Zhao Qiao, Leung Sara, Corbett Anita H, Meier Iris
Plant Cellular and Molecular Biology and Plant Biotechnology Center, The Ohio State University, Columbus, 43210, USA.
Plant Physiol. 2006 Mar;140(3):869-78. doi: 10.1104/pp.105.075499. Epub 2006 Jan 20.
Ran is a multifunctional small GTPase that is involved in nucleocytoplasmic transport, mitotic spindle assembly, and nuclear envelope formation. Nuclear import of Ran relies on a small RanGDP-binding protein, Nuclear Transport Factor 2 (NTF2). Three proteins are expressed in Arabidopsis (Arabidopsis thaliana) that show significant sequence similarity to human and yeast (Saccharomyces cerevisiae) NTF2. Here, we demonstrate that two of them, AtNTF2a and AtNTF2b, can functionally replace the essential NTF2 gene in yeast. Consistent with this finding, both AtNTF2a and AtNTF2b interact with yeast and Arabidopsis Ran. The third NTF2-related protein, AtNTL, does not functionally replace NTF2 in yeast. Similar to yeast NTF2-green fluorescent protein (GFP), AtNTF2a-GFP and AtNTF2b-GFP accumulate at the nuclear rim. The AtNTF2a E38K and E91K mutants, which fail to bind Ran, are not functional in yeast, indicating conservation of the requirement for these key amino acids in plants and yeast. AtNTF2a overexpression, but not AtNTF2aE38K overexpression, blocks nuclear import of a plant transcription factor in Nicotiana benthamiana leaves, indicating that excess AtNTF2a disrupts nuclear import in a Ran-binding-dependent manner. On the basis of these results, we propose that AtNTF2a and AtNTF2b function in Ran import in Arabidopsis and that nuclear import of Ran is functionally conserved in plants.
Ran是一种多功能小GTP酶，参与核质运输、有丝分裂纺锤体组装和核膜形成。Ran的核输入依赖于一种小的RanGDP结合蛋白——核运输因子2（NTF2）。拟南芥中表达的三种蛋白质与人和酵母（酿酒酵母）的NTF2具有显著的序列相似性。在这里，我们证明其中两种，AtNTF2a和AtNTF2b，能够在功能上替代酵母中必需的NTF2基因。与这一发现一致，AtNTF2a和AtNTF2b都与酵母和拟南芥的Ran相互作用。第三种与NTF2相关的蛋白质AtNTL在酵母中不能在功能上替代NTF2。与酵母NTF2-绿色荧光蛋白（GFP）类似，AtNTF2a-GFP和AtNTF2b-GFP在核边缘积累。不能结合Ran的AtNTF2a E38K和E91K突变体在酵母中无功能，表明植物和酵母中对这些关键氨基酸的需求具有保守性。AtNTF2a的过表达，而不是AtNTF2aE38K的过表达，会阻断本氏烟草叶片中植物转录因子的核输入，这表明过量的AtNTF2a以Ran结合依赖的方式破坏核输入。基于这些结果，我们提出AtNTF2a和AtNTF2b在拟南芥的Ran输入中发挥作用，并且Ran的核输入在植物中具有功能保守性。
