A 25-kilodalton ubiquitin carrier protein (E2) catalyzes multi-ubiquitin chain synthesis via lysine 48 of ubiquitin

Target protein multi-ubiquitination involving lysine 48 of ubiquitin (Ub) is known to occur during protein degradation in the ATP- and Ub-dependent proteolytic pathway (Chau, V., Tobias, J. W., Bachmair, A., Marriott, D., Ecker, D. J., Gonda, D. K., and Varshavsky, A. (1989) Science 243, 1576-1583). However, little is known about the enzymatic mechanism of multi-ubiquitination. We show that a purified Ub carrier protein, E2_25K, catalyzes multi-Ub chain synthesis from purified Ub. Incubation of E2_25K with Ub activating enzyme (E1), MgATP, and radiolabeled Ub (M_r = 8500) resulted in time-dependent appearance of a "ladder" of radiolabeled Ub conjugates with molecular masses of 8.5n kDa, where n = 1, 2, 3, 4. . . (up to at least n = 10). The kinetics of this conjugative process were consistent with Ub₂acting as a steady-state intermediate. The putative Ub₂ product of E2_25K catalysis was purified and cleaved with a partially purified isopeptidase preparation. The sole cleavage product (M_r = 8500) had a tryptic digest identical to that of authentic Ub, confirming that the original conjugate was Ub₂. Tryptic digestion of intact Ub₂ gave products consistent with the existence of an isopeptide linkage between the COOH terminus of one Ub and Lys-48 of the other; this structure was confirmed by sequence analysis of the unique Ub₂ tryptic fragment. Tryptic digestion of higher order Ub_n adducts (n ≥ 4) yielded fragments identical to those of Ub₂, indicating that E2_25K ligates successive Ub molecules primarily or exclusively via Lys-48. Although several other E2s supported synthesis of an apparent Ub₂ adduct of undetermined linkage, only E2_25K was capable of synthesizing multi-Ub chains from isolated Ub. Quantitative analysis of single turnovers showed that transfer from E2_25K-Ub to Ub and Ub₂ occurred with k₂ = 488 and 1170 M^-1 min^-1, respectively, at pH 7.3 and 37°C. These results show that increasing the number of Ub molecules in a chain increases susceptibility to further ubiquitination by E2_25K. Ub₂ was a good substrate for activation by E1 and was readily transferred to E2_25K. The labile E2_25K-Ub₂ adduct was catalytically active, and exhibited preference for Ub₂ (versus Ub) as acceptor. These results suggest that E2_25K may function as a multi-ubiquitinating enzyme in the Ub-dependent proteolytic pathway.