Priming and Extending: A UbcH5/Cdc34 E2 Handoff Mechanism for Polyubiquitination on a SCF Substrate

We describe a mechanistic model of polyubiquitination by the SCF^βTrCP2 E3 ubiquitin (Ub) ligase using human IκBα as a substrate. Biochemical reconstitution experiments revealed that the polyubiquitination of IκBα began with the action of the UbcH5 E2 Ub-conjugating enzyme, transferring a single Ub to IκBα K21/K22 rapidly and efficiently. Subsequently, the Cdc34 E2 functioned in the formation of polyubiquitin chains. It was determined that a Ub fused at IκBα K21 acts as a receptor, directing Cdc34 for rapid and efficient K48-linked Ub chain synthesis that depends on SCF^βTrCP2 and the substrate's N terminus. The IκBα-linked fusion Ub appears to mediate direct contacts with Cdc34 and the SCF's RING subcomplex. Taken together, these results suggest a role for the multifaceted interactions between the IκBα K21/K22-linked receptor Ub, the SCF's RING complex, and Cdc34∼S∼Ub in establishing the optimal orientation of the receptor Ub to drive conjugation.