Spit A, Hyland R H, Mellor E J, Casselton L A
Department of Plant Sciences, University of Oxford, South Parks Road, Oxford, OX1 3RB, United Kingdom.
Proc Natl Acad Sci U S A. 1998 May 26;95(11):6228-33. doi: 10.1073/pnas.95.11.6228.
The A mating type genes of the mushroom Coprinus cinereus encode two families of dissimilar homeodomain proteins (HD1 and HD2). The proteins heterodimerize when mating cells fuse to generate a transcriptional regulator that promotes expression of genes required for early steps in sexual development. In previous work we showed that heterodimerization brings together different functional domains of the HD1 and HD2 proteins; a potential activation domain at the C terminus of the HD1 protein and an essential HD2 DNA-binding motif. Two predicted nuclear localization signals (NLS) are present in the HD1 protein but none are in the HD2 protein. We deleted each NLS separately from an HD1 protein and showed that one (NLS1) is essential for normal heterodimer function. Fusion of the NLS sequences to the C terminus of an HD2 protein compensated for their deletion from the HD1 protein partner and permitted the two modified proteins to form a functional transcriptional regulator. The nuclear targeting properties of the A protein NLS sequences were demonstrated by fusing the region that encodes them to the bacterial uidA (beta-glucuronidase) gene and showing that beta-glucuronidase expression localized to the nuclei of onion epidermal cells. These observations lead to the proposal that heterodimerization regulates entry of the active transcription factor complex to the nucleus.
灰盖鬼伞蘑菇的A交配型基因编码两个不同的同源异型结构域蛋白家族（HD1和HD2）。当交配细胞融合时，这些蛋白会形成异源二聚体，从而产生一种转录调节因子，促进性发育早期步骤所需基因的表达。在之前的研究中，我们表明异源二聚化将HD1和HD2蛋白的不同功能域聚集在一起；HD1蛋白C末端的一个潜在激活域和一个必需的HD2 DNA结合基序。HD1蛋白中有两个预测的核定位信号（NLS），但HD2蛋白中没有。我们分别从HD1蛋白中删除了每个NLS，结果表明其中一个（NLS1）对于正常的异源二聚体功能至关重要。将NLS序列融合到HD2蛋白的C末端，弥补了它们从HD1蛋白伴侣中缺失的情况，并使这两种修饰后的蛋白能够形成功能性转录调节因子。通过将编码A蛋白NLS序列的区域与细菌uidA（β-葡萄糖醛酸酶）基因融合，并表明β-葡萄糖醛酸酶表达定位于洋葱表皮细胞核，证明了A蛋白NLS序列的核靶向特性。这些观察结果促使我们提出这样的假设：异源二聚化调节活性转录因子复合物进入细胞核。
