Yeast RAD6 encoded ubiquitin conjugating enzyme mediates protein degradation dependent on the N-end-recognizing E3 enzyme.

The RAD6 gene of Saccharomyces cerevisiae encodes a 20 kd ubiquitin conjugating (E2) enzyme that is required for DNA repair, DNA damage-induced mutagenesis, and sporulation. Here, we demonstrate a novel activity of RAD6 protein--its ability to mediate protein degradation dependent on the N-end-recognizing ubiquitin protein ligase (E3). In reaction mixtures containing E1, E3 and the ubiquitin specific protease from rabbit reticulocytes, RAD6 is as effective as mammalian E214k in E3 dependent ubiquitin--protein conjugate formation and subsequent protein degradation. The ubiquitin conjugating activity of RAD6 is required for these reactions as indicated by the ineffectiveness of the rad6 Ala88 and rad6 Val88 mutant proteins, which lack the ability to form a thioester adduct with ubiquitin and therefore do not conjugate ubiquitin to substrates. We also show that the highly acidic carboxyl-terminus of RAD6 is dispensable for the interaction with E3, and that purified S. cerevisiae E2(30k), product of the UBC1 gene, does not function with E3. These findings demonstrate a specific interaction between RAD6 and E3, and highlight the strong conservation of the ubiquitin conjugating system in eukaryotes. We suggest a function for RAD6 mediated E3 dependent protein degradation in sporulation, and discuss the possible role of this activity during vegetative growth.     

Ubiquitin conjugation by the yeast RAD6 and CDC34 gene products. Comparison to their putative rabbit homologs, E2(20K) AND E2(32K).

The recombinant yeast RAD6 and CDC34 gene products were expressed in Escherichia coli extracts and purified to apparent homogeneity. The physical and catalytic properties of RAD6 and CDC34 were similar but distinct from their putative rabbit reticulocyte homologs, E2(20k) and E2(32k), respectively. Like their reticulocyte counterparts, RAD6 and CDC34 are bifunctional enzymes competent in both ubiquitin:protein ligase (E3)-independent and E3-dependent conjugation reactions. RAD6 and E2(20k) exhibit marked specificity for the conjugation of core histones and catalyze the processive ligation of up to three ubiquitin moieties directly to such model substrates. RAD6 differed from its putative E2(20k) homolog in exhibiting simple saturation behavior in the kinetics of histone conjugation and in being unable to distinguish kinetically between core histones H2A and H2B, yielding identical values of kcat (1.9 min-1) and Km (20 microM). A slow rate of multiubiquitination involving formation of extended ubiquitin homopolymers on the histones was also observed with RAD6 and E2(20k). Comparison of conjugate patterns among native, reductively methylated, and K48R ubiquitin variants demonstrated that the linkage between ubiquitin moieties formed by E2(20k) and RAD6 was not through Lys-48 of ubiquitin, the site previously demonstrated as a strong signal for degradation of the target protein. In contrast, CDC34 differs from its putative homolog, E2(32k), in showing a specificity for conjugation to bovine serum albumin rather than to core histones. Both CDC34 and E2(32k) exhibit a marked kinetic selectivity for processive multiubiquitination via Lys-48 of ubiquitin. Calculations based on a model ubiquitin conjugation reaction indicated that E2(32k) and CDC34 preferentially catalyzed multiubiquitination over ligation of the polypeptide directly to target proteins. Formation of such multiubiquitin homopolymers by E2(32k) and CDC34 suggests these enzymes may commit their respective target proteins to degradation via an E3-independent pathway.     

Cloning of a 16-kDa ubiquitin carrier protein from wheat and Arabidopsis thaliana. Identification of functional domains by in vitro mutagenesis.

Ubiquitin carrier proteins (E2s) are involved in the covalent attachment of ubiquitin to a variety of cellular target proteins in eukaryotes. Here, we report the cloning of genes from wheat and Arabidopsis thaliana that encode 16-kDa E2s and a domain analysis of E2s by in vitro mutagenesis. The genes for E216kDa, which we have designated wheat and At UBC1, encode proteins that are only 33% identical (58% similar) with a 23-kDa E2 from wheat (encoded by the gene now designated wheat UBC4), but are 63% identical (82% similar) with the E2 encoded by the Saccharomyces cerevisiae DNA repair gene, RAD6. Unlike the proteins encoded by RAD6 and wheat UBC4, the UBC1 gene products lack acidic C-terminal domains extending beyond the conserved core of the proteins and are incapable of efficient in vitro ligation of ubiquitin to histones. From enzymatic analysis of the UBC1 and UBC4 gene products mutagenized in vitro, we have identified several domains important for E2 function, including the active site cysteine and N-terminal and C-terminal domains. Cysteine residues 88 and 85 in the UBC1 and UBC4 gene products, respectively, are necessary for formation of the ubiquitin-E2 thiol ester intermediate. Whereas the UBC1 gene product does not require its additional cysteine residue at position 116 for thiol ester formation, alteration of cysteine 143 in the UBC4 gene product greatly diminishes this ability. The N terminus of UBC1 contains two domains that affect activity: a proximal region containing hydroxylated and uncharged residues whose removal increases the rate of thiol ester formation and a distal tract rich in basic residues. Deletion or substitution of these basic residues with neutral residues diminishes the rate of thiol ester formation. We have demonstrated also that C-terminal extensions can function to confer substrate specificity to E2s. When the acidic extension was deleted from UBC4, the protein was unable to efficiently conjugate ubiquitin to histones in vitro. Furthermore, fusion of the UBC4 acidic extension to the C terminus of UBC1 resulted in a chimeric protein capable of efficient histone conjugation, as did fusion of short tracts of alternating aspartate and glutamate residues. This result suggests that the target protein specificity of E2s can be altered by the addition of appropriate C-terminal extensions, thus providing a way to modify the selectivity of the ubiquitin system.     

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.     

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.     

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.     

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.     

Regulation of the ubiquitin-conjugating enzyme hHR6A by CDK-mediated phosphorylation

Cell cycle progression in eukaryotes is mediated by phosphorylation of protein substrates by the cyclin-dependent kinases (CDKs). We screened a cDNA library by solid-phase phosphorylation and isolated hHR6A as a CDK2 substrate. hHR6A is the human homologue of the product of the Saccharomyces cerevisiae RAD6/UBC2 gene, a member of the family of ubiquitin-conjugating enzymes. hHR6A is phosphorylated in vitro by CDK-1 and -2 on Ser120, a residue conserved in all hHR6A homologues, resulting in a 4-fold increase in its ubiquitin-conjugating activity. In vivo, hHR6A phosphorylation peaks during the G2/M phase of cell cycle transition, with a concomitant increase in histone H2B ubiquitylation. Mutation of Ser120 to threonine or alanine abolished hHR6A activity, while mutation to aspartate to mimic phosphorylated serine increased hHR6A activity 3-fold. Genetic complementation studies in S.cerevisiae demonstrated that hHR6A Ser120 is critical for cellular proliferation. This is the first study to demonstrate regulation of UBC function by phosphorylation on a conserved residue and suggests that CDK-mediated phosphorylation of hHR6A is an important regulatory event in the control of cell cycle progression.     

Erythrocyte spectrin is an E2 ubiquitin conjugating enzyme

The involvement of red blood cell spectrin in the ubiquitination process was studied. Spectrin was found to form two ubiquitin-associated derivatives, a DTT-sensitive ubiquitin adduct and a DTT-insensitive conjugate, characteristic intermediate and final products of the ubiquitination reaction cascade. In addition to spectrin and ubiquitin, ubiquitin-activating enzyme (E1) and ATP were necessary and sufficient to form both the spectrin-ubiquitin adduct and conjugate. No exogenous ubiquitin-conjugating (E2) or ligase (E3) activities were required, suggesting that erythrocyte spectrin is an E2 ubiquitin-conjugating enzyme able to target itself. Both ubiquitin adduct and conjugate were linked to the alpha subunit of spectrin, suggesting that the ubiquitin-conjugating (UBC) domain and its target regions reside on the same subunit.     

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.     

Human HRD1 is an E3 ubiquitin ligase involved in degradation of proteins from the endoplasmic reticulum

The ubiquitin system plays an important role in endoplasmic reticulum (ER)-associated degradation of proteins that are misfolded, that fail to associate with their oligomerization partners, or whose levels are metabolically regulated. E3 ubiquitin ligases are key enzymes in the ubiquitination process as they recognize the substrate and facilitate coupling of multiple ubiquitin units to the protein that is to be degraded. The Saccharomyces cerevisiae ER-resident E3 ligase Hrd1p/Der3p functions in the metabolically regulated degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and additionally facilitates the degradation of a number of misfolded proteins from the ER. In this study we characterized the structure and function of the putative human orthologue of yeast Hrd1p/Der3p, designated human HRD1. We show that human HRD1 is a non-glycosylated, stable ER protein with a cytosolic RING-H2 finger domain. In the presence of the ubiquitin-conjugating enzyme UBC7, the RING-H2 finger has in vitro ubiquitination activity for Lys(48)-specific polyubiquitin linkage, suggesting that human HRD1 is an E3 ubiquitin ligase involved in protein degradation. Human HRD1 appears to be involved in the basal degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase but not in the degradation that is regulated by sterols. Additionally we show that human HRD1 is involved in the elimination of two model ER-associated degradation substrates, TCR-alpha and CD3-delta.     

Identification of determinants in E2 ubiquitin-conjugating enzymes required for hect E3 ubiquitin-protein ligase interaction

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.     

A novel protein modification pathway related to the ubiquitin system.

Ubiquitin conjugation is known to target protein substrates primarily to degradation by the proteasome or via the endocytic route. Here we describe a novel protein modification pathway in yeast which mediates the conjugation of RUB1, a ubiquitin-like protein displaying 53% amino acid identity to ubiquitin. We show that RUB1 conjugation requires at least three proteins in vivo. ULA1 and UBA3 are related to the N- and C-terminal domains of the E1 ubiquitin-activating enzyme, respectively, and together fulfil E1-like functions for RUB1 activation. RUB1 conjugation also requires UBC12, a protein related to E2 ubiquitin-conjugating enzymes, which functions analogously to E2 enzymes in RUB1-protein conjugate formation. Conjugation of RUB1 is not essential for normal cell growth and appears to be selective for a small set of substrates. Remarkably, CDC53/cullin, a common subunit of the multifunctional SCF ubiquitin ligase, was found to be a major substrate for RUB1 conjugation. This suggests that the RUB1 conjugation pathway is functionally affiliated to the ubiquitin-proteasome system and may play a regulatory role.     

A rabbit reticulocyte ubiquitin carrier protein that supports ubiquitin-dependent proteolysis (E214k) is homologous to the yeast DNA repair gene RAD6.

The two isoforms of the 14-kDa ubiquitin carrier protein (E2(14k)) are unique among rabbit E2s in efficiently supporting ubiquitin-protein ligase (E3)-mediated ubiquitination of proteins destined for degradation. To begin determining the structural basis for this property, we have isolated a cDNA encoding the predominant reticulocyte isoform of the E2 from a rabbit skeletal muscle library. The sequence predicts a protein of 152 amino acids with a molecular weight of 17,293. Expression of the cDNA in Escherichia coli and purification of the recombinant protein revealed an E2 with high affinity for E3 and ubiquitin activating enzyme (E1). The latter high affinity interaction appears to be between the ubiquitin charged form of E1 and the uncharged form of E2 and does not result in a stable complex between these two enzymes. The predicted sequence shows regions of strong homology with other sequenced E2s, suggesting that these regions may be involved in binding to E1 and/or in ubiquitin transfer from E1, functions common to all E2s. Surprisingly, the E2(14k)) sequence is markedly more similar to Saccharomyces cerevisiae RAD6 (69% identity) than to its proposed homologs UBC4/UBC5 (38% identity). The sequence is identical to that recently reported for a human 17-kDa E2 which can complement rad6 mutants thereby identifying rabbit E2(14k) as a RAD6 homologue. The biochemical properties of this previously uncharacterized human 17-kDa E2 are now defined and its misassignment as a homologue of rabbit E2(17k) is corrected. Our findings resolve current confusion regarding relationships among E2s and define yeast RAD6, rabbit E2(14k), and the human 17-kDa E2 as a subclass of E2s which biochemically support E3-mediated conjugation and ubiquitin-dependent proteolysis and physiologically play a role in DNA repair.     

Cloning and characterization of a Saccharomyces cerevisiae gene encoding a new member of the ubiquitin-conjugating protein family

Ubiquitin-conjugating enzymes (E2s), which participate in the post-translational conjugation of ubiquitin to proteins, are encoded by a multigene family in the yeast Saccharomyces cerevisiae. E2s function in a variety of cellular activities including intracellular proteolysis, DNA repair, sporulation, and cell cycle traverse. Here, we report the cloning and characterization of a new member of the yeast UBC gene family, UBC8. UBC8 encodes a 206-amino acid protein containing a highly acidic carboxyl terminus. The primary structure of the protein is similar to that of all other known E2s, with the highest homology being to the E2 (23 kDa) of wheat germ. Haploid strains in which the UBC8 gene is disrupted are viable, and the disruption does not produce any obvious phenotype. The UBC8 protein, produced in Escherichia coli, forms thiol ester adducts with ubiquitin and, apparently, diubiquitin, but does not transfer ubiquitin to histones.     

A ubiquitin conjugating enzyme encoded by African swine fever virus.

The post-translational modification of proteins by covalent attachment of ubiquitin occurs in all eukaryotes by a multi-step process. A family of E2 or ubiquitin conjugating (UBC) enzymes catalyse one step of this process and these have been implicated in several diverse regulatory functions. We report here the sequence of a gene encoded by African swine fever virus (ASFV) which has high homology with UBC enzymes. This ASFV encoded enzyme has UBC activity when expressed in Escherichia coli since it forms thiolester bonds with [125I]ubiquitin in the presence of purified ubiquitin activating enzyme (E1) and ATP, and subsequently transfers [125I]ubiquitin to specific protein substrates. These substrates include histones, ubiquitin and the UBC enzyme itself. The ASFV encoded UBC enzyme is similar in structure and enzyme activity to the yeast ubiquitin conjugating enzymes UBC2 and UBC3. This is the first report of a virus encoding a functionally active UBC enzyme and provides an example of the exploitation of host regulatory mechanisms by viruses.     

Human SHPRH suppresses genomic instability through proliferating cell nuclear antigen polyubiquitination

Differential modifications of proliferating cell nuclear antigen (PCNA) determine DNA repair pathways at stalled replication forks. In yeast, PCNA monoubiquitination by the ubiquitin ligase (E3) yRad18 promotes translesion synthesis (TLS), whereas the lysine-63-linked polyubiquitination of PCNA by yRad5 (E3) promotes the error-free mode of bypass. The yRad5-dependent pathway is important to prevent genomic instability during replication, although its exact molecular mechanism is poorly understood. This mechanism has remained totally elusive in mammals because of the lack of apparent RAD5 homologues. We report that a putative tumor suppressor gene, SHPRH, is a human orthologue of yeast RAD5. SHPRH associates with PCNA, RAD18, and the ubiquitin-conjugating enzyme UBC13 (E2) and promotes methyl methanesulfonate (MMS)-induced PCNA polyubiquitination. The reduction of SHPRH by stable short hairpin RNA increases sensitivity to MMS and enhances genomic instability. Therefore, the yRad5/SHPRH-dependent pathway is a conserved and fundamental DNA repair mechanism that protects the genome from genotoxic stress.     

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.     

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.     

Characterization of novel yeast RAD6 (UBC2) ubiquitin-conjugating enzyme mutants constructed by charge-to-alanine scanning mutagenesis.

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.