UC Irvine

Post-translational modification of proteins with ubiquitin plays a role in most biological processes. The key players of this system are the E3 ubiquitin ligases, which mediate substrate specific covalent attachment of ubiquitin. The SCF ubiquitin ligases are amongst best-understood E3 complexes. They are composed of Skp1, Cdc53/Cullin, Rbx1, and one of the multiple F-box proteins, which bind substrates and confer specificity to this complex. As these modular ligases share the same core components but differ in the identity of F-box proteins, the repertoire of SCF ligases present in a cell at any given time is thus governed by the abundance of different F-box proteins. Regulating F-box protein abundance is thus essential for normal cellular function and misregulation causes human diseases. In the studies presented here, we describe a novel mechanism governing maintenance of F-box protein homeostasis.

In the absence of substrates, the core SCF ligase ubiquitylates its F-box protein and targets it for degradation. Using the yeast F-box protein Met30 as a model system, we demonstrate presence of an additional mechanism for F-box protein degradation apart from autoubiquitylation. This pathway targets F-box proteins that are dissociated from Skp1, and plays a crucial role in limiting substrate shielding effects caused by association of excess F-box proteins with their substrates thereby inhibiting substrate recognition by fully assembled ligases. Characterization of this pathway shows that it is governed by the ubiquitin proteasome system and is mediated by a ligase dependent on Cdc53 but independent of Skp1 and for the first time illustrates uncoupling of Cdc53 and Skp1 function in yeast.

Furthermore, we show that Cic1 and the AAA ATPase Cdc48, along with its substrate recruiting factors Npl4/Ufd1 are involved in this mechanism of F-box protein degradation. Preliminary studies conducted to ascribe a role for these proteins in this process, suggest Cic1 as a unique factor that recruits ubiquitylated F-box proteins to Cdc48/Npl4/Ufd1 for targeting to proteasome for degradation.

Together, our findings shed light on the complexity of the ubiquitin system. They uncover a novel degradation pathway, suggest existence of yet unknown types of ligases, and introduce the concept of protein specificity factors for Cdc48.