Paschal B M, Delphin C, Gerace L
Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Proc Natl Acad Sci U S A. 1996 Jul 23;93(15):7679-83. doi: 10.1073/pnas.93.15.7679.
The use of permeabilized cell models to study nuclear protein import has led to the identification of cytosolic components of the import machinery, including the NLS receptor, p97, Ran/TC4, and nuclear transport factor 2 (NTF2). These proteins are required to reconstitute docking of transport ligand at the nuclear pore complex and subsequent translocation through the nuclear pore. However, a detailed molecular understanding of how these factors mediate protein import is lacking. Here we describe the results of solution and solid phase binding assays, which demonstrate that the small GTPase Ran/TC4 interacts directly with the cytosolic transport factors p97 and NTF2. By preloading recombinant Ran/TC4 with [gamma-32P]GTP or [3H]GDP, we show that the interactions with p97 and NTF2 are specific for the GTP- and GDP-bound forms, respectively. These data together with previous studies lead us to suggest that the interaction of the GTP-bound form of Ran/TC4 with p97 is linked to an early step in the nuclear protein import pathway and that the association of the GDP-bound form of Ran/TC4 with NTF2 helps define vectorial transport.
利用通透细胞模型研究核蛋白输入已鉴定出输入机制的胞质成分，包括核定位信号（NLS）受体、p97、Ran/TC4和核转运因子2（NTF2）。这些蛋白质是在核孔复合体处重建转运配体对接并随后通过核孔转运所必需的。然而，对于这些因子如何介导蛋白质输入，目前仍缺乏详细的分子层面理解。在此，我们描述了溶液和固相结合试验的结果，这些结果表明小GTP酶Ran/TC4直接与胞质转运因子p97和NTF2相互作用。通过用[γ-32P]GTP或[3H]GDP预加载重组Ran/TC4，我们发现与p97和NTF2的相互作用分别对结合GTP和GDP的形式具有特异性。这些数据以及先前的研究使我们推测，结合GTP的Ran/TC4形式与p97的相互作用与核蛋白输入途径中的早期步骤相关，而结合GDP的Ran/TC4形式与NTF2的结合有助于确定单向转运。
