Abstract
Cullins are members of a family of scaffold proteins that assemble multisubunit ubiquitin ligase complexes to confer substrate specificity for the ubiquitination pathway. Cullin3 (Cul3) forms a catalytically inactive BTB-Cul3-Rbx1 (BCR) ubiquitin ligase, which becomes functional upon covalent attachment of the ubiquitin homologue neural-precursor-cell-expressed and developmentally down regulated 8 (Nedd8) near the C terminus of Cul3. Current models suggest that Nedd8 activates cullin complexes by providing a recognition site for a ubiquitin-conjugating enzyme. Based on the following evidence, we propose that Nedd8 activates the BCR ubiquitin ligase by mediating the dimerization of Cul3. First, Cul3 is found as a neddylated heterodimer bound to a BTB domain-containing protein in vivo. Second, the formation of a Cul3 heterodimer is mediated by a Nedd8 molecule, which covalently attaches itself to one Cul3 molecule and binds to the winged-helix B domain at the C terminus of the second Cul3 molecule. Third, complementation experiments revealed that coexpression of two distinct nonfunctional Cul3 mutants can rescue the ubiquitin ligase function of the BCR complex. Likewise, a substrate of the BCR complex binds heterodimeric Cul3, suggesting that the Cul3 complex is active as a dimer. These findings not only provide insight into the architecture of the active BCR complex but also suggest assembly as a regulatory mechanism for activation of all cullin-based ubiquitin ligases.