Rhee Y, Gurel F, Gafni Y, Dingwall C, Citovsky V
Department of Biochemistry Cell Biology, Institute for Cell and Developmental Biology, State University of New York, Stony Brook, NY 1794-5215, USA.
Nat Biotechnol. 2000 Apr;18(4):433-7. doi: 10.1038/74500.
We have developed a simple genetic assay to detect active nuclear localization (NLS) and export signals (NES) on the basis of their function within yeast cells. The bacterial LexA protein was modified (mLexA) to abolish its intrinsic NLS and fused to the activation domain of the yeast Gal4p (Gal4AD) with or without the SV40 large T-antigen NLS. In the import assay, if a tested protein fused to mLexA-Gal4AD contains a functional NLS, it will enter the cell nucleus and activate the reporter gene expression. In the export assay, if a tested protein fused to mLexA-SV40 NLS-Gal4AD contains a functional NES, it will exit into the cytoplasm, decreasing the reporter gene expression. We tested this system with known NLS and NES and then used it to demonstrate a NES activity of the capsid protein of a plant geminivirus. This approach may help to identify, analyze, and select for proteins containing functional NLS and NES.
我们基于核定位信号（NLS）和核输出信号（NES）在酵母细胞内的功能，开发了一种简单的遗传检测方法来检测它们的活性。对细菌LexA蛋白进行了修饰（mLexA）以消除其内在的NLS，并将其与酵母Gal4p的激活结构域（Gal4AD）融合，融合时带有或不带有SV40大T抗原的NLS。在导入检测中，如果与mLexA-Gal4AD融合的被测蛋白含有功能性NLS，它将进入细胞核并激活报告基因的表达。在输出检测中，如果与mLexA-SV40 NLS-Gal4AD融合的被测蛋白含有功能性NES，它将进入细胞质，从而降低报告基因的表达。我们用已知的NLS和NES对该系统进行了测试，然后用它来证明一种植物双生病毒衣壳蛋白的NES活性。这种方法可能有助于识别、分析和筛选含有功能性NLS和NES的蛋白质。
