| We have explored the mechanisms of polyubiquitin chain assembly with reconstituted ubiquitination of IκBα and β-catenin by the Skp1-cullin 1βTrCP F-box protein (SCFβTrCP) E3 ubiquitin ligase complex. Competition experiments revealed that SCFβTrCP formed a stable complex with β-catenin and IκBα and that this E3-substrate platform engaged in dynamic interactions with the Cdc34 E2 ubiquitin conjugating enzyme for chain elongation. Using "elongation intermediates" containing β-catenin linked with ubiquitin chains of defined length, it was observed that a K48-linked ubiquitin chain of a length greater than four, but not its K63-linkage counterpart, slowed the rate of additional ubiquitin conjugation. Thus, the ubiquitin chain length and linkage impact kinetic rates of chain elongation. Given that K48-linked tetra-ubiquitin is packed into compact conformations due to extensive intra-chain interactions between ubiquitin subunits, this topology may limit the accessibility of SCFβTrCP/Cdc34 to the distal ubiquitin's K48 and result in slowed elongation. |
