Grundt Kirsten, Haga Ingvild V, Huitfeldt Henrik S, Ostvold Anne Carine
Department of Biochemistry, Institute of Basic Medical Sciences, University of Oslo, P.O. Box 1112, Blindern, 0317 Oslo, Norway.
Biochim Biophys Acta. 2007 Sep;1773(9):1398-406. doi: 10.1016/j.bbamcr.2007.05.013. Epub 2007 Jun 2.
Immunofluorescence analyses show that the vertebrate specific and DNA-binding protein NUCKS is distributed throughout the cytoplasm in mitotic cells and targeted to the reforming nuclei in late telophase of the cell cycle. Computer analysis of the primary structure of NUCKS revealed the presence of two regions of highly charged, basic residues, which were identified as potential nuclear localization signals (NLSs). One of these signals (NLS1) is highly conserved between the species investigated, and fits to the description of being a classical bipartite NLS. The other amino acid motif (NLS2) is less conserved and does not constitute a classical bipartite NLS consensus sequence. We have shown that each of the two putative NLSs is capable of translocating green fluorescent protein (GFP) into the nucleus. The highly conserved NLS1 is monopartite, resembling the signals of c-Myc and RanBP3. Surprisingly, a natural occurring splice variant of NUCKS lacking 40 amino acids including NLS1, is not capable of translocating a corresponding NUCKS-GFP fusion protein into the nucleus, indicating that NLS1 is the main nuclear localization signal in NUCKS. This is also confirmed by site-directed mutagenesis of the full-length protein. By GFP-immunoprecipitation and GST-pull down experiments, we show that NUCKS binds to importin alpha3 and importin alpha5 in vitro, suggesting that the nuclear targeting of NUCKS follows a receptor-mediated and energy-dependent import mechanism.
免疫荧光分析表明，脊椎动物特有的DNA结合蛋白NUCKS在有丝分裂细胞中分布于整个细胞质中，并在细胞周期末期定位于重新形成的细胞核。对NUCKS一级结构的计算机分析揭示了存在两个高度带电的碱性残基区域，这两个区域被确定为潜在的核定位信号（NLSs）。其中一个信号（NLS1）在所研究的物种之间高度保守，符合经典二分核定位信号的描述。另一个氨基酸基序（NLS2）保守性较低，不构成经典二分核定位信号的共有序列。我们已经表明，两个假定的核定位信号中的每一个都能够将绿色荧光蛋白（GFP）转运到细胞核中。高度保守的NLS1是单分的，类似于c-Myc和RanBP3的信号。令人惊讶的是，一种天然存在的缺失包括NLS1在内的40个氨基酸的NUCKS剪接变体，不能将相应的NUCKS-GFP融合蛋白转运到细胞核中，这表明NLS1是NUCKS中的主要核定位信号。全长蛋白的定点诱变也证实了这一点。通过GFP免疫沉淀和GST下拉实验，我们表明NUCKS在体外与输入蛋白α3和输入蛋白α5结合，这表明NUCKS的核靶向遵循受体介导的能量依赖输入机制。
