cerevisiae SCF(Cdc4) is a prototype of RING-type SCF
E3s, which recruit substrates for polyubiquitination by the Cdc34
ubiquitin-conjugating enzyme. Current models propose that Cdc34 ubiquitinates
the substrate while remaining bound to the RING domain. In contrast, we found
that the formation of a ubiquitin thiol ester regulates the Cdc34/SCF(Cdc4) binding equilibrium by increasing the dissociation
rate constant, with only a minor effect on the association rate. By using a
F72VCdc34 mutant with increased affinity for the RING domain, we demonstrate
that release of ubiquitin-charged Cdc34-S - Ub from the RING is essential for
ubiquitination of the SCF(Cdc4)-bound substrate Sic1.
Release of ubiquitin-charged E2 from E3 prior to ubiquitin transfer is a
previously unrecognized step in ubiquitination, which can explain both the
modification of multiple lysines on the recruited substrate and the extension
of polyubiquitin chains. We discuss implications of
this finding for function of other ubiquitin ligases.
PMID: 13678584 [PubMed - indexed for MEDLINE]