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 beta TrCP2 E3 ubiquitin (Ub) ligase using human I kappa B alpha as a substrate. Biochemical reconstitution experiments revealed that the polyubiquitination of I kappa B alpha began with the action of the UbcH5 E2 Ub-conjugating enzyme, transferring a single Ub to I kappa B alpha K21/K22 rapidly and efficiently. Subsequently, the Cdc34 E2 functioned in the formation of polyubiquitin chains. It was determined that a Ub fused at I kappa B alpha K21 acts as a receptor, directing Cdc34 for rapid and efficient K48-linked Ub chain synthesis that depends on SCF beta TrCP2 and the substrate's N terminus. The I kappa B alpha-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 I kappa B alpha K21/K22-linked receptor Ub, the SCF's RING complex, and Cdc34 similar to S similar to Ub in establishing the optimal orientation of the receptor Ub to drive conjugation.