Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Conecticut 06520-8104.
Plant Cell. 2014 Feb;26(2):695-711. doi: 10.1105/tpc.113.119974. Epub 2014 Feb 21.
Members of the DDB1-CUL4-associated factors (DCAFs) family directly bind to DAMAGED DNA BINDING PROTEIN1 (DDB1) and function as the substrate receptors in CULLIN4-based E3 (CUL4) ubiquitin ligases, which regulate the selective ubiquitination of proteins. Here, we describe a DCAF protein, ABD1 (for ABA-hypersensitive DCAF1), that negatively regulates abscisic acid (ABA) signaling in Arabidopsis thaliana. ABD1 interacts with DDB1 in vitro and in vivo, indicating that it likely functions as a CUL4 E3 ligase substrate receptor. ABD1 expression is induced by ABA, and mutations in ABD1 result in ABA- and NaCl-hypersensitive phenotypes. Loss of ABD1 leads to hyperinduction of ABA-responsive genes and higher accumulation of the ABA-responsive transcription factor ABA INSENSITIVE5 (ABI5), hypersensitivity to ABA during seed germination and seedling growth, enhanced stomatal closure, reduced water loss, and, ultimately, increased drought tolerance. ABD1 directly interacts with ABI5 in yeast two-hybrid assays and associates with ABI5 in vivo by coimmunoprecipitation, and the interaction was found in the nucleus by bimolecular fluorescence complementation. Furthermore, loss of ABD1 results in a retardation of ABI5 degradation by the 26S proteasome. Taken together, these data suggest that the DCAF-CUL4 E3 ubiquitin ligase assembled with ABD1 is a negative regulator of ABA responses by directly binding to and affecting the stability of ABI5 in the nucleus.
DDB1-CUL4 相关因子 (DCAFs) 家族的成员直接与 DAMAGED DNA BINDING PROTEIN1 (DDB1) 结合,并作为 CULLIN4 基 E3 (CUL4) 泛素连接酶中的底物受体发挥作用,调节蛋白质的选择性泛素化。在这里,我们描述了一种 DCAF 蛋白,ABD1(ABA 超敏感 DCAF1 的缩写),它负调控拟南芥中的脱落酸 (ABA) 信号。ABD1 在体外和体内与 DDB1 相互作用,表明它可能作为 CUL4 E3 连接酶底物受体发挥作用。ABD1 的表达受 ABA 诱导,ABD1 突变导致 ABA 和 NaCl 超敏表型。ABD1 的缺失导致 ABA 反应基因的过度诱导和 ABA 反应转录因子 ABI5 的更高积累,导致种子萌发和幼苗生长期间对 ABA 敏感,增强了气孔关闭,减少了水分流失,最终提高了耐旱性。ABD1 在酵母双杂交测定中直接与 ABI5 相互作用,并通过共免疫沉淀在体内与 ABI5 相关联,并且通过双分子荧光互补在核内发现了这种相互作用。此外,ABD1 的缺失导致 ABI5 通过 26S 蛋白酶体的降解延迟。总之,这些数据表明,与 ABD1 组装的 DCAF-CUL4 E3 泛素连接酶通过直接结合并影响核内 ABI5 的稳定性,是 ABA 反应的负调节剂。