Molecular interactions of a soluble gibberellin receptor, GID1, with a rice DELLA protein, SLR1, and gibberellin
Molecular interactions of a soluble gibberellin receptor, GID1, with a rice DELLA protein, SLR1, and gibberellin
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DOI：
10.1105/tpc.106.043729
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发表时间：
2007-07-01
期刊：
影响因子：
11.6
通讯作者：
Matsuokaa, Makoto
中科院分区：
文献类型：
--
作者：
Ueguchi-Tanaka, Miyako;Nakajima, Masatoshi;Matsuokaa, Makoto
赤霉素不敏感 DWARF1 (GID1) 编码可溶性赤霉素 (GA) 受体，与激素敏感脂肪酶 (HSL) 具有序列相似性。此前，酵母双杂交 (Y2H) 测定表明 GID1-GA 复合物直接与 SLENDER RICE1 (SLR1) 相互作用，SLENDER RICE1 (SLR1) 是 GA 信号传导中的一种 DELLA 阻遏蛋白。在这里，我们通过下拉和双分子荧光互补 (BiFC) 实验证明，GA 依赖性 GID1-SLR1 相互作用也发生在植物中。在 Y2H 测定中发现 GA4 对 GID1 具有最高的亲和力，并且是植物中 GA 最有效的形式。使用 Y2H、凝胶过滤和 BiFC 方法对 SLR1 的结构域进行分析表明，SLR1 的 DELLA 和 TVHYNP 结构域是 GID1-SLR1 相互作用所必需的。为了确定 GID1 对 GA 和 SLR1 相互作用的重要区域，我们使用了许多不同的 GID1 突变版本，例如自发突变 GID1、N 端和 C 端截短的 GID1 以及用 Ala 替换保守氨基酸的诱变 GID1 蛋白。对 SLR1 相互作用重要的氨基酸残基与分散在整个 GID1 分子中的 GA 结合所需的残基完全重叠。当我们将这些残基绘制在通过与 HSL 三级结构类比预测的 GID1 结构上时，许多残基位于与底物结合袋和盖相对应的区域。此外，SLR1 稳定了 GA-GID1 相互作用。基于这些观察，我们提出了 GA、GID1 和 SLR1 之间相互作用的分子模型。
GIBBERELLIN INSENSITIVE DWARF1 ( GID1) encodes a soluble gibberellin ( GA) receptor that shares sequence similarity with a hormone- sensitive lipase (HSL). Previously, a yeast two-hybrid (Y2H) assay revealed that the GID1-GA complex directly interacts with SLENDER RICE1 (SLR1), a DELLA repressor protein in GA signaling. Here, we demonstrated, by pulldown and bimolecular fluorescence complementation (BiFC) experiments, that the GA-dependent GID1-SLR1 interaction also occurs in planta. GA4 was found to have the highest affinity to GID1 in Y2H assays and is the most effective form of GA in planta. Domain analyses of SLR1 using Y2H, gel filtration, and BiFC methods revealed that the DELLA and TVHYNP domains of SLR1 are required for the GID1-SLR1 interaction. To identify the important regions of GID1 for GA and SLR1 interactions, we used many different mutant versions of GID1, such as the spontaneous mutant GID1s, N- and C-terminal truncated GID1s, and mutagenized GID1 proteins with conserved amino acids replaced with Ala. The amino acid residues important for SLR1 interaction completely overlapped the residues required for GA binding that were scattered throughout the GID1 molecule. When we plotted these residues on the GID1 structure predicted by analogy with HSL tertiary structure, many residues were located at regions corresponding to the substrate binding pocket and lid. Furthermore, the GA-GID1 interaction was stabilized by SLR1. Based on these observations, we proposed a molecular model for interaction between GA, GID1, and SLR1.