Hieda M, Tachibana T, Fukumoto M, Yoneda Y
Department of Cell Biology and Neuroscience, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka and Institute for Molecular and Cellular Biology, Osaka University, 1-3 Yamada-oka, Suita, Osaka 565-0871, Japan.
J Biol Chem. 2001 May 18;276(20):16824-32. doi: 10.1074/jbc.M008299200. Epub 2001 Feb 21.
U1A is a component of the uracil-rich small nuclear ribonucleoprotein. The molecular mechanism of nuclear import of U1A was investigated in vivo and in vitro. When recombinant deletion mutants of U1A are injected into the BHK21 cell cytoplasm, the nuclear localization signal (NLS) of U1A is found in the N-terminal half of the central domain (residues 100-144 in mouse U1A). In an in vitro assay, it was found that the U1A-NLS accumulated in only a portion of the nuclei in the absence of cytosolic extract. In contrast, the addition of importin alpha/beta and Ran induced the uniform nuclear accumulation of U1A-NLS in all cells. Furthermore, U1A was found to bind the C-terminal portion of importin alpha. In addition, the in vitro nuclear migration of full-length U1A was found to be exclusively dependent on importin alpha/beta and Ran. Moreover, in living cells, the full-length U1A accumulated in the nucleus in a Ran-dependent manner, and nuclear accumulation was inhibited by the importin beta binding domain of importin alpha. These results suggest that the nuclear import of U1A is mediated by at least two distinct pathways, an importin alpha/beta and Ran-dependent and an -independent pathway in permeabilized cells, and that the latter pathway may be suppressed in intact cells.
U1A是富含尿嘧啶的小核核糖核蛋白的一个组成部分。在体内和体外研究了U1A的核输入分子机制。当将U1A的重组缺失突变体注射到BHK21细胞质中时，发现U1A的核定位信号（NLS）位于中央结构域的N端一半（小鼠U1A中的第100 - 144位氨基酸）。在体外试验中，发现在没有胞质提取物的情况下，U1A - NLS仅在部分细胞核中积累。相反，添加输入蛋白α/β和Ran可诱导U1A - NLS在所有细胞中均匀地向核内积累。此外，发现U1A与输入蛋白α的C端部分结合。另外，发现全长U1A的体外核迁移完全依赖于输入蛋白α/β和Ran。而且，在活细胞中，全长U1A以Ran依赖性方式在细胞核中积累，并且核积累受到输入蛋白α的输入蛋白β结合结构域的抑制。这些结果表明，U1A的核输入至少由两条不同的途径介导，一条是输入蛋白α/β和Ran依赖性途径，另一条是通透细胞中的非依赖性途径，并且后一条途径在完整细胞中可能受到抑制。
