In silico modeling of the cryptic E2∼ubiquitin-binding site of E6-associated protein (E6AP)/UBE3A reveals the mechanism of polyubiquitin chain assembly.

To understand the mechanism for assembly of Lys48-linked polyubiquitin degradation signals, we previously demonstrated that the E6AP/UBE3A ligase harbors two functionally distinct E2∼ubiquitin-binding sites: a high-affinity Site 1 required for E6AP Cys820∼ubiquitin thioester formation and a canonical Site 2 responsible for subsequent chain elongation. Ordered binding to Sites 1 and 2 is here revealed by observation of UbcH7∼ubiquitin-dependent substrate inhibition of chain formation at micromolar concentrations. To understand substrate inhibition, we exploited the PatchDock algorithm to model in silico UbcH7∼ubiquitin bound to Site 1, validated by chain assembly kinetics of selected point mutants. The predicted structure buries an extensive solvent-excluded surface bringing the UbcH7∼ubiquitin thioester bond within 6 Šof the Cys820 nucleophile. Modeling onto the active E6AP trimer suggests that substrate inhibition arises from steric hindrance between Sites 1 and 2 of adjacent subunits. Confirmation that Sites 1 and 2 function in trans was demonstrated by examining the effect of E6APC820A on wild-type activity and single-turnover pulse-chase kinetics. A cyclic proximal indexation model proposes that Sites 1 and 2 function in tandem to assemble thioester-linked polyubiquitin chains from the proximal end attached to Cys820 before stochastic en bloc transfer to the target protein. Non-reducing SDS-PAGE confirms assembly of the predicted Cys820-linked 125I-polyubiquitin thioester intermediate. Other studies suggest that Glu550 serves as a general base to generate the Cys820 thiolate within the low dielectric binding interface and Arg506 functions to orient Glu550 and to stabilize the incipient anionic transition state during thioester exchange.

The active form of E6-associated protein (E6AP)/UBE3A ubiquitin ligase is an oligomer.

Employing 125I-polyubiquitin chain formation as a functional readout of ligase activity, biochemical and biophysical evidence demonstrates that catalytically active E6-associated protein (E6AP)/UBE3A is an oligomer. Based on an extant structure previously discounted as an artifact of crystal packing forces, we propose that the fully active form of E6AP is a trimer, analysis of which reveals a buried surface of 7508Å2 and radially symmetric interacting residues that are conserved within the Hect (homologous to E6AP C terminus) ligase superfamily. An absolutely conserved interaction between Phe(727) and a hydrophobic pocket present on the adjacent subunit is critical for trimer stabilization because mutation disrupts the oligomer and decreases kcat 62-fold but fails to affect E2 ubiquitin binding or subsequent formation of the Hect domain Cys(820) ubiquitin thioester catalytic intermediate. Exogenous N-acetylphenylalanylamide reversibly antagonizes Phe(727)-dependent trimer formation and catalytic activity (Ki12 mM), as does a conserved-helical peptide corresponding to residues 474­490 of E6A Pisoform 1 (Ki22M) reported to bind the hydrophobic pocket of other Hect ligases, presumably blocking Phe(727) intercalation and trimer formation. Conversely, oncogenic human papillomavirus-16/18 E6 protein significantly enhances E6AP catalytic activity by promoting trimer formation (Kactivation 1.5 nM) through the ability of E6 to form homodimers. Recombinant E6 protein additionally rescues the kcat defect of the Phe(727) mutation and that of a specific loss-of-function Angelman syndrome mutation that promotes trimer destabilization. The present findings codify otherwise disparate observations regarding the mechanism of E6AP and related Hect ligases in addition to suggesting therapeutic approaches for modulating ligase activity.

Tripartite motif ligases catalyze polyubiquitin chain formation through a cooperative allosteric mechanism.

Ligation of polyubiquitin chains to proteins is a fundamental post-translational modification, often resulting in targeted degradation of conjugated proteins. Attachment of polyubiquitin chains requires the activities of an E1 activating enzyme, an E2 carrier protein, and an E3 ligase. The mechanism by which polyubiquitin chains are formed remains largely speculative, especially for RING-based ligases. The tripartite motif (TRIM) superfamily of ligases functions in many cellular processes including innate immunity, cellular localization, development and differentiation, signaling, and cancer progression. The present results show that TRIM ligases catalyze polyubiquitin chain formation in the absence of substrate, the rates of which can be used as a functional readout of enzyme function. Initial rate studies under biochemically defined conditions show that TRIM32 and TRIM25 are specific for the Ubc5 family of E2-conjugating proteins and, along with TRIM5α, exhibit cooperative kinetics with respect to Ubc5 concentration, with submicromolar [S]0.5 and Hill coefficients of 3-5, suggesting they possess multiple binding sites for their cognate E2-ubiquitin thioester. Mutation studies reveal a second, non-canonical binding site encompassing the C-terminal Ubc5α-helix. Polyubiquitin chain formation requires TRIM subunit oligomerization through the conserved coiled-coil domain, but can be partially replaced by fusing the catalytic domain to GST to promote dimerization. Other results suggest that TRIM32 assembles polyubiquitin chains as a Ubc5-linked thioester intermediate. These results represent the first detailed mechanistic study of TRIM ligase activity and provide a functional context for oligomerization observed in the superfamily.

ISG15 disrupts cytoskeletal architecture and promotes motility in human breast cancer cells.

The interferon-stimulated gene 15 (ISG15) pathway is highly elevated in breast cancer; however, very little is known about how the ISG15 pathway contributes to breast tumorigenesis. In the current study, using the gene disruption approach, we demonstrate that both ISG15 and UbcH8 (ISG15-specific conjugating enzyme) disrupt F-actin architecture and formation of focal adhesions in ZR-75-1 breast cancer cells. In addition, ISG15 and UbcH8 promote breast cancer cell migration. We also demonstrate that ISG15 inhibits ubiquitin/26S proteasome-mediated turnover of proteins implicated in tumor cell motility, invasion and metastasis. Together, our results suggest that the aberrant activation of the ISG15 pathway confers a motile phenotype to breast cancer cells by disrupting cell architecture and stabilizing proteins involved in cell motility, invasion and metastasis. Because the cellular architecture is conserved and the ISG15 pathway is constitutively activated in tumor cells of different lineages, it is reasonable to assume that our observations in breast cancer must hold true for many other tumors.

A novel role for ATM in regulating proteasome-mediated protein degradation through suppression of the ISG15 conjugation pathway.

Ataxia Telangiectasia (A-T) is an inherited immunodeficiency disorder wherein mutation of the ATM kinase is responsible for the A-T pathogenesis. Although the precise role of ATM in A-T pathogenesis is still unclear, its function in responding to DNA damage has been well established. Here we demonstrate that in addition to its role in DNA repair, ATM also regulates proteasome-mediated protein turnover through suppression of the ISG15 pathway. This conclusion is based on three major pieces of evidence: First, we demonstrate that proteasome-mediated protein degradation is impaired in A-T cells. Second, we show that the reduced protein turnover is causally linked to the elevated expression of the ubiquitin-like protein ISG15 in A-T cells. Third, we show that expression of the ISG15 is elevated in A-T cells derived from various A-T patients, as well as in brain tissues derived from the ATM knockout mice and A-T patients, suggesting that ATM negatively regulates the ISG15 pathway. Our current findings suggest for the first time that proteasome-mediated protein degradation is impaired in A-T cells due to elevated expression of the ISG15 conjugation pathway, which could contribute to progressive neurodegeneration in A-T patients.

Ser(120) of Ubc2/Rad6 regulates ubiquitin-dependent N-end rule targeting by E3{alpha}/Ubr1.

In CHO cells, CDK1/2-dependent phosphorylation of Ubc2/Rad6 at Ser(120) stimulates its ubiquitin conjugating activity and can be replicated by a S120D point mutant (Sarcevic, B., Mawson, A., Baker, R. T., and Sutherland, R. L. (2002) EMBO J. 21, 2009-2018). In contrast, we find that ectopic expression of wild type Ubc2b but not Ubc2bS120D or Ubc2bS120A in T47D human breast cancer cells specifically stimulates N-end rule-dependent degradation but not the Ubc2-independent unfolded protein response pathway, indicating that the former is E2 limiting in vivo and likely down-regulated by Ser(120) phosphorylation, as modeled by the S120D point mutation. In vitro kinetic analysis shows the in vivo phenotype of Ubc2bS120D and Ubc2bS120A is not due to differences in activating enzyme-catalyzed E2 transthiolation. However, the Ser(120) mutants possess marked differences in their abilities to support in vitro conjugation by the N-end rule-specific E3α/Ubr1 ligase that presumably accounts for their in vivo effects. Initial rate kinetics of human E3α-catalyzed conjugation of the human α-lactalbumin N-end rule substrate shows Ubc2bS120D is 20-fold less active than wild type E2, resulting from an 8-fold increase in K(m) and a 2.5-fold decrease in V(max), the latter reflecting a decreased ability to support the initial step in target protein conjugation; Ubc2bS120A is 8-fold less active than wild type E2 due almost exclusively to a decrease in V(max), reflecting a defect in polyubiquitin chain elongation. These studies suggest a mechanism for the integrated regulation of diverse ubiquitin-dependent signaling pathways through E2 phosphorylation that yields differential effects on its cognate ligases.

Activation of ubiquitin and ubiquitin-like proteins.

Attachment of ubiquitin and ubiquitin-like proteins to cellular targets represents a fundamental regulatory strategy within eukaryotes and exhibits remarkably pleiotropic effects on cell function. These posttranslational modifications share a common mechanism comprised of three steps: an activating enzyme to couple ATP hydrolysis to formation of a high-energy intermediate at the carboxyl terminus of ubiquitin or the ubiquitin-like protein, a ligase to couple aminolysis of the activated polypeptide to formation of the new peptide bond and a carrier protein to link the two half reactions. The activating enzymes play pivotal roles in defining pathway specificity for ubiquitin or the ubiquitin-like protein and for target protein specificity in charging the cognate carrier protein supporting downstream ligation steps. Therefore, the family of activating enzymes are critical components of cell regulation that have only recently been recognized as important pharmacological targets.

RANBP2 is an allosteric activator of the conventional kinesin-1 motor protein, KIF5B, in a minimal cell-free system.

The association of cargoes to kinesins is thought to promote kinesin activation, yet the validation of such a model with native cargoes is lacking because none is known to activate kinesins directly in an in vitro system of purified components. The RAN-binding protein 2 (RANBP2), through its kinesin-binding domain (KBD), associates in vivo with kinesin-1, KIF5B/KIF5C. Here, we show that KBD and its flanking domains, RAN GTPase-binding domains 2 and 3 (RBD2/RBD3), activate the ATPase activity of KIF5B approximately 30-fold in the presence of microtubules and ATP. The activation kinetics of KIF5B by RANBP2 is biphasic and highly cooperative. Deletion of one of its RBDs lowers the activation of KIF5B threefold and abolishes cooperativity. Remarkably, RBD2-KBD-RBD3 induces unfolding and modest activation of KIF5B in the absence of microtubules. Hence, RANBP2 is the first native and positive allosteric activator known to jump-start and boost directly the activity of a kinesin.

The hinge region between two ubiquitin-like domains destabilizes recombinant ISG15 in solution.

Interferon-stimulated gene (ISG) 15 mediates antiviral responses and also is upregulated within the endometrium in response to the developing embryo during early pregnancy. Structurally, ISG15 resembles two ubiquitin domains (30% identical) that are separated by a hinge region. Recombinant (r) bovISG15 is not stable in solution. It was hypothesized that the hinge region contributed to the instability of rbovISG15. Within 24 h of dialysis, rbovISG15 formed complexes as detected by reducing and denaturing SDS-PAGE. However, chemical perturbations of cysteine prevented formation of rbovISG15 complexes over time. Furthermore, a site-directed mutant of rbovISG15 (Cys80Ser) was isomeric and more stable than rbovISG15. Neither wild-type nor mutant rbovISG15 was able to interact with the ISG15 E1 initiating enzyme, UBE1L, in an in vitro pull-down assay. Ovine (ov) ISG15 has three additional amino acids within the hinge region that were hypothesized to increase stability and the degree of interaction with UBE1L because of increased separation of the ubiquitin-like domains. Over time in solution, rovISG15 the level of rovISG15 secondary structure was diminished, whereas the Cys80Ser rovISG15 structure did not change. A GST-Cys80Ser rovISG15 fusion protein had increased structural stability and enhanced protein-protein interaction with UBE1L after dialysis for 48 h, when compared to the GST-rovISG15 fusion protein or rbovISG15. Models of bovISG15, Cys80Ser bovISG15, and ovISG15 were constructed, which confirmed that the hinge region between the two ubiquitin domains destabilizes rbovISG15 in solution.

Demonstration of ubiquitin thiolester formation of UBE2Q2 (UBCi), a novel ubiquitin-conjugating enzyme with implantation site-specific expression.

We recently identified a differentially expressed gene in implantation stage rabbit endometrium encoding a new member of the ubiquitin-conjugating enzyme family designated UBE2Q2 (also known as UBCi). Its unusually high molecular mass, novel N-terminus extension, and highly selective pattern of mRNA expression suggest a specific function in implantation. This study analyzes its relationship to the E2 ubiquitin-conjugating enzyme superfamily, investigates its enzymatic activity, and examines its localization in implantation site endometrium. Construction of a dendrogram indicated that UBE2Q2 is homologous to the UBC2 family of enzymes, and isoforms are present in a broad range of species. In vitro enzymatic assays of ubiquitin thiolester formation demonstrated that UBE2Q2 is a functional ubiquitin-conjugating enzyme. The Km for transfer of ubiquitin thiolester from E1 to UBE2Q2 is 817 nM compared to 100 nM for other E2 paralogs; this suggests that the unique amino terminal domain of UBE2Q2 confers specific functional differences. Affinity-purified antibodies prepared with purified recombinant UBE2Q2 showed that the protein was undetectable by immunoblot analysis in endometrial lysates from estrous and Day 6(3/4) pregnant (blastocyst attachment stage) rabbits but was expressed in both mesometrial and antimesometrial implantation site endometrium of Day 8 pregnant animals. No expression was detected in adjacent interimplantion sites. Immunohistochemistry demonstrated UBE2Q2 expression exclusively in mesometrial and antimesometrial endometrial luminal epithelial cells of the Day 8 implantation chamber. Immunohistochemical localization of ubiquitin mirrored UBE2Q2 expression, with low-to-undetectable levels in implantation sites of Day 6(3/4) pregnant endometrium but high levels in luminal epithelial cells of Day 8 pregnant endometrium. This implantation site-specific expression of UBE2Q2 in luminal epithelial cells could play major roles in orchestrating differentiation events through the modification of specific protein substrates.

Pleiotropic effects of ATP.Mg2+ binding in the catalytic cycle of ubiquitin-activating enzyme.

Conjugation of ubiquitin and other Class 1 ubiquitin-like polypeptides to specific protein targets serves diverse regulatory functions in eukaryotes. The obligatory first step of conjugation requires ATP-coupled activation of the ubiquitin-like protein by members of a superfamily of evolutionarily related enzymes. Kinetic and equilibrium studies of the human ubiquitin-activating enzyme (HsUba1a) reveal that mutations within the ATP.Mg(2+) binding site have remarkably pleiotropic effects on the catalytic phenotype of the enzyme. Mutation of Asp(576) or Lys(528) results in dramatically impaired binding affinities for ATP.Mg(2+), a shift from ordered to random addition in co-substrate binding, and a significantly reduced rate of ternary complex formation that shifts the rate-limiting step to ubiquitin adenylate formation. Mutations at neither position affect the affinity of HsUbc2b binding; however, differences in k(cat) values determined from ternary complex formation versus HsUbc2b transthiolation suggest that binding of the E2 enhances the rate of bound ubiquitin adenylate formation. These results confirm that Asp(576) and Lys(528) are important for ATP.Mg(2+) binding but are essential catalytic groups for ubiquitin adenylate transition state stabilization. The latter mechanistic effect explicates the observed loss-of-function phenotype associated with mutation of residues paralogous to Asp(576) within the activating enzymes for other ubiquitin-like proteins.

Proteasome-dependent down-regulation of activated Stat5A in the nucleus.

A broad spectrum of cytokines can activate the signal transducer and activator of transcription 5 (Stat5) by inducing a single tyrosine phosphorylation of the molecule. Although the process of Stat5 activation has been well studied, the mechanism by which it is inactivated is not fully understood. We demonstrate that the proteasome inhibitor MG132, but not the nuclear export inhibitor leptomycin B (LMB), stabilizes active nuclear Stat5A, whereas MG132 only partially stabilizes active cytoplasmic Stat5A. Importantly, ubiquitinated Stat5A is detected in the nucleus and the polyubiquitination of active Stat5A is K48 linked, a linkage type targeting proteins for degradation. Ubiquitination of Stat5A is recapitulated in a cell-free system, and Ubc5 is identified as the E2-conjugating enzyme for Stat5A ubiquitination. Interestingly, phosphorylation of Stat5A per se is not required for ubiquitination. Finally, C-terminal deletion analysis of Stat5A localizes the amphipathic region of amino acids 751-762 as a ubiquitination signal, possibly representing an E3 recognition motif. Taken together, these results demonstrate that the down-regulation of nuclear and cytoplasmic active Stat5A is differentially regulated. In the nucleus, ubiquitin/proteasome-mediated protein degradation is the dominant mechanism for the down-regulation of active Stat5A, whereas in the cytoplasm, protein tyrosine phasphatase is a major player in the down-regulation of active Stat5A.

Elevated expression of ISG15 in tumor cells interferes with the ubiquitin/26S proteasome pathway.

IFN-stimulatory gene factor 15 (ISG15) is a ubiquitin-like protein, which is conjugated to many cellular proteins. However, its role in protein degradation is unclear. Here, we show that ISG15 is highly elevated and extensively conjugated to cellular proteins in many tumors and tumor cell lines. The increased levels of ISG15 in tumor cells were found to be associated with decreased levels of polyubiquitinated proteins. Specific knockdown of ISG15 expression using ISG15-specific small interfering RNA (siRNA) was shown to increase the levels of polyubiquitinated proteins, suggesting an antagonistic role of ISG15 in regulating ubiquitin-mediated protein turnover. Moreover, siRNA-mediated down-regulation of the major E2 for ISG15 (UbcH8), which blocked the formation of ISG15 protein conjugates, also increased the levels of polyubiquitinated proteins. Together, our results suggest that the ISG15 pathway, which is deregulated during tumorigenesis, negatively regulates the ubiquitin/proteasome pathway by interfering with protein polyubiquitination/degradation.

Involvement of the ubiquitin pathway in decreasing Ku70 levels in response to drug-induced apoptosis.

Ku70 plays an important role in DNA damage repair and prevention of cell death. Previously, we reported that apoptosis caused a decrease in cellular Ku70 levels. In this study, we analyzed the mechanism of how Ku70 levels decrease during drug-induced apoptosis. In HeLa cells, staurosporin (STS) caused a decrease in Ku70 levels without significantly affecting Ku70 mRNA levels. We found that Ku70 protein was highly ubiquitinated in various cell types, such as HeLa, HEK293T, Dami (a megakaryocytic cell line), endothelial, and rat kidney cells. An increase in ubiquitinated Ku70 protein was observed in apoptotic cells, and proteasome inhibitors attenuated the decrease in Ku70 levels in apoptotic cells. These results suggest that the ubiquitin-proteasome proteolytic pathway plays a role in decreasing Ku70 levels in apoptotic cells. Ku70 forms a heterodimer with Ku80, which is required for the DNA repair activity of Ku proteins. We also found that Ku80 levels decreased in apoptotic cells and that Ku80 is a target of ubiquitin. Ubiquitinated Ku70 was not found in the Ku70-Ku80 heterodimer, suggesting that modification by ubiquitin inhibits Ku heterodimer formation. We propose that the ubiquitin-dependent modification of Ku70 plays an important role in the control of cellular levels of Ku70.

Lys6-modified ubiquitin inhibits ubiquitin-dependent protein degradation.

Ubiquitin plays essential roles in various cellular processes; therefore, it is of keen interest to study the structure-function relationship of ubiquitin itself. We investigated the modification of Lys(6) of ubiquitin and its physiological consequences. Mass spectrometry-based peptide mapping and N-terminal sequencing demonstrated that, of the 7 Lys residues in ubiquitin, Lys(6) was the most readily labeled with sulfosuccinimidobiotin. Lys(6)-biotinylated ubiquitin was incorporated into high molecular mass ubiquitin conjugates as efficiently as unmodified ubiquitin. However, Lys(6)-biotinylated ubiquitin inhibited ubiquitin-dependent proteolysis, as conjugates formed with Lys(6)-biotinylated ubiquitin were resistant to proteasomal degradation. Ubiquitins with a mutation of Lys(6) had similar phenotypes as Lys(6)-biotinylated ubiquitin. Lys(6) mutant ubiquitins (K6A, K6R, and K6W) also inhibited ATP-dependent proteolysis and caused accumulation of ubiquitin conjugates. Conjugates formed with K6W mutant ubiquitin were also resistant to proteasomal degradation. The dominant-negative effect of Lys(6)-modified ubiquitin was further demonstrated in intact cells. Overexpression of K6W mutant ubiquitin resulted in accumulation of intracellular ubiquitin conjugates, stabilization of typical substrates for ubiquitin-dependent proteolysis, and enhanced susceptibility to oxidative stress. Taken together, these results show that Lys(6)-modified ubiquitin is a potent and specific inhibitor of ubiquitin-mediated protein degradation.

Glutathione S-transferase pull-down assays using dehydrated immobilized glutathione resin.

We have developed an affinity-precipitation technique to facilitate conducting glutathione S-transferase (GST) pull-down assays. The dehydrated immobilized glutathione resin format, when combined with microcentrifuge spin columns, is a powerful tool that enables the simultaneous performance of resin hydration, the binding of the GST fusion protein, and the pull-down step with the appropriate protein partner in a semihigh-throughput fashion (multiple samples processed at the same time). The entire assay process is shortened and recovery is enhanced when coupled with a spin-column format, providing a convenient way to study protein-protein interactions. We successfully tested the resin format/technique in three common pull-down applications utilizing radiolabeled, overexpressed, and activated endogenous interacting protein partners.

Nitric oxide inhibits H2O2-induced transferrin receptor-dependent apoptosis in endothelial cells: Role of ubiquitin-proteasome pathway.

We investigated here the mechanism of cytoprotection of nitric oxide (*NO) in bovine aortic endothelial cells treated with H2O2. NONOates were used as *NO donors that released *NO slowly at a well defined rate in the extracellular and intracellular milieus. H2O2-mediated intracellular dichlorofluorescein fluorescence and apoptosis were enhanced by the transferrin receptor (TfR)-mediated iron uptake. *NO inhibited the TfR-mediated iron uptake, dichlorofluorescein fluorescence, and apoptosis in H2O2-treated cells. *NO increased the proteasomal activity and degradation of nitrated TfR via ubiquitination. Nomega-nitro-L-arginine methyl ester, a nonspecific inhibitor of endogenous *NO biosynthesis, decreased the trypsin-like activity of 26S proteasome. *NO, by activating proteolysis, mitigates TfR-dependent iron uptake, dichlorodihydrofluorescein oxidation, and apoptosis in H2O2-treated bovine aortic endothelial cells. The relevance of biological nitration on redox signaling is discussed.

Biochemical and functional properties of the full-length cation-dependent mannose 6-phosphate receptor expressed in Pichia pastoris.

A glycosylation-deficient, full-length cation-dependent mannose 6-phosphate receptor (CD-MPR) containing a yeast signal sequence was expressed in Pichia pastoris using the constitutive promoter of the PGAP gene. The membrane-bound receptor was solubilized using detergents and purified by pentamannosyl phosphate-agarose affinity chromatography. Equilibrium binding studies identified a binding affinity of 2 nM for the lysosomal enzyme, beta-glucuronidase. To probe the linkage specificity of the recombinant CD-MPR, inhibition binding studies were conducted using non-phosphorylated oligomannoses which demonstrated that Manalpha1,2Man exhibits a 4-fold higher inhibition than Manalpha1,3Man and Manalpha1,6Man. The receptor was capable of associating into oligomeric forms and enzymatic deglycosylation revealed the presence of high-mannose sugars at the single potential N-glycosylation site. Mass spectrometric analysis revealed that the receptor was palmitoylated at the two potential cysteines in its cytoplasmic domain. In conclusion, the full-length CD-MPR produced in P. pastoris is structurally and functionally suitable for crystallization studies.

Protein interactions within the N-end rule ubiquitin ligation pathway.

Rate studies have been employed as a reporter function to probe protein-protein interactions within a biochemically defined reconstituted N-end rule ubiquitin ligation pathway. The concentration dependence for E1-catalyzed HsUbc2b/E2(14kb) transthiolation is hyperbolic and yields K(m) values of 102 +/- 13 nm and 123 +/- 19 nm for high affinity binding to rabbit and human E1/Uba1 orthologs. Competitive inhibition by the inactive substrate and product analogs HsUbc2bC88A (K(i) = 104 +/- 15 nm) and HsUbc2bC88S-ubiquitin oxyester (K(i) = 169 +/- 17 nm), respectively, indicates that the ubiquitin moiety contributes little to E1 binding. Under conditions of rate-limiting E3alpha-catalyzed conjugation to human alpha-lactalbumin, HsUbc2b-ubiquitin thiolester exhibits a K(i) of 54 +/- 18 nm and is competitively inhibited by the substrate analog HsUbc2bC88S-ubiquitin oxyester (K(i) = 66 +/- 29 nm). In contrast, the ligase product analog HsUbc2bC88A exhibits a K(i) of 440 +/- 55 nm with respect to the wild type HsUbc2b-ubiquitin thiolester, demonstrating that ubiquitin binding contributes to the ability of E3alpha to discriminate between substrate and product E2. A survey of E1 and E2 isoform distribution in selected cell lines demonstrates that Ubc2 isoforms are the predominant intracellular ubiquitin carrier protein. Intracellular levels of E1 and Ubc2 are micromolar and approximately equal based on in vitro quantitation by stoichiometric (125)I-ubiquitin thiolester formation. Comparison of intracellular E1 and Ubc2 pools with the corresponding ubiquitin pools reveals that most of the free ubiquitin in cells is present as thiolesters to the components of the conjugation pathways. The present data represent the first comprehensive analysis of protein interactions within a ubiquitin ligation pathway.