Schultz T F, Medina J, Hill A, Quatrano R S
Department of Biology and Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3280, USA.
Plant Cell. 1998 May;10(5):837-47. doi: 10.1105/tpc.10.5.837.
Protein-DNA complexes were formed when nuclear extracts from embryogenic rice suspension cultures or maize embryos were incubated with an abscisic acid-VIVIPAROUS1 (VP1) response element (Em1a) from the Em promoter. Monoclonal antibodies generated to GF14, a 14-3-3 protein from plants, resulted in gel retardation of the Em1a-protein complexes. Antibodies generated to the C and N termini of GF14 detected protein isoforms in rice nuclear and cytoplasmic extracts, but no differences in distribution of the GF14 isoforms were recognized between the nucleus and cytoplasm or when abscisic acid-treated and untreated tissues were compared. When recombinant GF14 fusion proteins from rice were added to nuclear extracts, novel complexes were formed that required the dimerization domain of GF14. Chemical cross-linking showed that GF-14 interacted with the basic leucine zipper factor EmBP1, which binds specifically to Em1a, and with VP1, which transactivates Em through Em1a. GF14 proteins from rice were shown to interact with VP1 in yeast through the dimerization domain of GF14. Our results indicated that GF14 interacts with both site-specific DNA binding proteins (i.e., EmBP1) and tissue-specific regulatory factors (i.e., VP1) and may provide a structural link between VP1 and the Em1a transcriptional complex.
当将来自胚性水稻悬浮培养物或玉米胚的核提取物与来自Em启动子的脱落酸-胎萌蛋白1（VP1）反应元件（Em1a）一起温育时，会形成蛋白质-DNA复合物。针对植物中的一种14-3-3蛋白GF14产生的单克隆抗体导致Em1a-蛋白质复合物出现凝胶阻滞现象。针对GF14的C端和N端产生的抗体在水稻核提取物和细胞质提取物中检测到了蛋白质异构体，但在细胞核与细胞质之间，或者在比较脱落酸处理和未处理的组织时，未发现GF14异构体的分布存在差异。当将来自水稻的重组GF14融合蛋白添加到核提取物中时，会形成需要GF14二聚化结构域的新型复合物。化学交联表明，GF-14与特异性结合Em1a的碱性亮氨酸拉链因子EmBP1以及通过Em1a反式激活Em的VP1相互作用。水稻中的GF14蛋白通过GF14的二聚化结构域在酵母中与VP1相互作用。我们的结果表明，GF14与位点特异性DNA结合蛋白（即EmBP1）和组织特异性调节因子（即VP1）都相互作用，并且可能在VP1和Em1a转录复合物之间提供结构联系。
