Interacting proteins and differences in nuclear transport reveal specific functions for the NAP1 family proteins in plants
Interacting proteins and differences in nuclear transport reveal specific functions for the NAP1 family proteins in plants
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DOI：
10.1104/pp.105.060509
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发表时间：
2005-07-01
期刊：
影响因子：
7.4
通讯作者：
Shen, WH
中科院分区：
文献类型：
--
作者：
Dong, AW;Liu, ZQ;Shen, WH
核小体组装蛋白1(NAP1)从酵母到人类都是保守的，它促进了核小体作为组蛋白伴侣蛋白的体外组装。然而，许多NAP1蛋白被报道定位在细胞质中，这与它们在细胞核中的功能不同。我们首先通过鉴定相互作用伙伴和直接检测绿色荧光蛋白标记蛋白的定位，研究了烟草和水稻NAP1家族蛋白的亚细胞定位。通过软霉素B处理烟草细胞和核输出信号的诱变，我们证明了NICTA；NAP1；1和Orysa；NAP1；1在细胞质和细胞核之间穿梭。结合烟草NAP1蛋白与组蛋白H_2A和H_2B结合的证明，我们的结果支持目前的模型，并提供了额外的证据，证明NAP1作为组蛋白伴侣蛋白的功能在植物中似乎是保守的。此外，我们还发现烟草NAP1蛋白与微管蛋白和有丝分裂周期蛋白NICTA；CYCB1；1相互作用，提示NAP1在微管动力学中发挥作用。有趣的是，尽管它们与上述NAP1蛋白有很高的同源性，但其他三个烟草蛋白和Orysa；NAP1；2并不显示核质穿梭，仅定位于细胞质。此外，缺少典型核定位信号序列的Orysa；NAP1；3定位于细胞质和细胞核。最后，我们发现只有Orysa；NAP1；3在体外可以被酪蛋白激酶2α磷酸化。然而，通过突变研究证明，这种磷酸化并不是导致水稻；NAP1；3核进口的原因。总之，我们的结果为阐明NAP1家族蛋白在植物中发挥作用的分子机制提供了重要的一步。
Nucleosome assembly protein 1 (NAP1) is conserved from yeast to human and facilitates the in vitro assembly of nucleosomes as a histone chaperone. Inconsistent with their proposed function in the nucleus, however, many NAP1 proteins had been reported to localize in the cytoplasm. We investigated the subcellular localization of tobacco ( Nicotiana tabacum) and rice ( Oryza sativa) NAP1 family proteins first by identification of interacting partners and by direct examination of the localization of green fluorescent protein-tagged proteins. Through treatment of tobacco cells with leptomycin B and mutagenesis of nuclear export signal, we demonstrated that Nicta; NAP1; 1 and Orysa; NAP1; 1 shuttle between the cytoplasm and the nucleus. Together with the demonstration that tobacco NAP1 proteins bind histone H2A and H2B, our results support the current model and provide additional evidence that function of NAP1 as histone chaperones appears to be conserved in plants. In addition, we show that tobacco NAP1 proteins interact with tubulin and the mitotic cyclin Nicta; CYCB1; 1, suggesting a role for NAP1 in microtubule dynamics. Interestingly, in spite of their high homology with the above NAP1 proteins, the other three tobacco proteins and Orysa; NAP1; 2 did not show nucleocytoplasmic shuttling and were localized only in the cytoplasm. Moreover, Orysa; NAP1; 3 that lacks a typical nuclear localization signal sequence was localized in both the cytoplasm and the nucleus. Finally, we show that only Orysa; NAP1; 3 could be phosphorylated by casein kinase 2 alpha in vitro. However, this phosphorylation was not responsible for nuclear import of Orysa; NAP1; 3 as being demonstrated through mutagenesis studies. Together, our results provide an important step toward elucidating the molecular mechanism of function of the NAP1 family proteins in plants.