Separate SCFCDC4 recognition elements target Cdc6 for proteolysis in S phase and mitosis

The Cdc6 DNA replication initiation factor is targeted for ubiquitin-mediat ed proteolysis by the E3 ubiquitin ligase SCFCDC4 from the end of G(1) phas e until mitosis in the budding yeast Saccharomyces cerevisiae. Here we desc ribe a dominant-negative CDC6 mutant that, when overexpressed, arrests the cell cycle by inhibiting cyclin-dependent kinases (CDKs) and, thus, prevent s passage through mitosis. This mutant protein inhibits CDKs more efficient ly than wild-type Cdc6, in part because it is completely refractory to SCFC DC4- mediated proteolysis late in the cell cycle and consequently accumulat es to high levels. The mutation responsible for this phenotype destroys a p utative CDK phosphorylation site near the middle of the Cdc6 primary amino acid sequence. We show that this site lies within a novel Cdc4-interacting domain distinct from a Cdc4-interacting site identified previously near the N-terminus of the protein. We show that both sites can target Cdc6 for pro teolysis in late G(1)/early S phase whilst only the newly identified site c an target Cdc6 for proteolysis during mitosis.