title: Separate SCF(CDC4) recognition elements target Cdc6 for proteolysis in S phase and mitosis.
abstract: The Cdc6 DNA replication initiation factor is targeted for ubiquitin-mediated proteolysis by the E3 ubiquitin ligase SCF(CDC4) from the end of G1phase until mitosis in the budding yeast Saccharomyces cerevisiae. Here we describe a dominant-negative CDC6 mutant that, when overexpressed, arrests the cell cycle by inhibiting cyclin-dependent kinases (CDKs) and, thus, prevents passage through mitosis. This mutant protein inhibits CDKs more efficiently than wild-type Cdc6, in part because it is completely refractory to SCF(CDC4)-mediated proteolysis late in the cell cycle and consequently accumulates to high levels. The mutation responsible for this phenotype destroys a putative 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 proteolysis in late G1/early S phase whilst only the newly identified site can target Cdc6 for proteolysis during mitosis.
authors.affiliation: ICRF Clare Hall Laboratories, South Mimms EN6 3LD, UK.
authors.forename: G
authors.initials: G
authors.lastname: Perkins
authors.forename: L S
authors.initials: LS
authors.lastname: Drury
authors.forename: J F
authors.initials: JF
authors.lastname: Diffley
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D017931:DNA Primers; D044783:F-Box Proteins; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D005690:Galactose / Q000378:metabolism; D005838:Genotype; D005947:Glucose / Q000378:metabolism; D008938:Mitosis / Q000502:physiology; D015739:Nocodazole / Q000494:pharmacology; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D016196:S Phase / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000235:genetics / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013489:Suppression, Genetic; D044767:Ubiquitin-Protein Ligases
chemical_list: C080443:CDC4 protein, S cerevisiae; C066503:CDC6 protein, S cerevisiae; D018797:Cell Cycle Proteins; D017931:DNA Primers; D044783:F-Box Proteins; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D044767:Ubiquitin-Protein Ligases; D018844:Cyclin-Dependent Kinases; D005947:Glucose; D015739:Nocodazole; D005690:Galactose