Separate SCF(CDC4) recognition elements target Cdc6 for proteolysis in S phase and mitosis

The Cdc6 DNA replication initiation factor is targeted for ubiquitin-mediated proteolysis by the E3 ubiquitin ligase SCF(CDC4) from the end of G(1) phase 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 G(1)/early S phase whilst only the newly identified site can target Cdc6 for proteolysis during mitosis.

在出芽酵母酿酒酵母（Saccharomyces cerevisiae）中，Cdc6 DNA复制起始因子（Cdc6 DNA replication initiation factor）会在G1期晚期至有丝分裂阶段，被E3泛素连接酶SCF(CDC4)靶向介导泛素依赖型蛋白水解。本研究报道了一种显性负性CDC6突变体：当其过表达时，可通过抑制细胞周期蛋白依赖性激酶（cyclin-dependent kinases, CDKs）阻滞细胞周期，进而阻断细胞通过有丝分裂期。该突变体蛋白对CDKs的抑制效率高于野生型Cdc6，部分原因在于其在细胞周期晚期完全不受SCF(CDC4)介导的蛋白水解影响，因此能够大量积累。导致该表型的突变破坏了Cdc6一级氨基酸序列中部附近的一个推定CDK磷酸化位点。本研究证实，该位点位于一个全新的Cdc4互作结构域内，该结构域与此前在该蛋白N端附近鉴定出的Cdc4互作位点不同。本研究表明，这两个位点均可在G1期晚期/S期早期介导Cdc6的蛋白水解，而仅新鉴定出的该位点能够在有丝分裂期对Cdc6进行靶向蛋白水解。