Protein Interactions within the N-end Rule Ubiquitin Ligation Pathway

Abstract

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...

Conservation in the Mechanism of Nedd8 Activation by the Human AppBp1-Uba3 Heterodimer

“…Ubiquitin-activating enzyme exhibits an analogous substrate inhibition at a similar range of ubiquitin concentrations that reflects ordered substrate binding (29 (41). Transthiolation Kinetics for AppBp1-Uba3 HeterodimerWork from our laboratory has recently shown that initial rates for the transfer of 125 I-ubiquitin thiol ester from the E1 ternary complex to various E2 isozymes can be used as a facile kinetic assay for determining the intrinsic K d of substrate binding (38). Fig.…”

“…The reactions were incubated for 1 min at 37°C to reach thermal equilibrium prior to the addition of radiolabeled Nedd8. After 30 s, the reactions were quenched by the addition of an equal volume of 4% SDS sample buffer and the proteins were resolved by non-reducing SDS-PAGE on 12% (w/v) acrylamide gels (37,38). The gels were dried and the 125 I-Nedd8 thiol esters were visualized by autoradiography.…”

“…The gels were dried and the 125 I-Nedd8 thiol esters were visualized by autoradiography. Absolute amounts of Ubc12-125 I-Nedd8 thiol ester formed were determined by ␥-counting of excised bands (37,38).…”

“…HsUbc12 Transthiolation Kinetic Assays-Initial rates of AppBp1-Uba3 heterodimer-catalyzed transthiolation were assayed by monitoring formation of HsUbc12-125 I-Nedd8 as described previously for HsUbc2b (38). Reactions of 25 l contained 50 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , 1 mM DTT, 0.5 IU fast protein liquid chromatographypurified inorganic pyrophosphatase, 0.5 nM AppBp1-Uba3, and the indicated concentrations of ATP, 125 I-Nedd8 (about 10,000 cpm/pmol), and recombinant HsUbc12.…”

Mechanistic Studies on Activation of Ubiquitin and Di-ubiquitin-like Protein, FAT10, by Ubiquitin-like Modifier Activating Enzyme 6, Uba6

“…The "free ubiquitin" levels described are most likely an overestimation because the data were obtained under reducing conditions and so ubiquitin that is covalently attached as a thioester to E1s, E2s, and E3s within a cell would be counted as free ubiquitin. Siepmann et al (37) found that most of the free ubiquitin in cells is incorporated as thioesters to components of conjugation pathways. In terms of competing with FAT10 for charging, it is the available free ubiquitin that is not incorporated as thioester that directly competes with FAT10 for binding to Uba6.…”