title: Evaluation of a diffusion-driven mechanism for substrate ubiquitination by the SCF-Cdc34 ubiquitin ligase complex.
abstract: Release of ubiquitin-charged Cdc34 from the SCF ubiquitin ligase followed by diffusion-driven collision with substrate has been proposed to underlie ubiquitination of the canonical SCF substrate Sic1. Cdc34 F72V, reported to be defective in dissociation from SCF, served as key validation. Here, we test predictions of this "hit-and-run" hypothesis. We find that Cdc34 F72V is generally defective in SCF-mediated activation but, contrary to expectation, does not compete with wild-type Cdc34 in vitro or in vivo and can fulfill the physiological role of Cdc34 with only moderate delay in Sic1 turnover. Whereas a hit-and-run mechanism might explain how Cdc34 can transfer ubiquitin to the ends of growing ubiquitin chains on SCF-bound substrates, molecular modeling suggests that an E2 docked to SCF can do so without dissociating. We propose that interactions between Cdc34 approximately Ub and SCF directly activate ubiquitin transfer within a substrate-SCF-Cdc34 approximately Ub ternary complex.
authors.affiliation: Howard Hughes Medical Institute, Division of Biology, 156-29, California Institute of Technology, 1200 East California Boulevard, Pasadena, California 91125, USA. mpetroski@rigel.com <mpetroski@rigel.com>
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Petroski
authors.forename: Gary
authors.initials: G
authors.lastname: Kleiger
authors.forename: Raymond J
authors.initials: RJ
authors.lastname: Deshaies
mesh_terms: D064173:Anaphase-Promoting Complex-Cyclosome; D002478:Cells, Cultured; D004058:Diffusion; D008954:Models, Biological; D008958:Models, Molecular; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D019089:Stem Cell Factor / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes / Q000378:metabolism*; D014452:Ubiquitins / Q000378:metabolism*
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D019089:Stem Cell Factor; D014452:Ubiquitins; C577449:CDC34 protein, S cerevisiae; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome