Identification of dynamin as an interactor of rice GIGANTEA by tandem affinity purification (TAP)
Identification of dynamin as an interactor of rice GIGANTEA by tandem affinity purification (TAP)
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DOI：
10.1093/pcp/pcn019
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发表时间：
2008-03-01
影响因子：
4.9
通讯作者：
Shimamoto, Ko
中科院分区：
文献类型：
--
作者：
Abe, Makoto;Fujiwara, Masayuki;Shimamoto, Ko
GIGANTEA（GI）、CONSTANS（CO）和FLOWARDINLOCUS T（FT）调节拟南芥的光周期开花。在水稻中，OsGI、Hd1和Hd3a分别被鉴定为GI、CO和FT的直向同源基因，也是重要的开花调控基因。虽然GI在开花和生物钟中都有作用，但我们对其生化功能的了解仍然有限。在这项研究中，我们通过使用串联亲和纯化（TAP）方法纯化新的OsGI相互作用蛋白。TAP方法已被有效地用于许多模式物种中，以分离与目标蛋白相互作用的蛋白质。然而，在植物中，TAP方法仅在少数研究中使用，并且以前没有通过该方法分离出新的蛋白质。我们产生了转基因水稻植物和细胞培养表达TAP标记版本的OsGI。经过两步纯化程序后，通过质谱分析相互作用的蛋白质。包括发动蛋白在内的7个蛋白被鉴定为OsGI相互作用蛋白。OsGI与发动蛋白的相互作用通过使用Myc标记的OsGI版本的免疫共沉淀来验证。此外，对拟南芥动力蛋白突变体的分析表明，虽然突变体的开花时间与野生型植物的开花时间没有不同，但在突变体中观察到气生莲座丛表型。我们还发现，OsGI是目前在细胞核和胞质溶胶通过Western印迹分析和瞬时测定。这些结果表明，TAP方法是有效的分离新的蛋白质，与目标蛋白在植物中的相互作用。
GIGANTEA (GI), CONSTANS (CO) and FLOWERING LOCUS T (FT) regulate photoperiodic flowering in Arabidopsis. In rice, OsGI, Hd1 and Hd3a were identified as orthologs of GI, CO and FT, respectively, and are also important regulators of flowering. Although GI has roles in both flowering and the circadian clock, our understanding of its biochemical functions is still limited. In this study, we purified novel OsGI-interacting proteins by using the tandem affinity purification (TAP) method. The TAP method has been used effectively in a number of model species to isolate proteins that interact with proteins of interest. However, in plants, the TAP method has been used in only a few studies, and no novel proteins have previously been isolated by this method. We generated transgenic rice plants and cell cultures expressing a TAP-tagged version of OsGI. After a two-step purification procedure, the interacting proteins were analyzed by mass spectrometry. Seven proteins, including dynamin, were identified as OsGI-interacting proteins. The interaction of OsGI with dynamin was verified by co-immunoprecipitation using a myc-tagged version of OsGI. Moreover, an analysis of Arabidopsis dynamin mutants indicated that although the flowering times of the mutants were not different from those of wild-type plants, an aerial rosette phenotype was observed in the mutants. We also found that OsGI is present in both the nucleus and the cytosol by Western blot analysis and by transient assays. These results indicate that the TAP method is effective for the isolation of novel proteins that interact with target proteins in plants.