Analysis of transport mechanism and regulation through the recycling of transport factors.

1.我们初步研究了活细胞内β的核进口和核出口，发现其核出口依赖于能源的存在，而其进口是以不依赖于能源的方式发生的。在洋地黄素通透性的无细胞体系中分析，通过胞浆的分级研究，我们发现Importinβ的核输出不仅被小的GTP酶RAN刺激，而且还被70 kDa的热休克同源蛋白(Hsc70)刺激。缺失ATPase的点突变hsc70不能刺激Importinβ的核输出。我们的结果表明，额外的因素参与了Importinβ的循环，并且这种刺激依赖于ATPase的活性。2.β-Catenin是一个典型的分子，它可以通过核孔复合体以促进的方式进行双向转移。我们对β-连环蛋白的核进出口进行了检验，发现进出口所需的β-连环蛋白区域基本重叠。然而，不同的核质穿梭分子如进口素β和GAS，不同程度地阻断了β-连环蛋白的核进出口。在诱导转运因子循环的条件下，也观察到了不同的作用。这些数据表明，β-连环蛋白对其进出口显示出重叠的序列要求，但通过鼻咽癌的双向运动是通过不同的分子相互作用进行的。3.我们发现Importinα/β介导的核输入途径在热休克反应中下调，这是由于Importinα的核滞留和循环抑制所致。这种对运输的调节表明了一种新的调节机制，用于细胞适应环境变化，如热休克。

1.We have initially examined nuclear import and export of importin β in living cells, and found that its nuclear export depends on the presence of energy sources, whereas its import occurs in an energy-independent manner. Analysis in digitonin-permeabilized cell-free system and through the fractionation studies of cytosol, we found that nuclear export of importin β is stimulated not only by small GTPase Ran but also by 70kDa heat-shock cognate protein (hsc70). Point mutated hsc70 lacking ATPase did not stimulate nuclear export of importin β. Our results indicate that additional factor is involved in recycling of importin β and the stimulation is dependent on ATPase activity. 2.β-catenin is an example of a typical molecule that can be bi-directionally translocated through nuclear pore complexes (NPC) on its own in a facilitated manner. We examined the nuclear import and export of β-catenin, and showed that region of β-catenin required for import and export substantially overlapped. However, different nucleocytoplasmic shuttling molecules such as importin β and GAS, differentially blocked nuclear import and export of β-catenin. The different effect was also observed under the condition that induces recycling of transport factor. These data indicates that β-catenin shows an overlapping sequence requirement for its import and export, but that bi-directional movement through the NPC proceeds through distinct molecular interactions. 3.We found that importin α/β mediated nuclear import pathway is down-regulated in response to heat-shock, which was caused by nuclear retention and recycling inhibition of importin α. Such regulation of transport suggests a new regulatory mechanism for the adaptation of cells to environmental changes, such as heat-shock.