Transport of galectin-3 between the nucleus and cytoplasm. I. Conditions and signals for nuclear import
Transport of galectin-3 between the nucleus and cytoplasm. I. Conditions and signals for nuclear import
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DOI：
10.1093/glycob/cwj088
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发表时间：
2006-07-01
期刊：
影响因子：
4.3
通讯作者：
Arnoys, Eric J.
中科院分区：
文献类型：
--
作者：
Davidson, Peter J.;Li, Su-Yin;Arnoys, Eric J.
Galectin-3是一种参与Pre-mRNA剪接的因子，穿梭于细胞核和细胞质之间。我们已经设计了一个表达融合蛋白的载体，该融合蛋白包括：(A)作为定位报告的绿色荧光蛋白，(B)细菌麦芽糖结合蛋白，以增加报告多肽的大小，以及(C)Galectin-3，我们希望对其序列进行剖析，以寻找对核定位至关重要的氨基酸残基。在转基因小鼠3T3成纤维细胞中，全长Galectin-3(残基1-263)融合蛋白主要定位于细胞核。该结构的突变体含有氨基端Galectin-3多肽的截短，通过128个残基保留了核定位；因此，氨基端的一半对于核进口是不必要的。相同结构的突变，包括从羧基末端截断，表现出核定位的丧失。这种影响从残数259处的截断开始观察到，并在残数253处截断时观察到完整的效果。ITLT(残基253-256)序列的定点突变表明，核输入依赖于IXLT类型的核定位序列，该序列首先在果蝇蛋白Dsh(杂乱)中发现。在Galectin-3多肽中，这个核定位序列的活性受到邻近的富含亮氨酸的核输出信号的调控。
Galectin-3, a factor involved in the splicing of pre-mRNA, shuttles between the nucleus and the cytoplasm. We have engineered a vector that expresses the fusion protein containing the following: (a) green fluorescent protein as a reporter of localization, (b) bacterial maltose-binding protein to increase the size of the reporter polypeptide, and (c) galectin-3, whose sequence we wished to dissect in search of amino acid residues vital for nuclear localization. In mouse 3T3 fibroblasts transfected with this expression construct, the full-length galectin-3 (residues 1-263) fusion protein was localized predominantly in the nucleus. Mutants of this construct, containing truncations of the galectin-3 polypeptide from the amino terminus, retained nuclear localization through residue 128; thus, the amino-terminal half was dispensable for nuclear import. Mutants of the same construct, containing truncations from the carboxyl terminus, showed loss of nuclear localization. This effect was observed beginning with truncation at residue 259, and the full effect was seen with truncation at residue 253. Site-directed mutagenesis of the sequence ITLT (residues 253-256) suggested that nuclear import was dependent on the IXLT type of nuclear localization sequence, first discovered in the Drosophila protein Dsh (dishevelled). In the galectin-3 polypeptide, the activity of this nuclear localization sequence is modulated by a neighboring leucine-rich nuclear export signal.