Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut 06520-8104, USA.
Plant Cell. 2010 Jun;22(6):1716-32. doi: 10.1105/tpc.109.073783. Epub 2010 Jun 4.
To elucidate potential roles of CUL4-DDB1-DWD (for Cullin 4-Damaged DNA Binding1-DDB1 binding WD40) E3 ligases in abscisic acid (ABA) signaling, we examined ABA sensitivities of T-DNA mutants of a number of Arabidopsis thaliana DWD genes, which encode substrate receptors for CUL4 E3 ligases. Mutants in two DWD genes, DWA1 and DWA2 (DWD hypersensitive to ABA1 and 2), had ABA-hypersensitive phenotypes. Both proteins interacted with DDB1 in yeast two-hybrid assays and associated with DDB1 and CUL4 in vivo, implying they could form CUL4-based complexes. Several ABA-responsive genes were hyperinduced in both mutants, and the ABA-responsive transcription factors ABA INSENSITIVE 5 (ABI5) and MYC2 accumulated to high levels in the mutants after ABA treatment. Moreover, ABI5 interacted with DWA1 and DWA2 in vivo. Cell-free degradation assays showed ABI5 was degraded more slowly in dwa1 and dwa2 than in wild-type cell extracts. Therefore, DWA1 and/or DWA2 may be the substrate receptors for a CUL4 E3 ligase that targets ABI5 for degradation. Our data indicate that DWA1 and DWA2 can directly interact with each other, and their double mutants exhibited enhanced ABA and NaCl hypersensitivities, implying they can act together. This report thus describes a previously unknown heterodimeric cooperation between two independent substrate receptors for CUL4-based E3 ligases.
为了阐明 CUL4-DDB1-DWD（用于 Cullin 4-Damaged DNA Binding1-DDB1 结合 WD40）E3 连接酶在脱落酸（ABA）信号转导中的潜在作用，我们研究了拟南芥 DWD 基因的 T-DNA 突变体对 ABA 的敏感性，这些基因编码 CUL4 E3 连接酶的底物受体。两个 DWD 基因（DWA1 和 DWA2）的突变体表现出 ABA 超敏表型。这两种蛋白在酵母双杂交实验中相互作用，并与体内的 DDB1 和 CUL4 相关联，表明它们可以形成基于 CUL4 的复合物。在这两个突变体中，几个 ABA 响应基因被高度诱导，并且在 ABA 处理后，ABA 不敏感转录因子 ABI5 和 MYC2 在突变体中积累到高水平。此外，ABI5 在体内与 DWA1 和 DWA2 相互作用。无细胞降解测定表明，在 dwa1 和 dwa2 中 ABI5 的降解速度比在野生型细胞提取物中慢。因此，DWA1 和/或 DWA2 可能是 CUL4 E3 连接酶的底物受体，该酶将 ABI5 作为降解的靶标。我们的数据表明，DWA1 和 DWA2 可以直接相互作用，它们的双突变体表现出增强的 ABA 和 NaCl 超敏性，表明它们可以协同作用。因此，本报告描述了以前未知的 CUL4 基 E3 连接酶的两个独立底物受体之间的异二聚体合作。
