Immink Richard G H, Gadella Theodorus W J, Ferrario Silvia, Busscher Marco, Angenent Gerco C
Plant Research International, P.O. Box 16, 6700 AA Wageningen, The Netherlands.
Proc Natl Acad Sci U S A. 2002 Feb 19;99(4):2416-21. doi: 10.1073/pnas.042677699.
Over the last decade, the yeast two-hybrid system has become the tool to use for the identification of protein-protein interactions and recently, even complete interactomes were elucidated by this method. Nevertheless, it is an artificial system that is sensitive to errors resulting in the identification of false-positive and false-negative interactions. In this study, plant MADS box transcription factor interactions identified by yeast two-hybrid systems where studied in living plant cells by a technique based on fluorescence resonance energy transfer (FRET). Petunia MADS box proteins were fused to either cyan fluorescent protein or yellow fluorescent protein and transiently expressed in protoplasts followed by FRET-spectral imaging microscopy and FRET-fluorescence lifetime imaging microscopy to detect FRET and hence protein-protein interactions. All petunia MADS box heterodimers identified in yeast were confirmed in protoplasts. However, in contrast to the yeast two-hybrid results, homodimerization was demonstrated in plant cells for three petunia MADS box proteins. Heterodimers were identified between the ovule-specific MADS box protein FLORAL BINDING PROTEIN 11 and members of the petunia FLORAL BINDING PROTEIN 2 subfamily, which are also expressed in ovules, suggesting that these dimers play a role in ovule development. Furthermore, the role of dimerization in translocation of MADS box protein dimers to the nucleus is demonstrated, and the nuclear localization signal of MADS box proteins has been mapped to the N-terminal region of the MADS domain by means of mutant analyses.
在过去十年中，酵母双杂交系统已成为用于鉴定蛋白质-蛋白质相互作用的工具，最近，甚至通过该方法阐明了完整的相互作用组。然而，它是一个对错误敏感的人工系统，可能导致假阳性和假阴性相互作用的鉴定。在本研究中，通过基于荧光共振能量转移（FRET）的技术，在活的植物细胞中研究了酵母双杂交系统鉴定的植物MADS盒转录因子相互作用。将矮牵牛MADS盒蛋白与青色荧光蛋白或黄色荧光蛋白融合，并在原生质体中瞬时表达，随后通过FRET光谱成像显微镜和FRET荧光寿命成像显微镜检测FRET，从而检测蛋白质-蛋白质相互作用。在酵母中鉴定的所有矮牵牛MADS盒异二聚体在原生质体中得到了证实。然而，与酵母双杂交结果相反，在植物细胞中证实了三种矮牵牛MADS盒蛋白的同二聚化。在胚珠特异性MADS盒蛋白FLORAL BINDING PROTEIN 11与也在胚珠中表达的矮牵牛FLORAL BINDING PROTEIN 2亚家族成员之间鉴定到了异二聚体，这表明这些二聚体在胚珠发育中起作用。此外，还证明了二聚化在MADS盒蛋白二聚体转运到细胞核中的作用，并且通过突变分析将MADS盒蛋白的核定位信号定位到MADS结构域的N端区域。
