A SnapShot of Ubiquitin Chain Elongation LYSINE 48-TETRA-UBIQUITIN SLOWS DOWN UBIQUITINATION

Background: Polyubiquitin chains are signaling polypeptides altering the fate of substrates. Results: A Lys-48-ubiquitin chain of a length greater than four, but not its Lys-63 linkage counterparts, slowed the rate of additional ubiquitin conjugation. Conclusion: The ubiquitin chain length and linkage may impact kinetic rates of chain elongation. Significance: Our findings suggest a self-restraining mechanism that limits Lys-48-polyubiquitination. We have explored the mechanisms of polyubiquitin chain assembly with reconstituted ubiquitination of IB and -catenin by the Skp1-cullin 1-TrCP F-box protein (SCFTrCP) E3 ubiquitin (Ub) ligase complex. Competition experiments revealed that SCFTrCP formed a complex with IB and that the Nedd8 modified E3-substrate platform engaged in dynamic interactions with the Cdc34 E2 Ub conjugating enzyme for chain elongation. Using elongation intermediates containing -catenin linked with Ub chains of defined length, it was observed that a Lys-48-Ub chain of a length greater than four, but not its Lys-63 linkage counterparts, slowed the rate of additional Ub conjugation. Thus, the Ub chain length and linkage impact kinetic rates of chain elongation. Given that Lys-48-tetra-Ub is packed into compact conformations due to extensive intrachain interactions between Ub subunits, this topology may limit the accessibility of SCFTrCP/Cdc34 to the distal Ub Lys-48 and result in slowed elongation.