Identification of a novel cytoplasmic protein that specifically binds to nuclear localization signal motifs
Identification of a novel cytoplasmic protein that specifically binds to nuclear localization signal motifs
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DOI：
10.1074/jbc.273.11.6183
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发表时间：
1998-03-13
影响因子：
4.8
通讯作者：
Lee, WH
中科院分区：
文献类型：
--
作者：
Li, S;Ku, CY;Lee, WH
蛋白质主动转运入核是由靶蛋白的经典核定位信号（NLS）与NLS受体（importin）复合物之间的相互作用介导的。这种核转运系统在真核生物中受到高度调节和保守，对细胞存活至关重要。利用含有两个NLS基序的BRCA 1片段作为酵母双杂交筛选的诱饵，我们分离出了四个克隆，其中一个是importin alpha。在这里，我们的特征之一，BRAP 2，这是一个新的基因，并作为2-羟化酶mRNA在人类乳腺上皮细胞和一些，但不是所有的小鼠组织中表达。分离的全长cDNA编码一种新的蛋白质，含有600个氨基酸残基，pI 6.04。BRAP 2蛋白质的中部和C-末端区域分别存在C2 H2锌指和亮氨酸七肽重复序列的特征基序，BRAP 2还与来自酿酒酵母（Saccharomycescerevisiae）的假设蛋白质具有显著的同源性，尤其是在锌指区域。针对与谷胱甘肽S-转移酶融合的BRAP 2的C-末端区域制备的抗体特异性识别分子大小为68 kDa的细胞蛋白，与体外翻译蛋白的大小一致。细胞BRAP 2主要是细胞质的，并结合BRCA 1的NLS基序，在酵母双杂交试验和谷胱甘肽S-转移酶下拉试验中，与输入素α的特异性相似。其他基序，如SV 40大T抗原NLS基序和分裂素中发现的二分NLS基序也被BRAP 2识别。类似地，BRAP 2的酵母同系物也在体外结合这些NLS基序。这些结果表明BRAP 2可能作为胞质滞留蛋白发挥功能，并在调节核蛋白的转运中发挥作用。
Active transport of proteins into the nucleus is mediated by interaction between the classical nuclear localization signals (NLSs) of the targeted proteins and the NLS receptor (importin) complex. This nuclear transport system is highly regulated and conserved in eukaryotes and is essential for cell survival. Using a fragment of BRCA1 containing the two NLS motifs as a bait for yeast two-hybrid screening, we have isolated four clones, one of which is importin alpha. Here we characterize one of the other clones identified, BRAP2, which is a novel gene and expressed as a 2-kilobase mRNA in human mammary epithelial cells and some but not all tissues of mice. The isolated full-length cDNA encodes a novel protein containing 600 amino acid residues with pI 6.04. Characteristic motifs of C2H2 zinc fingers and leucine heptad repeats are present in the middle and C-terminal regions of the protein, respectively, BRAP2 also shares significant homology with a hypothetical protein from yeast Saccharomyces cerevisiae, especially in the zinc finger region. Antibodies prepared against the C-terminal region of BRAP2 fused to glutathione S-transferase specifically recognize a cellular protein with a molecular size of 68 kDa, consistent with the size of the in vitro translated protein. Cellular BRAP2 is mainly cytoplasmic and binds to the NLS motifs of BRCA1 with similar specificity to that of importin alpha in both two-hybrid assays in yeast and glutathione S-transferase pull-down assays in vitro. Other motifs such as the SV40 large T antigen NLS motif and the bipartite NLS motif found in mitosin are also recognized by BRAP2. Similarly, the yeast homolog of BRAP2 also binds to these NLS motifs in vitro. These results imply that BRAP2 may function as a cytoplasmic retention protein and play a role in regulating transport of nuclear proteins.