Fluorescence cross-correlation analyses of the molecular interaction between an Aux/IAA protein, MSG2/IAA19, and protein-protein interaction domains of auxin response factors of Arabidopsis expressed in HeLa cells
Fluorescence cross-correlation analyses of the molecular interaction between an Aux/IAA protein, MSG2/IAA19, and protein-protein interaction domains of auxin response factors of Arabidopsis expressed in HeLa cells
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DOI：
10.1093/pcp/pcj080
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发表时间：
2006-08-01
影响因子：
4.9
通讯作者：
Yamamoto, Kotaro T.
中科院分区：
文献类型：
--
作者：
Muto, Hideki;Nagao, Issei;Yamamoto, Kotaro T.
由于生长素可以通过使用生长素反应因子（ARF）及其Aux/IAA阻遏物的组合来引发许多发育反应，因此以定量方式确定两个蛋白质家族之间的相互作用是重要的。我们在HeLa细胞中瞬时表达了拟南芥ARF、MP/ARF 5和NPH 4/ARF 7以及MSG 2/IAA 19的C-末端蛋白质-蛋白质相互作用结构域（CTD），并将其与荧光蛋白融合，并用荧光交叉相关光谱（FCCS）测定了它们的分子相互作用。MSG 2与MP-CTD和NPH 4-CTD之间几乎完全相关。对于MSG 2同源二聚体、NPH 4-CTD同源二聚体和MP-CTD/NPH 4-CTD异源二聚体，发现约20%的关联。MP-CTD的同型结合可能弱于MSG 2。MSG 2定位于HeLa细胞的细胞质区室中，而其定位于植物细胞的细胞核中。MSG 2和ARF-CTD之间的异型相互作用强于每种同型相互作用的事实似乎是生长素严格控制ARF转录活性的分子基础。这些结果也表明，FCCS是有用的检查蛋白质-蛋白质相互作用，特别是转录调控。
Since auxin may elicit numerous developmental responses by the use of a combination of auxin response factors (ARFs) and their Aux/IAA repressors, it is important to determine the interaction between the two protein families in a quantitative manner. We transiently expressed the C-terminal protein-protein interaction domains (CTDs) of Arabidopsis ARFs, MP/ARF5 and NPH4/ARF7, and MSG2/IAA19, fused to fluorescent proteins in HeLa cells, and determined their molecular interactions with fluorescence cross-correlation spectroscopy (FCCS). Almost complete association was found between MSG2 and MP-CTD and between MSG2 and NPH4-CTD. Approximately 20% association was found for MSG2 homodimers, NPH4-CTD homodimers and MP-CTD/NPH4-CTD heterodimers. Homotypic binding of MP-CTD may be weaker than that of MSG2. MSG2 was localized in cytoplasmic compartments in HeLa cells, whereas it was localized in the nuclei in plant cells. The fact that the heterotypic interaction between MSG2 and ARF-CTDs is stronger than each of the homotypic interactions appears to be the molecular basis for tight control of the transcriptional activity of ARFs by auxin. These results also show that FCCS is useful to examine protein-protein interactions especially for transcriptional regulators.