Abstract
Ubiquitin-conjugating enzymes (E2s) have a dominant role in determining which of the seven lysine residues of ubiquitin is used for polyubiquitination. Here we show that tethering of a substrate to an E2 enzyme in the absence of an E3 ubiquitin ligase is sufficient to promote its ubiquitination, whereas the type of the ubiquitin conjugates and the identity of the target lysine on the substrate are promiscuous. In contrast, when an E3 enzyme is introduced, a clear decision between mono- and polyubiquitination is made, and the conjugation type as well as the identity of the target lysine residue on the substrate becomes highly specific. These features of the E3 can be further regulated by auxiliary factors as exemplified by MDMX (Murine Double Minute X). In fact, we show that this interactor reconfigures MDM2-dependent ubiquitination of p53. Based on several model systems, we propose that although interaction with an E2 is sufficient to promote substrate ubiquitination the E3 molds the reaction into a specific, physiologically relevant protein modification.
Highlights
Targeting of most substrates to the 26 S proteasome requires covalent marking with polyubiquitin chains
Global in vivo analysis of the specific lysine residues engaged in polyubiquitination indicated that all seven lysine residues of ubiquitin participate in ubiquitin reticulum protein; RFP, red fluorescent protein; Ub, ubiquitin; BARD-BRCA, BRCA-associated really interesting new gene (RING) domain-breast cancer; Ni-NTA, nickel-nitrilotriacetic acid; AA, amino acid; Murine Double Minute 2 (MDM2), murine double minute 2; DBD, DNA binding domain
B and C, both the wild type- and the mutant-bound RFP-E2s promote ubiquitination in an untagged E2-dependent manner. These results demonstrate that homodimerization of E2s is sufficient to promote autoubiquitination and that ubiquitination occurs in a mechanism that requires the recruitment of an additional E2 molecule (e.g. RFP linked to an active site mutant E2 was ubiquitinated as well as RFP linked to an active E2)
Summary
Targeting of most substrates to the 26 S proteasome requires covalent marking with polyubiquitin chains. This substrate was used in a ubiquitination assay in the presence of various E2s but did not undergo ubiquitination, demonstrating that the E2 enzymes are recruited through the Ub domain to the RFP-Ub fusion proteins (supplemental Fig. S4).
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