ATP-dependent conjugation of ubiquitin to protein targets is currently recognized to mediate a variety of cellular processes by signaling selective degradation of the latter through the 26 S proteasome pathway, reviewed most recently in Refs. 1 and 2. Among the cellular targets serving as substrates for this unique post-translational modification are various proteins exhibiting either constitutive or conditional short half-lives including cyclins (3-8), various oncoproteins (9 -12), p53 (13-15), transcriptional factors (16 -18), and proteins of abnormal structure (19 -21). In all cases, the signal for ubiquitination probably requires transient exposure of one or more lysines that can serve as sites for recognition and attachment of the polypeptide. For certain targets, enhanced steric accessibility of sensitive lysines arising by minute conformational changes (22)(23)(24) or more global folding transitions (21, 25) may be accompanied by unmasking of specific amino-terminal residues that dispose the protein to recognition by relevant isopeptide ligases (E3) that confer specificity (4, 26 -30). In the case of cyclins, discrete recognition signals are conserved among related isoforms and within unrelated proteins (6, 31), although the precise mechanism by which these sequences contribute to substrate recognition by relevant conjugating enzymes has not been well elucidated.Attachment of single ubiquitin moieties to target proteins effects a modest rate of degradation by the 26 S proteasome (32-34); however, more robust signals for degradative targeting require subsequent formation of multiubiquitin homopolymers by chain elongation from the initial polypeptide conjugate (32,33,35). Considerable recent work has demonstrated that these multiubiquitin chains are formed by a repeating structure in which the carboxyl terminus of each ubiquitin is linked to Lys-48 of the preceding ubiquitin (33,35). The crystal structure of the resulting multiubiquitin chain exhibits considerable packing order and symmetry that is thought essential for recognition by the S5 subunit of the regulatory complex capping the 26 S proteasome (36,37). This model is supported by mutagenesis studies identifying essential ubiquitin residues required for both multiubiquitin chain binding to S5 and for subsequent degradative targeting (38).Ubiquitin-mediated proteolysis has been most extensively studied in yeast and rabbit reticulocytes. Within these systems, the almost quantitative inhibition of ATP-dependent degradation accompanying substitution of rmUb 1 or UbK48R for wild type polypeptide demonstrates that a significant fraction of degradative flux proceeds through conjugated intermediates bearing Lys-48-linked multiubiquitin chains since neither rmUb nor UbK48R supports chain elongation (33,35). However, mounting evidence supports the existence of multiubiq-

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 E3␣-catalyzed conjugation to human ␣-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 E3␣ 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.The majority of short-lived cellular proteins are targeted for degradation by the 26 S proteasome in response to assembly of degradation signals on their surface comprising chains of ubiquitin moieties covalently linked through specific lysine residues (1). Target protein specificity for this process is determined in part by a large family of diverse ubiquitin-protein isopeptide ligases (E3) 1 that recognize specific features of the native structure (2-4), transposable trans-acting amino acid sequences (5-7), or exposed regions of non-native conformation (8). The activated ubiquitin required to drive isopeptide bond formation is donated by specific ubiquitin carrier proteins (E2/ Ubc), also termed ubiquitin-conjugating enzymes, in which the ubiquitin carboxyl terminus is bound as a thiolester to a conserved cysteine (9, 10). The apparent specificity of different isopeptide ligases for recognizing a single or limited number of E2 isozymes accounts for the large cohort of related carrier protein isoforms (9 -11). Some E2 moieties may contribute to the substrate specificity of their cognate E3 isozyme because they are able to associate with targets in the absence of ligase (12)(13)(14). In addition, a subset of E2 isozymes catalyzes formation of polyubiquiti...

The developmentally programmed cell death of abdominal intersegmental muscles in the tobacco hawk-moth Manduca sexta is coincident with a 10-fold induction of the polyubiquitin gene as a hormonally regulated event (Schwartz, L. M., Myer, A., Kosz, L., Engelstein, M., and Maier, C. (1990) Neuron 5, 411-419). Solid phase immunochemical assays measuring intersegmental muscle pools of free and conjugated ubiquitin reveal that the induction of polyubiquitin mRNA is accompanied by a proportional increase in total ubiquitin polypeptide. Ubiquitin conjugate pools increase 10-fold at eclosion, during which loss of muscle protein mass is maximum. A smaller but measurable increase in ubiquitin conjugates is observed earlier in pupal development coincident with a modest enhanced degradation of myofibrillar proteins. Accumulation of ubiquitin conjugates is accompanied by induction in the pathway for polypeptide ligation, including the activating enzyme (E1), several carrier protein (E2) isoforms, and ubiquitin:protein isopeptide ligase (E3). Both accumulation of ubiquitin polypeptide and the enzymes of the conjugation pathway are subject to regulation by declining titers of the insect molting hormone 20-hydroxyecdysone, which signals onset of programmed cell death in the intersegmental muscles. Thus, programmed cell death within the intersegmental muscles is accomplished in part by stimulation of the ubiquitin-mediated degradative pathway through a coordinated induction of ubiquitin and the enzymes responsible for its conjugation to yield proteolytic intermediates. This suggests enzymes required for ubiquitin conjugation may represent additional genes recruited for developmentally programmed death.

The N-end rule relates the amino terminus to the rate of degradation through the ubiquitin/26 S proteasome pathway. Proteins bearing basic (type 1) or large hydrophobic (type 2) amino termini are assumed to be targeted through this pathway by their higher affinity for binding to the responsible E3 ligase compared with proteins bearing other residues (type 3). Paradoxically, a significant fraction of eukaryotic protein degradation occurs through the N-end rule pathway, although the majority of cellular proteins are type 3 substrates. We have exploited specific interactions between ubiquitin carrier proteins (E2/Ubc) and their cognate E3 ligases to purify for the first time the mammalian N-end rule ligase E3␣/Ubr1 to near homogeneity. In vitro studies show that E3␣ forms lysine 48-linked polyubiquitin degradation signals on type 1-3 substrates and is absolutely dependent on Ubc2/Rad6 orthologs. Biochemically defined kinetic studies show that the basis of N-end rule specificity is a k cat rather than the K m effect originally proposed, since all three substrate classes show similar binding affinities (K m ϳ5 M) but V max values that are 100-and 50-fold greater for type 1 and 2 versus type 3 model substrates, respectively. In addition, the N-end rule dipeptides lysylalanine and phenylalanylalanine are general noncompetitive inhibitors for E3␣-catalyzed ubiquitination of type 1-3 substrates rather than typespecific competitive inhibitors as predicted. These observations are consistent with a model in which the N-end rule effect reflects substrate binding-induced transitions in E3␣ to a catalytically competent conformer, the equilibrium for which depends on the identity of the amino terminus or the presence of basic or hydrophobic surface features. The model reconciles conflicts between specific predictions and empirical observations relating N-end rule targeting in addition to explicating the efficacy of selected dipeptides as potent in vivo inhibitors of this pathway.