Zlopasa Livija, Brachner Andreas, Foisner Roland
Max F. Perutz Laboratories (MFPL), Department of Medical Biochemistry, Medical University of Vienna, Vienna Biocenter (VBC), Vienna, Austria.
BMC Cell Biol. 2016 Jun 1;17(1):23. doi: 10.1186/s12860-016-0102-z.
Ankyrin repeats and LEM domain containing protein 1 (Ankle1) belongs to the LEM protein family, whose members share a chromatin-interacting LEM motif. Unlike most other LEM proteins, Ankle1 is not an integral protein of the inner nuclear membrane but shuttles between the nucleus and the cytoplasm. It contains a GIY-YIG-type nuclease domain, but its function is unknown. The mammalian genome encodes only one other GIY-YIG domain protein, termed Slx1. Slx1 has been described as a resolvase that processes Holliday junctions during homologous recombination-mediated DNA double strand break repair. Resolvase activity is regulated in a spatial and temporal manner during the cell cycle. We hypothesized that Ankle1 may have a similar function and its nucleo-cytoplasmic shuttling may contribute to the regulation of Ankle1 activity. Hence, we aimed at identifying the domains mediating Ankle1 shuttling and investigating whether cellular localization is affected during DNA damage response.
Sequence analysis predicts the presence of two canonical nuclear import and export signals in Ankle1. Immunofluorescence microscopy of cells expressing wild-type and various mutated Ankle1-fusion proteins revealed a C-terminally located classical monopartite nuclear localization signal and a centrally located CRM1-dependent nuclear export signal that mediate nucleo-cytoplasmic shuttling of Ankle1. These sequences are also functional in heterologous proteins. The predominant localization of Ankle1 in the cytoplasm, however, does not change upon induction of several DNA damage response pathways throughout the cell cycle.
We identified the domains mediating nuclear import and export of Ankle1. Ankle1's cellular localization was not affected following DNA damage.
锚蛋白重复序列和含LEM结构域蛋白1（Ankle1）属于LEM蛋白家族，其成员共享一个与染色质相互作用的LEM基序。与大多数其他LEM蛋白不同，Ankle1不是内核膜的整合蛋白，而是在细胞核和细胞质之间穿梭。它包含一个GIY - YIG型核酸酶结构域，但其功能尚不清楚。哺乳动物基因组仅编码另一种GIY - YIG结构域蛋白，称为Slx1。Slx1已被描述为一种在同源重组介导的DNA双链断裂修复过程中处理霍利迪连接体的解离酶。解离酶活性在细胞周期中以空间和时间方式受到调节。我们假设Ankle1可能具有类似功能，其核质穿梭可能有助于调节Ankle1的活性。因此，我们旨在鉴定介导Ankle1穿梭的结构域，并研究在DNA损伤反应期间细胞定位是否受到影响。
序列分析预测Ankle1中存在两个典型的核输入和输出信号。对表达野生型和各种突变的Ankle1融合蛋白的细胞进行免疫荧光显微镜检查，发现了一个位于C末端的经典单部分核定位信号和一个位于中央的依赖CRM1的核输出信号，它们介导Ankle1的核质穿梭。这些序列在异源蛋白中也具有功能。然而，在整个细胞周期中诱导几种DNA损伤反应途径后，Ankle1在细胞质中的主要定位并未改变。
我们鉴定了介导Ankle1核输入和输出的结构域。DNA损伤后Ankle1的细胞定位未受影响。
