Liu Cong, Poitelea Marius, Watson Adam, Yoshida Shu-hei, Shimoda Chikashi, Holmberg Christian, Nielsen Olaf, Carr Antony M
Genome Damage and Stability Centre, University of Sussex, Brighton, UK.
EMBO J. 2005 Nov 16;24(22):3940-51. doi: 10.1038/sj.emboj.7600854. Epub 2005 Oct 27.
Cullin-4 forms a scaffold for multiple ubiquitin ligases. In Schizosaccharomyces pombe, the Cullin-4 homologue (Pcu4) physically associates with Ddb1 and the COP9 signalosome (CSN). One target of this complex is Spd1. Spd1 regulates ribonucleotide reductase (RNR) activity. Spd1 degradation during S phase, or following DNA damage of G2 cells, results in the nuclear export of the small RNR subunit. We demonstrate that Cdt2, an unstable WD40 protein, is a regulatory subunit of Pcu4-Ddb1-CSN ubiquitin ligase. cdt2 deletion stabilises Spd1 and prevents relocalisation of the small RNR subunit from the nucleus to the cytoplasm. cdt2+ is periodically transcribed by the Cdc10/DSC1 transcription factor during S phase and transiently transcribed following DNA damage of G2 cells, corresponding to Spd1 degradation profiles. Cdt2 co-precipitates with Spd1, and Cdt2 overexpression results in constitutive Spd1 degradation. We propose that Cdt2 incorporation into the Pcu4-Ddb1-CSN complex prompts Spd1 targeting and subsequent degradation and that Cdt2 is a WD40 repeat adaptor protein for Cullin-4-based ubiquitin ligase.
Cullin-4为多种泛素连接酶形成一个支架。在粟酒裂殖酵母中，Cullin-4同源物（Pcu4）与Ddb1及COP9信号体（CSN）发生物理关联。该复合物的一个靶标是Spd1。Spd1调节核糖核苷酸还原酶（RNR）的活性。在S期或G2期细胞DNA损伤后Spd1的降解，会导致小RNR亚基的核输出。我们证明，不稳定的WD40蛋白Cdt2是Pcu4-Ddb1-CSN泛素连接酶的一个调节亚基。cdt2缺失会使Spd1稳定，并阻止小RNR亚基从细胞核重新定位到细胞质。cdt2⁺在S期由Cdc10/DSC1转录因子周期性转录，并在G2期细胞DNA损伤后短暂转录，这与Spd1的降解模式相对应。Cdt2与Spd1共沉淀，且Cdt2过表达导致Spd1组成型降解。我们提出，Cdt2掺入Pcu4-Ddb1-CSN复合物会促使Spd1被靶向并随后降解，并且Cdt2是基于Cullin-4的泛素连接酶的WD40重复衔接蛋白。
