Abstract
We have explored the mechanisms of polyubiquitin chain assembly with reconstituted ubiquitination of IκBα and β-catenin by the Skp1-cullin 1-βTrCP F-box protein (SCF(βTrCP)) E3 ubiquitin (Ub) ligase complex. Competition experiments revealed that SCF(βTrCP) formed a complex with IκBα 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 β-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 SCF(βTrCP)/Cdc34 to the distal Ub Lys-48 and result in slowed elongation.
Highlights
Polyubiquitin chains are signaling polypeptides altering the fate of substrates
The optimal concentration of sodium glutamate was found at 0.1 M, a condition in which a majority of the IB␣ 1–54 input substrate was converted to high molecular weight products in the presence of both UbcH5c and Cdc34 (Fig. 1, C and D)
Polyubiquitin chains act as signaling polypeptides that upon attachment to a target protein can either commit a substrate to proteolytic destruction or, alternatively, change the substrate protein function
Summary
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. 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 Nedd modified E3-substrate platform engaged in dynamic interactions with the Cdc 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. 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/Cdc to the distal Ub Lys-48 and result in slowed elongation
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