Novel Multiubiquitin Chain Linkages Catalyzed by the Conjugating Enzymes E2EPF and RAD6 Are Recognized by 26 S Proteasome Subunit 5

Abstract

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-

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

“…To determine whether human erythrocyte AppBp1-Uba3 heterodimer forms a similar Nedd8 ternary complex, the stoichiometry of Nedd8 [ 3 H]adenylate and Uba3-125 I-Nedd8 thiol ester was determined in parallel with a quantity of human erythrocyte Uba1 ubiquitinactivating enzyme that produced a silver-stained band following SDS-PAGE resolution of the same intensity as affinity purified erythrocyte HsUba3. 32 PP i exchange kinetics have been used to demonstrate that ubiquitinactivating enzyme proceeds through an ordered addition mechanism for which ATP is the leading and ubiquitin the trailing substrate (29). Human AppBp1-Uba3 heterodimer catalyzes an analogous ATP: 32 (Fig.…”

“…32 PP i exchange kinetics have been used to demonstrate that ubiquitinactivating enzyme proceeds through an ordered addition mechanism for which ATP is the leading and ubiquitin the trailing substrate (29). Human AppBp1-Uba3 heterodimer catalyzes an analogous ATP: 32 (Fig. 2).…”

“…Protein expression was induced by rapidly increasing the cultures to the non-permissive temperature of 42°C (32,33,35). After 2 h of induction at 42°C, cells from 2 liters of LB culture containing 100 g/ml ampicillin were collected by centrifugation at 5,000 ϫ g for 15 min then lysed by passage through a French press.…”

“…Bovine ubiquitin was purchased from Sigma and purified to homogeneity as described previously (32). Homogeneous wild-type ubiquitin, the recombinant ubiquitin mutant UbR72L (33), and recombinant human Nedd8 were radiolabeled by the chloramine-T method using carrier-free Na 125 I obtained from PerkinElmer Life Sciences (34 Cloning, Expression, and Purification of Human Recombinant Nedd8 -Nedd8 was cloned from a HeLa cell cDNA library by PCR amplification using 5Ј and 3Ј primers flanking the coding sequence that contained NdeI or EcoRI restriction sites, respectively.…”

Identification of a physiological E2 module for the human anaphase-promoting complex

“…In a similar manner, addition of a peptide comprising the C terminus of Ube2S (CTP) blocks the activity of Ube2S on APC/C. Ube2S catalyzes the formation of K11-linked ubiquitin-dimers independently of APC/C (22). This activity depends on the active-site Cys of Ube2S, but is not affected by deletion of its C terminus (Fig.…”