Link between the ubiquitin conjugation system and the ISG15 conjugation system: ISG15 conjugation to the UbcH6 ubiquitin E2 enzyme

ISG15 is a ubiquitin-like protein that is upregulated on treatment with interferon. ISG15 is considered to be covalently conjugated to cellular proteins through a sequential reaction similar to that of the ubiquitin conjugation system consisting of E1/E2/E3 enzymes: UBE1L and UbcH8 have been reported to function as E1 and E2 enzymes, respectively, for ISG15 conjugation. Several cellular proteins have been identified as targets for ISG15 conjugation, but the roles of ISG15 conjugation remain unclear. In this study, we found that UbcH6 and UbcH8, E2 enzymes for ubiquitin conjugation, are covalently modified by ISG15. We also found that UbcH6 is capable of forming a thioester intermediate with ISG15 through Cys131. We determined that the Lys136 residue near the catalytic site Cys131 is the ISG15 conjugation site in UbcH6. We isolated ISG15-modified and unmodified UbcH6 proteins, and analyzed their abilities to form thioester intermediates with ubiquitin. A ubiquitin thioester intermediate was detected in the case of unmodified UbcH6, but not in that of ISG15-modified UbcH6, strongly suggesting that ISG15 conjugation to UbcH6 suppresses its ubiquitin E2 enzyme activity. Thus, we provide evidence for a link between the ubiquitin conjugation system and the ISG15 conjugation system.     

The ubiquitin-conjugating enzymes UbcH7 and UbcH8 interact with RING finger/IBR motif-containing domains of HHARI and H7-AP1.

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.     

E2-c-Cbl recognition is necessary but not sufficient for ubiquitination activity

The E2 ubiquitin-conjugating enzymes UbcH7 and UbcH5B both show specific binding to the RING (really interesting new gene) domain of the E3 ubiquitin-protein ligase c-Cbl, but UbcH7 hardly supports ubiquitination of c-Cbl and substrate in a reconstituted system. Here, we found that neither structural changes nor subtle differences in the E2-E3 interaction surface are possible explanations for the functional specificity of UbcH5B and UbcH7 in their interaction with c-Cbl. The quick transfer of ubiquitin from the UbcH5B∼Ub thioester to c-Cbl or other ubiquitin acceptors suggests that UbcH5B might functionally be a relatively pliable E2 enzyme. In contrast, the UbcH7∼Ub thioester is too stable to transfer ubiquitin under our assay conditions, indicating that UbcH7 might be a more specific E2 enzyme. Our results imply that the interaction specificity between c-Cbl and E2 is required but not sufficient for transfer of ubiquitin to potential targets.     

UBE1L2, a novel E1 enzyme specific for ubiquitin

UBE1 is known as the human ubiquitin-activating enzyme (E1), which activates ubiquitin in an ATP-dependent manner. Here, we identified a novel human ubiquitin-activating enzyme referred to as UBE1L2, which also shows specificity for ubiquitin. The UBE1L2 sequence displays a 40% identity to UBE1 and also contains an ATP-binding domain and an active site cysteine conserved among E1 family proteins. UBE1L2 forms a covalent link with ubiquitin in vitro and in vivo, which is sensitive to reducing conditions. In an in vitro polyubiquitylation assay, recombinant UBE1L2 could activate ubiquitin and transfer it onto the ubiquitin-conjugating enzyme UbcH5b. Ubiquitin activated by UBE1L2 could be used for ubiquitylation of p53 by MDM2 and supported the autoubiquitylation of the E3 ubiquitin ligases HectH9 and E6-AP. The UBE1L2 mRNA is most abundantly expressed in the testis, suggesting an organ-specific regulation of ubiquitin activation.     

The N-Terminal Extension of UBE2E Ubiquitin-Conjugating Enzymes Limits Chain Assembly

Protein ubiquitylation depends upon the concerted action of ubiquitin-conjugating enzymes (E2s) and ubiquitin ligases (E3s). All E2s have a conserved ubiquitin-conjugating (UBC) domain but many have variable extensions N- and C-terminal to the UBC domain. For many E2s, the function of the extension is not well understood. Here, we show that the N-terminal extension of the UBE2E proteins regulates formation of polyubiquitin chains by the processive UBC domain. Target proteins are therefore monoubiquitylated by full-length UBE2E, whereas the UBC domain alone polyubiquitylates proteins. Although the N-terminal extension of UBE2E1 is largely disordered in solution, these residues have a critical role in limiting chain building, and when fused to the highly processive E2, UBE2D2, ubiquitylation is limited. For some E2s, interaction of ubiquitin with the ‘backside’ of the UBC domain promotes polyubiquitylation. However, interaction of ubiquitin with the backside of the UBC domain of UBE2E1 does not appear to be important for processivity. This study underscores the importance of studying full-length E2 proteins and not just the highly conserved core domain.     

E2~Ub conjugates regulate the kinase activity of Shigella effector OspG during pathogenesis

Pathogenic bacteria introduce effector proteins directly into the cytosol of eukaryotic cells to promote invasion and colonization. OspG, a Shigella spp. effector kinase, plays a role in this process by helping to suppress the host inflammatory response. OspG has been reported to bind host E2 ubiquitin‐conjugating enzymes activated with ubiquitin (E2~Ub), a key enzyme complex in ubiquitin transfer pathways. A co‐crystal structure of the OspG/UbcH5c~Ub complex reveals that complex formation has important ramifications for the activity of both OspG and the UbcH5c~Ub conjugate. OspG is a minimal kinase domain containing only essential elements required for catalysis. UbcH5c~Ub binding stabilizes an active conformation of the kinase, greatly enhancing OspG kinase activity. In contrast, interaction with OspG stabilizes an extended, less reactive form of UbcH5c~Ub. Recognizing conserved E2 features, OspG can interact with at least ten distinct human E2s~Ub. Mouse oral infection studies indicate that E2~Ub conjugates act as novel regulators of OspG effector kinase function in eukaryotic host cells.     

Genome analysis and functional characterization of the E2 and RING-type E3 ligase ubiquitination enzymes of Arabidopsis

Attachment of ubiquitin to substrate proteins is catalyzed by the three enzymes E1, E2 (ubiquitin conjugating [UBC]), and E3 (ubiquitin ligase). Forty-one functional proteins with a UBC domain and active-site cysteine are predicted in the Arabidopsis (Arabidopsis thaliana) genome, which includes four that are predicted or shown to function with ubiquitin-like proteins. Only nine were previously characterized biochemically as ubiquitin E2s. We obtained soluble protein for 22 of the 28 uncharacterized UBCs after expression in Escherichia coli and demonstrated that 16 function as ubiquitin E2s. Twelve, plus three previously characterized ubiquitin E2s, were also tested for the ability to catalyze ubiquitination in vitro in the presence of one of 65 really interesting new gene (RING) E3 ligases. UBC22, UBC19-20, and UBC1-6 had variable levels of E3-independent activity. Six UBCs were inactive with all RINGs tested. Closely related UBC8, 10, 11, and 28 were active with the largest number of RING E3s and with all RING types. Expression analysis was performed to determine whether E2s or E3s were expressed in specific organs or under specific environmental conditions. Closely related E2s show unique patterns of expression and most express ubiquitously. Some RING E3s are also ubiquitously expressed; however, others show organ-specific expression. Of all the organs tested, RING mRNAs are most abundant in floral organs. This study demonstrates that E2 diversity includes examples with broad and narrow specificity toward RINGs, and that most ubiquitin E2s are broadly expressed with each having a unique spatial and developmental pattern of expression.     

The role of the ubiquitin ligase E6-AP in human papillomavirus E6-mediated degradation of PDZ domain-containing proteins

The E6 oncoprotein of human papillomaviruses associated with cervical cancer targets the tumor suppressor p53 and several other cellular proteins including the human homologs of Dlg and Scribble for degradation via the ubiquitin-proteasome system. Similar to p53 degradation, E6-induced degradation of Scribble is mediated by the ubiquitin ligase E6-AP. In contrast, degradation of Dlg in vitro and within cells has been reported to be independent of E6-AP, suggesting that the E6 oncoprotein has the ability to interact with ubiquitin ligases other than E6-AP. Furthermore, the ability of the E6 oncoprotein to interact with these yet unidentified ubiquitin ligases may be shared by the E6 protein of so-called low risk human papillomaviruses that are not associated with cervical cancer. In this study, we used the RNA interference technology and mouse embryo fibroblasts derived from E6-AP-deficient mice to obtain information about the identity of the ubiquitin ligase(s) involved in E6-mediated degradation of Dlg. We report that, within cells, E6-mediated degradation of Dlg depends on the presence of functional E6-AP and provide evidence that the E6 protein of low risk human papillomaviruses functionally interacts with E6-AP. Based on these data, we propose that, in general, the proteolytic properties of human papillomavirus E6 proteins are mediated by interaction with E6-AP.     

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.     

mGrb10 interacts with Nedd4.

We have utilized the yeast two-hybrid system to identify proteins interacting with mouse Grb10, an adapter protein known to interact with both the insulin and the insulin-like growth factor-I receptors. We have isolated a mouse cDNA clone containing the C2 domain of mouse Nedd4, a ubiquitin protein ligase (E3) that also contains a hect (homologous to the E6-AP carboxyl-terminus) domain and three WW domains. The interaction with Grb10 in the two-hybrid system was confirmed using the full-length Nedd4, and it was abolished by deleting the last 148 amino acids of Grb10, a region that includes the SH2 domain and the newly identified BPS domain. The interaction between Grb10 and Nedd4 was also reproduced in vivo in mouse embryo fibroblasts, where endogenous Nedd4 co-immunoprecipitated constitutively with both the endogenous and an overexpressed Grb10. This interaction was Ca(2+)-independent. Grb10 interacting with Nedd4 was not ubiquitinated in vivo, raising the possibility that this interaction may be used to target other proteins, like tyrosine kinase receptors, for ubiquitination.     

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.     

Itch WW Domains Inhibit Its E3 Ubiquitin Ligase Activity by Blocking E2-E3 Ligase Trans-thiolation

Nedd4-family E3 ubiquitin ligases regulate an array of biologic processes. Autoinhibition maintains these catalytic ligases in an inactive state through several mechanisms. However, although some Nedd4 family members are activated by binding to Nedd4 family-interacting proteins (Ndfips), how binding activates E3 function remains unclear. Our data reveal how these two regulatory processes are linked functionally. In the absence of Ndfip1, the Nedd4 family member Itch can bind an E2 but cannot accept ubiquitin onto its catalytic cysteine. This is because Itch is autoinhibited by an intramolecular interaction between its HECT (homologous to the E6-AP carboxy terminus domain) and two central WW domains. Ndfip1 binds these WW domains to release the HECT, allowing trans-thiolation and Itch catalytic activity. This molecular switch also regulates the closely related family member WWP2. Importantly, multiple PY motifs are required for Ndfip1 to activate Itch, functionally distinguishing Ndfips from single PY-containing substrates. These data establish a novel mechanism for control of the function of a subfamily of Nedd4 E3 ligases at the level of E2-E3 trans-thiolation.     

Noncanonical E2 variant-independent function of UBC13 in promoting checkpoint protein assembly

The E2 ubiquitin-conjugating enzyme UBC13 plays pivotal roles in diverse biological processes. Recent studies have elucidated that UBC13, in concert with the E3 ubiquitin ligase RNF8, propagates the DNA damage signal via a ubiquitylation-dependent signaling pathway. However, mechanistically how UBC13 mediates its role in promoting checkpoint protein assembly and its genetic requirement for E2 variants remain elusive. Here we provide evidence to support the idea that the E3 ubiquitin ligase complex RNF8-UBC13 functions independently of E2 variants and is sufficient in facilitating ubiquitin conjugations and accumulation of DNA damage mediator 53BP1 at DNA breaks. The RNF8 RING domain serves as the molecular platform to anchor UBC13 at the damaged chromatin, where localized ubiquitylation events allow sustained accumulation of checkpoint proteins. Intriguingly, we found that only a group of RING domains derived from E3 ubiquitin ligases, which have been shown to interact with UBC13, enabled UBC13-mediated FK2 and 53BP1 focus formation at DNA breaks. We propose that the RNF8 RING domain selects and loads a subset of UBC13 molecules, distinct from those that exist as heterodimers, onto sites of double-strand breaks, which facilitates the amplification of DNA damage signals.     

A novel UbcH10-binding protein facilitates the ubiquitinylation of cyclin B in vitro

UbcH10 is known to act as a ubiquitin-conjugating enzyme (E2) for anaphase-promoting complex/cyclosome. Since some E2s support different ubiquitin ligases (E3), it is possible that UbcH10 interacts with other proteins. We cloned a novel protein named H10BH by using a yeast two-hybrid screening method with UbcH10 as bait. The carboxyl terminus of H10BH showed a weak homology to the HECT (homologous to E6-AP carboxyl terminus) domain, which is conserved in one of the families of E3. H10BH bound UbcH10, and the amino acid sequence between 235 and 257 was necessary for this binding. H10BH showed a self-ubiquitinylation activity in a HECT-like sequence-dependent manner. The carboxyl terminal half (amino acids 188-389) showed stronger activity than the full-length H10BH. Furthermore, the carboxyl terminal half of H10BH was able to bind cyclin B and ubiquitinylate cyclin B in vitro. These results suggest that H10BH functions as an E3 using UbcH10 for its E2.     

N-Terminally extended human ubiquitin-conjugating enzymes (E2s) mediate the ubiquitination of RING-finger proteins, ARA54 and RNF8.

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).     

Ubiquitin manipulation by an E2 conjugating enzyme using a novel covalent intermediate

Degradation of misfolded and damaged proteins by the 26 S proteasome requires the substrate to be tagged with a polyubiquitin chain. Assembly of polyubiquitin chains and subsequent substrate labeling potentially involves three enzymes, an E1, E2, and E3. E2 proteins are key enzymes and form a thioester intermediate through their catalytic cysteine with the C-terminal glycine (Gly76) of ubiquitin. This thioester intermediate is easily hydrolyzed in vitro and has eluded structural characterization. To overcome this, we have engineered a novel ubiquitin-E2 disulfide-linked complex by mutating Gly76 to Cys76 in ubiquitin. Reaction of Ubc1, an E2 from Saccharomyces cerevisiae, with this mutant ubiquitin resulted in an ubiquitin-E2 disulfide that could be purified and was stable for several weeks. Chemical shift perturbation analysis of the disulfide ubiquitin-Ubc1 complex by NMR spectroscopy reveals an ubiquitin-Ubc1 interface similar to that for the ubiquitin-E2 thioester. In addition to the typical E2 catalytic domain, Ubc1 contains an ubiquitin-associated (UBA) domain, and we have utilized NMR spectroscopy to demonstrate that in this disulfide complex the UBA domain is freely accessible to non-covalently bind a second molecule of ubiquitin. The ability of the Ubc1 to bind two ubiquitin molecules suggests that the UBA domain does not interact with the thioester-bound ubiquitin during polyubiquitin chain formation. Thus, construction of this novel ubiquitin-E2 disulfide provides a method to characterize structurally the first step in polyubiquitin chain assembly by Ubc1 and its related class II enzymes.     

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.     

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.