Release of ubiquitin-charged Cdc34-S - Ub from the RING domain is essential for ubiquitination of the SCF(Cdc4)-bound substrate Sic1. Cell 2003 Sep 05;114(5):611-22
Date
09/19/2003Pubmed ID
13678584DOI
10.1016/s0092-8674(03)00641-xScopus ID
2-s2.0-0141522457 (requires institutional sign-in at Scopus site) 82 CitationsAbstract
The S. 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.
Author List
Deffenbaugh AE, Scaglione KM, Zhang L, Moore JM, Buranda T, Sklar LA, Skowyra DMESH terms used to index this publication - Major topics in bold
Anaphase-Promoting Complex-CyclosomeBlotting, Western
Cell Cycle Proteins
Chromatography, Gel
Cyclin-Dependent Kinase Inhibitor Proteins
F-Box Proteins
Flow Cytometry
Kinetics
Ligases
Lysine
Models, Biological
Mutation
Protein Binding
Protein Structure, Tertiary
Recombinant Proteins
Saccharomyces cerevisiae
Saccharomyces cerevisiae Proteins
Stem Cell Factor
Time Factors
Ubiquitin
Ubiquitin-Conjugating Enzymes
Ubiquitin-Protein Ligase Complexes
Ubiquitin-Protein Ligases