Functional isolation of novel nuclear proteins showing a variety of subnuclear localizations
Functional isolation of novel nuclear proteins showing a variety of subnuclear localizations
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DOI：
10.1105/tpc.104.028456
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发表时间：
2005-02-01
期刊：
影响因子：
11.6
通讯作者：
Kurata, N
中科院分区：
文献类型：
--
作者：
Moriguchi, K;Suzuki, T;Kurata, N
核蛋白在基因组不稳定性、器官发育阶段和通过基因表达的生理反应的基本调节中起关键作用。虽然核蛋白已被证明占酵母总蛋白质的约四分之一，但没有有效的方法来鉴定新的核蛋白已被应用于植物。在这项研究中，尝试分离核蛋白的水稻，并确定了几个新的核蛋白显示各种亚核定位。核运输陷阱（NTT）系统是一种改良的双杂交系统，它从水稻NTT cDNA文库中分离出许多核蛋白。通过将蛋白质基因子集的绿色荧光蛋白融合构建体瞬时引入洋葱（Allium，cepa）细胞中来确认分离的蛋白质的核定位。这些蛋白质中的大多数，包括新的蛋白质和最初被归类为细胞质蛋白质的蛋白质，被发现定位在细胞核中。未知蛋白质的详细表征揭示了各种亚核定位，表明它们可能与染色质和核基质的病灶或斑点状分布。有的还表现为细胞核和细胞质的双重分布。在新的蛋白质组分中，通过免疫染色进一步鉴定了一种蛋白质在水稻特定器官中的染色质相关定位。因此，可以使用NTT方法分离具有染色质或基质缔合能力的多种新的核结构蛋白，其通过影响某些器官发育或细胞反应性而在核组织中是重要的。由于转录调节因子以外的核蛋白在植物中很少被表征，例如基质蛋白和发育特异性染色质蛋白，它们的鉴定和随后的表征可以为由核组织控制的全基因组调控机制提供重要信息。
Nuclear proteins play key roles in the fundamental regulation of genome instability, the phases of organ development, and physiological responsiveness through gene expression. Although nuclear proteins have been shown to account for approximately one-fourth of total proteins in yeast, no efficient method to identify novel nuclear proteins has been applied to plants. In this study, a trial to isolate nuclear proteins in rice was attempted, and several novel nuclear proteins showing a variety of subnuclear localizations were identified. The nuclear transportation trap (NTT) system, which is a modified two-hybrid system, isolated many nuclear proteins from rice (Oryza sativa) NTT cDNA libraries. Nuclear localization of the isolated proteins was confirmed by transient introduction of green fluorescent protein fusion constructs for a subset of protein genes into onion (Allium, cepa) cells. The majority of these proteins, including novel proteins and proteins initially categorized as cytoplasmic proteins, were revealed to be localized in the nucleus. Detailed characterization of unknown proteins revealed various subnuclear localizations, indicating their possible association with chromatin and the nuclear matrix with a foci or speckle-like distribution. Some also showed dual distribution in the nucleus and cytoplasm. In the novel protein fraction, a protein was further identified for its chromatin-associated localization in a specific organ of rice by immunostaining. Thus, a variety of novel nuclear architectural proteins with chromatin or matrix associating abilities, which are important in nuclear organization by influencing certain organ developments or cell responsiveness, can be isolated using the NTT method. Because nuclear proteins other than transcription regulators have rarely been characterized in plants, such as matrix proteins and development-specific chromatin proteins, their identification and subsequent characterization could provide important information for genome-wide regulatory mechanisms controlled by nuclear organization.