Lee Jae-Hoon, Terzaghi William, Gusmaroli Giuliana, Charron Jean-Benoit F, Yoon Hye-Jin, Chen Haodong, He Yizhou Joseph, Xiong Yue, Deng Xing Wang
Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Conecticut 06520-8104, USA.
Plant Cell. 2008 Jan;20(1):152-67. doi: 10.1105/tpc.107.055418. Epub 2008 Jan 25.
A subset of WD40 proteins that contain a DWD motif (for DDB1 binding WD40) is reported to act as substrate receptors for DDB1-CUL4-ROC1 (for Damaged DNA Binding 1-Cullin 4-Regulator of Cullins 1) based E3 ubiquitin ligases in humans. Here, we report 85 Arabidopsis thaliana and 78 rice (Oryza sativa) proteins containing the conserved 16-amino acid DWD motif. We show by yeast two-hybrid and in vivo coimmunoprecipitation that 11 Arabidopsis DWD proteins directly interact with DDB1 and thus may serve as substrate receptors for the DDB1-CUL4 machinery. We further examine whether the DWD protein PRL1 (for Pleiotropic Regulatory Locus 1) may act as part of a CUL4-based E3 ligase. PRL1 directly interacts with DDB1, and prl1 and cul4cs mutants exhibited similar phenotypes, including altered responses to a variety of stimuli. Moreover, AKIN10 (for Arabidopsis SNF1 Kinase Homolog 10) was degraded more slowly in cell extracts of prl1 and cul4cs than in cell extracts of the wild type. Thus, both genetic and biochemical analyses support the conclusion that PRL1 is the substrate receptor of a CUL4-ROC1-DDB1-PRL1 E3 ligase involved in the degradation of AKIN10. This work adds a large new family to the current portfolio of plant E3 ubiquitin ligases.
据报道，含有DWD基序（用于DDB1结合的WD40）的WD40蛋白亚群在人类中作为基于DDB1 - CUL4 - ROC1（损伤DNA结合蛋白1 - 库林4 - 库林调节因子1）的E3泛素连接酶的底物受体发挥作用。在此，我们报告了85种拟南芥和78种水稻（Oryza sativa）中含有保守的16个氨基酸DWD基序的蛋白质。我们通过酵母双杂交和体内共免疫沉淀表明，11种拟南芥DWD蛋白直接与DDB1相互作用，因此可能作为DDB1 - CUL4机制的底物受体。我们进一步研究DWD蛋白PRL1（多效性调控位点1）是否可能作为基于CUL4的E3连接酶的一部分发挥作用。PRL1直接与DDB1相互作用，prl1和cul4cs突变体表现出相似的表型，包括对多种刺激的反应改变。此外，与野生型细胞提取物相比，AKIN10（拟南芥SNF1激酶同源物10）在prl1和cul4cs细胞提取物中的降解更慢。因此，遗传和生化分析均支持PRL1是参与AKIN10降解的CUL4 - ROC1 - DDB1 - PRL1 E3连接酶的底物受体这一结论。这项工作为植物E3泛素连接酶的现有组合增添了一个庞大的新家族。
