Protein-protein interaction studies provide valuable insights into cellular signaling. Brassinosteroid (BR) signaling is initiated by the hormone-binding receptor Brassinosteroid Insensitive 1 (BRI1) and its co-receptor BRI1 Associated Kinase 1 (BAK1). BRI1 and BAK1 were shown to interact independently with the Receptor-Like Protein 44 (RLP44), which is implicated in BRI1/BAK1-dependent cell wall integrity perception. To demonstrate the proposed complex formation of BRI1, BAK1 and RLP44, we established three-fluorophore intensity-based spectral Förster resonance energy transfer (FRET) and FRET-fluorescence lifetime imaging microscopy (FLIM) for living plant cells. Our evidence indicates that RLP44, BRI1 and BAK1 form a ternary complex in a distinct plasma membrane nanodomain. In contrast, although the immune receptor Flagellin Sensing 2 (FLS2) also forms a heteromer with BAK1, the FLS2/BAK1 complexes are localized to other nanodomains. In conclusion, both three-fluorophore FRET approaches provide a feasible basis for studying the in vivo interaction and sub-compartmentalization of proteins in great detail.

中文翻译：

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三荧光团 FRET 能够分析活植物细胞中的三元蛋白关联

蛋白质-蛋白质相互作用研究为细胞信号传导提供了有价值的见解。油菜素类固醇 (BR) 信号由激素结合受体油菜素类固醇不敏感 1 (BRI1) 及其共同受体 BRI1 相关激酶 1 (BAK1) 启动。BRI1 和 BAK1 与受体样蛋白 44 (RLP44) 独立相互作用，这与 BRI1/BAK1 依赖性细胞壁完整性感知有关。为了证明所提议的 BRI1、BAK1 和 RLP44 的复合物形成，我们为活植物细胞建立了基于三荧光团强度的光谱 Förster 共振能量转移 (FRET) 和 FRET 荧光寿命成像显微镜 (FLIM)。我们的证据表明 RLP44、BRI1 和 BAK1 在不同的质膜纳米域中形成三元复合物。相比之下，虽然免疫受体鞭毛蛋白传感 2 (FLS2) 也与 BAK1 形成异聚体，但 FLS2/BAK1 复合物定位于其他纳米域。总之，这两种三荧光团 FRET 方法都为详细研究蛋白质的体内相互作用和亚区室化提供了可行的基础。