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 Ub2 acting as a steady-state intermediate. The putative Ub2 product of F2(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 F2(25K)-Ub to Ub and Ub2 occurred with k = 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.
|Original language||English (US)|
|Number of pages||8|
|Journal||Journal of Biological Chemistry|
|State||Published - Dec 1 1990|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology