encodes a ubiquitin-conjugating enzyme, is required for DNA repair, DNA damage-induced mutagenesis and sporulation. To evaluate the biological relevance of the thioester adduct between RAD6 protein and ubiquitin, formed as an obligatory, transient intermediate during ubiquitin conjugation to substrates, we altered cysteine 88 in RAD6 to serine. Esterification with ubiquitin occurs at serine 88 in the mutant protein, but conjugation of ubiquitin to the test substrate histone H2A is inactivated. Phenotypically, strains harboring the rad6 Ser88 allele are indistinguishable from rad6 deletion (rad6 delta) mutant cells. These findings argue against ligation of ubiquitin at cysteine 88 acting as a functional switch of a cryptic biochemical activity in RAD6.     

Role of the conserved carboxy-terminal α-helix of Rad6p in ubiquitination and DNA repair

RAD6 in the yeast Saccharomyces cerevisiae encodes a ubiquitin-conjugating enzyme essential for DNA repair as well as for a number of other biological processes. It is believed that the functions of Rad6p require the ubiquitination of target proteins, but its substrates as well as other interacting proteins are largely unknown. Rad6p homologues of higher eukaryotes have a number of amino acid residues in the C-terminal α-helix, which are conserved from yeast to man but are absent from most other yeast ubiquitin-conjugating enzymes (Ubcs). This specific conservation suggests that the C-terminal a-helix is important for the unique activities of the Rad6p family of Ubcs. We have investigated the effects of mutating this highly conserved region on the ubiquitination of model substrates in vitro and on error-free DNA repair in vivo. C-terminal point and deletion mutants of Rad6p differentially affected its in vitro activity on various substrates, raising the possibility that Rad6p interacts with its substrates in vivo by similar mechanisms. The distal part of the C-terminal u-helix is also essential for error-free DNA repair in vivo. Overexpression of Rad18p, a single-stranded DNA-binding protein that also interacts with Rad6p, alleviates the DNA repair defects of the C-terminal α-helix mutants to different degrees. This indicates that the C-terminal α-helix of Rad6p mediates its interaction with Rad18p, an essential step in DNA repair. Models of Rad6p action propose that its ubiquitination function is followed by proteolysis of unknown ubiquitinated targets. Mutants affecting several functions of the 26S proteasome retain wild-type capacity for error-free DNA repair. This raises the possibility that ubiquitination by Rad6p in DNA repair does not target proteins for proteasomal degradation.     

RAD6 Promotes Homologous Recombination Repair by Activating the Autophagy-Mediated Degradation of Heterochromatin Protein HP1

Efficient DNA double-strand break (DSB) repair is critical for the maintenance of genome stability. Unrepaired or misrepaired DSBs cause chromosomal rearrangements that can result in severe consequences, such as tumorigenesis. RAD6 is an E2 ubiquitin-conjugating enzyme that plays a pivotal role in repairing UV-induced DNA damage. Here, we present evidence that RAD6 is also required for DNA DSB repair via homologous recombination (HR) by specifically regulating the degradation of heterochromatin protein 1α (HP1α). Our study indicates that RAD6 physically interacts with HP1α and ubiquitinates HP1α at residue K154, thereby promoting HP1α degradation through the autophagy pathway and eventually leading to an open chromatin structure that facilitates efficient HR DSB repair. Furthermore, bioinformatics studies have indicated that the expression of RAD6 and HP1α exhibits an inverse relationship and correlates with the survival rate of patients.     

Ubiquitin-conjugating enzymes UBC4 and UBC5 mediate selective degradation of short-lived and abnormal proteins.

Ubiquitin-conjugating enzymes catalyse the covalent attachment of ubiquitin to target proteins. Members of this enzyme family are involved in strikingly diverse cellular functions: UBC2 (RAD6) is central to DNA repair, UBC3 (CDC34) is involved in cell cycle control. We have cloned the genes for two novel ubiquitin-conjugating enzymes, UBC4 and UBC5, from the yeast Saccharomyces cerevisiae. These enzymes mediate selective degradation of short-lived and abnormal proteins. UBC4 and UBC5 are closely related in sequence and complementing in function. Expression of UBC4 and UBC5 genes is heat inducible. UBC4 and UBC5 enzymes generate high mol. wt ubiquitin-protein conjugates in vivo consistent with previous studies which suggested that attachment of multiple ubiquitin molecules to proteolytic substrates is required for their selective degradation. UBC4 and UBC5 enzymes comprise a major part of total ubiquitin-conjugation activity in stressed cells. Turnover of short-lived proteins and canavanyl-peptides but not of long-lived proteins is markedly reduced in ubc4ubc5 mutants. Loss of UBC4 and UBC5 activity impairs cell growth, leads to inviability at elevated temperatures or in the presence of an amino acid analog, and induces the stress response.     

Determinants of RING-E2 fidelity for Hrd1p, a membrane-anchored ubiquitin ligase

A critical aspect of E3 ubiquitin ligase function is the selection of a particular E2 ubiquitin-conjugating enzyme to accomplish ubiquitination of a substrate. We examined the requirements for correct E2-E3 specificity in the RING-H2 ubiquitin ligase Hrd1p, an ER-localized protein known to use primarily Ubc7p for its function. Versions of Hrd1p containing the RING motif from homologous E3s were unable to carry out Hrd1p function, revealing a requirement for the specific Hrd1p RING motif in vivo. An in vitro assay revealed that these RING motifs were sufficient to function as ubiquitin ligases, but that they did not display the E2 specificity predicted from in vivo results. We further refined the in vitro assay of Hrd1p function by demanding not only ubiquitin ligase activity, but also specific activity that recapitulated both the E2 specificity and RING selectivity observed in vivo. Doing so revealed that correct E2 engagement by Hrd1p required the presence of portions of the Hrd1p soluble cytoplasmic domain outside the RING motif, the placement of the Hrd1p ubiquitin ligase in the ER membrane, and presentation of Ubc7p in the cytosolic context. We confirmed that these conditions supported the ubiquitination of Hrd1p itself, and the transfer of ubiquitin to the prototype substrate Hmg2p-GFP, validating Hrd1p self-ubiquitination as a viable assay of ligase function.     

Requirement of RAD5 and MMS2 for postreplication repair of UV-damaged DNA in Saccharomyces cerevisiae

UV lesions in the template strand block the DNA replication machinery. Genetic studies of the yeast Saccharomyces cerevisiae have indicated the requirement of the Rad6-Rad18 complex, which contains ubiquitin-conjugating and DNA-binding activities, in the error-free and mutagenic modes of damage bypass. Here, we examine the contributions of the REV3, RAD30, RAD5, and MMS2 genes, all of which belong to the RAD6 epistasis group, to the postreplication repair of UV-damaged DNA. Discontinuities, which are formed in DNA strands synthesized from UV-damaged templates, are not repaired in the rad5Delta and mms2Delta mutants, thus indicating the requirement of the Rad5 protein and the Mms2-Ubc13 ubiquitin-conjugating enzyme complex in this repair process. Some discontinuities accumulate in the absence of RAD30-encoded DNA polymerase eta (Poleta) but not in the absence of REV3-encoded DNA Polzeta. We concluded that replication through UV lesions in yeast is mediated by at least three separate Rad6-Rad18-dependent pathways, which include mutagenic translesion synthesis by Polzeta, error-free translesion synthesis by Poleta, and postreplication repair of discontinuities by a Rad5-dependent pathway. We suggest that newly synthesized DNA possessing discontinuities is restored to full size by a "copy choice" type of DNA synthesis which requires Rad5, a DNA-dependent ATPase, and also PCNA and Poldelta. The possible roles of the Rad6-Rad18 and the Mms2-Ubc13 enzyme complexes in Rad5-dependent damage bypass are discussed.     

A novel ubiquitination factor, E4, is involved in multiubiquitin chain assembly

Proteins modified by multiubiquitin chains are the preferred substrates of the proteasome. Ubiquitination involves a ubiquitin-activating enzyme, E1, a ubiquitin-conjugating enzyme, E2, and often a substrate-specific ubiquitin-protein ligase, E3. Here we show that efficient multiubiquitination needed for proteasomal targeting of a model substrate requires an additional conjugation factor, named E4. This protein, previously known as UFD2 in yeast, binds to the ubiquitin moieties of preformed conjugates and catalyzes ubiquitin chain assembly in conjunction with E1, E2, and E3. Intriguingly, E4 defines a novel protein family that includes two human members and the regulatory protein NOSA from Dictyostelium required for fruiting body development. In yeast, E4 activity is linked to cell survival under stress conditions, indicating that eukaryotes utilize E4-dependent proteolysis pathways for multiple cellular functions.     

Two different classes of E2 ubiquitin-conjugating enzymes are required for the mono-ubiquitination of proteins and elongation by polyubiquitin chains with a specific topology

RING (really interesting new gene) and U-box E3 ligases bridge E2 ubiquitin-conjugating enzymes and substrates to enable the transfer of ubiquitin to a lysine residue on the substrate or to one of the seven lysine residues of ubiquitin for polyubiquitin chain elongation. Different polyubiquitin chains have different functions. Lys(48)-linked chains target proteins for proteasomal degradation, and Lys(63)-linked chains function in signal transduction, endocytosis and DNA repair. For this reason, chain topology must be tightly controlled. Using the U-box E3 ligase CHIP [C-terminus of the Hsc (heat-shock cognate) 70-interacting protein] and the RING E3 ligase TRAF6 (tumour-necrosis-factor-receptor-associated factor 6) with the E2s Ubc13 (ubiquitin-conjugating enzyme 13)-Uev1a (ubiquitin E2 variant 1a) and UbcH5a, in the present study we demonstrate that Ubc13-Uev1a supports the formation of free Lys(63)-linked polyubiquitin chains not attached to CHIP or TRAF6, whereas UbcH5a catalyses the formation of polyubiquitin chains linked to CHIP and TRAF6 that lack specificity for any lysine residue of ubiquitin. Therefore the abilities of these E2s to ubiquitinate a substrate and to elongate polyubiquitin chains of a specific topology appear to be mutually exclusive. Thus two different classes of E2 may be required to attach a polyubiquitin chain of a particular topology to a substrate: the properties of one E2 are designed to mono-ubiquitinate a substrate with no or little inherent specificity for an acceptor lysine residue, whereas the properties of the second E2 are tailored to the elongation of a polyubiquitin chain using a defined lysine residue of ubiquitin.     

Rice ubiquitin-conjugating enzyme OsUBC26 is essential for immunity to the blast fungus Magnaporthe oryzae

The functions of ubiquitin-conjugating enzymes (E2) in plant immunity are not well understood. In this study, OsUBC26, a rice ubiquitin-conjugating enzyme, was characterized in the defence against Magnaporthe oryzae. The expression of OsUBC26 was induced by M. oryzae inoculation and methyl jasmonate treatment. Both RNA interference lines and CRISPR/Cas9 null mutants of OsUBC26 reduced rice resistance to M. oryzae. WRKY45 was down-regulated in OsUBC26 null mutants. In vitro E2 activity assay indicated that OsUBC26 is an active ubiquitin-conjugating enzyme. Yeast two-hybrid assays using OsUBC26 as bait identified the RING-type E3 ligase UCIP2 as an interacting protein. Coimmunoprecipitation assays confirmed the interaction between OsUBC26 and UCIP2. The CRISPR/Cas9 mutants of UCIP2 also showed compromised resistance to M. oryzae. Yeast two-hybrid screening using UCIP2 as bait revealed that APIP6 is a binding partner of UCIP2. Moreover, OsUBC26 working with APIP6 ubiquitinateds AvrPiz-t, an avirulence effector of M. oryzae, and OsUBC26 null mutation impaired the proteasome degradation of AvrPiz-t in rice cells. In summary, OsUBC26 plays important roles in rice disease resistance by regulating WRKY45 expression and working with E3 ligases such as APIP6 to counteract the effector protein AvrPiz-t from M. oryzae.     

Asymmetric nature of two subunits of RAD18, a RING-type ubiquitin ligase E3, in the human RAD6A-RAD18 ternary complex

RAD18, a RING-type ubiquitin ligase (E3) that plays an essential role in post-replication repair, possesses distinct domains named RING, UBZ, SAP and the RAD6-binding domain (R6BD) and forms a dimer. RAD6, an ubiquitin-conjugating enzyme (E2), stably associates with R6BD in the C-terminal portion. In this study, we established a method to distinguish between the two subunits of RAD18 by introduction of different tags, and analyzed mutant complexes. Our results, surprisingly, demonstrate that RAD6A and RAD18 form a ternary complex, RAD6A-(RAD18)(2) and the presence of only one R6BD in the two RAD18 subunits is sufficient for ternary complex formation and the ligase activity. Interestingly, ligase activity of a mutant dimer lacking both R6BDs is not restored even with large amounts of RAD6A added in solution, suggesting a requirement for precise juxtaposition via interaction with R6BD. We further show that mutations in both subunits of either RING or SAP, but not UBZ, strongly reduce ligase activity, although inactivation in only one of two subunits is without effect. These results suggest an asymmetric nature of the two RAD18 subunits in the complex.