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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Imaging of single-molecule translocation through nuclear pore complexes.
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Imaging of single-molecule translocation through nuclear pore complexes.

机译：通过核孔复合体的单分子移位成像。

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摘要

Nuclear pore complexes (NPCs) mediate bidirectional transport of proteins, RNAs, and ribonucleoprotein complexes across the double-membrane nuclear envelope. In vitro studies with purified transport cofactors have revealed a general scheme of cofactor-dependent transport energetically driven by the G protein Ran. However, the size and complexity of NPCs have made it difficult to clearly define the loci and kinetics of the cofactor-NPC interactions required for transport. We now report the use of single-molecule fluorescence microscopy to directly monitor a model protein substrate undergoing transport through NPCs in permeabilized cells. This substrate, NLS-2xGFP, interacts with NPCs for an average of 10 +/- 1 ms during transport. However, because the maximum nuclear accumulation rate of NLS-2xGFP was measured to be at least approximately 10(3) molecules per NPC per s, NPCs must be capable of transporting at least approximately 10 substrate molecules simultaneously. Molecular tracking reveals that substrate molecules spend most of their transit time randomly moving in the central pore of the NPC and that the rate-limiting step is escape from the central pore.
机译：核孔复合物（NPC）介导跨双膜核膜的蛋白质，RNA和核糖核蛋白复合物的双向运输。纯化的转运辅因子的体外研究揭示了由G蛋白Ran能量驱动的辅因子依赖性转运的一般方案。但是，NPC的大小和复杂性使得很难清楚地定义转运所需的辅因子-NPC相互作用的基因座和动力学。我们现在报告使用单分子荧光显微镜直接监测通过透化细胞中的NPC进行转运的模型蛋白底物。在运输过程中，此底物NLS-2xGFP与NPC相互作用的平均时间为10 +/- 1毫秒。但是，由于NLS-2xGFP的最大核积累速率被测量为每NPC每秒至少约10（3）个分子，因此NPC必须能够同时传输至少约10个底物分子。分子跟踪显示底物分子大部分时间都在NPC的中央孔中随机移动，而限速步骤则从中央孔中逸出。

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