Chatterjee S, Stochaj U
Physiology Department, McGill University, Montreal, QC, Canada.
Biotechniques. 1998 Apr;24(4):668-74. doi: 10.2144/98244rr04.
We describe an experimental system to study nucleocytoplasmic diffusion of proteins in living HeLa cells. To localize proteins to the nucleus, substrates were created that contain a nuclear localization sequence fused to Aequorea victoria green fluorescent protein (GFP). Transiently and stably transfected HeLa cells were used for these assays. A protein of 29-kDa molecular mass that harbors GFP and the bipartite Xenopus nucleoplasmin nuclear localization sequence (NLS) accumulates efficiently in nuclei of HeLa cells. However, in the absence of active facilitated nuclear import, the reporter protein exits the nucleus and equilibrates between nucleus and cytoplasm. We define different conditions that promote the diffusion of small nuclear proteins across the nuclear envelope of mammalian culture cells. Our results set the stage to analyze the competence of nuclear pore complexes for nucleocytoplasmic diffusion of macromolecules in living cells.
我们描述了一个用于研究蛋白质在活的HeLa细胞中核质扩散的实验系统。为了将蛋白质定位到细胞核，构建了含有与维多利亚水母绿色荧光蛋白（GFP）融合的核定位序列的底物。这些实验使用了瞬时和稳定转染的HeLa细胞。一种分子量为29 kDa、带有GFP和非洲爪蟾核质蛋白双功能核定位序列（NLS）的蛋白质能有效地在HeLa细胞的细胞核中积累。然而，在没有活跃的促进性核输入的情况下，报告蛋白会离开细胞核并在细胞核和细胞质之间达到平衡。我们定义了不同的条件来促进小核蛋白穿过哺乳动物培养细胞核膜的扩散。我们的结果为分析核孔复合体在活细胞中对大分子核质扩散的能力奠定了基础。
