| Ubiquitination regulates a host of biological processes. A cascade of enzymes is required for the covalent attachment of the small protein ubiquitin to other proteins. Among them, the E3 ubiquitin ligases are the focus of the current research because they interact directly with substrate proteins and so mediate substrate specificity of the process. A large class of ubiquitin ligases is formed by the SCF (Skp1-cullin-Fbox) complexes. It is currently believed that the F-box protein directly binds the substrate and thereby confers specificity to the ubiquitination reaction. Our study demonstrates that substrate recruitment can also be achieved by interaction of the F-box subunit with a member of a protein complex, which positions a binding partner such that it will be ubiquitinated by the SCF ligase without forming a direct contact with the F-box protein. Specifically we demonstrate that the transcriptional activator Met4 binds to the ubiquitin ligase SCFMet30 and recruits its cofactors to the ligase to regulate their ubiquitination and degradation.;The transcription factor Met4 and its cofactors, Cbf1, Met28, Met32 and Met31 control expression of a subset of genes encoding proteins involved in the biosynthesis of sulfur-containing metabolites, such as methionine, cysteine, glutathione, and S-adenosylmethionine. SCFMET30 regulates the activity of Met4 via proteolysis-independent ubiquitination. We show here that three of the Met4 cofactors, Cbf1, Met32, and Met31 are regulated by two degradation pathways. When bound to Met4, degradation of these three cofactors is mediated by SCFMet30. In contrast, free Cbf1, Met32, and Met31, which are not bound to Met4, are degraded by a second degradation pathway.;Our research focused on Met32 degradation because its degradation is crucial for cell cycle progression. We demonstrate in vivo and in vitro that Met32 interaction with SCFMet30 depends on Met4, and that Met32 ubiquitination requires Met4. Genetic analyses indicate that the other Met4 cofactors Cbf1 and Met31 are ubiquitinated by a similar mechanism, suggesting that Met4 mediates substrate specificity for its own cofactors as a component of SCFMet30-Met4. This mechanism allows selective and coordinated regulation of the entire Met4-transcription complex by the ubiquitin ligase SCFMet30.;Our findings add an additional layer to our understanding of substrate selection by E3 ubiquitin ligases and provide a general model for coordinated regulation of components in multisubunit protein complexes. |
