Sumioka Akio, Saito Yuhki, Sakuma Megumi, Araki Yoichi, Yamamoto Tohru, Suzuki Toshiharu
Laboratory of Neuroscience, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Exp Cell Res. 2008 Mar 10;314(5):1155-62. doi: 10.1016/j.yexcr.2007.12.006. Epub 2007 Dec 14.
The X11/MINT family proteins are adaptor scaffolding proteins involved in formation of multiprotein complexes, and trafficking and metabolism of membrane proteins such as the beta-amyloid precursor protein. We found that a significant portion of X11L and X11L2 are recovered in nuclear fraction of mouse brain homogenates. EGFP-X11s were not detected in the nucleus of N2a neuroblastoma cells; however, administration of leptomycin B (LMB) induced substantial nuclear accumulation of EGFP-X11L and EGFP-X11L2, while EGFP-X11 showed little accumulation. Fluorescence loss in photobleaching (FLIP) analysis indicated that EGFP-X11L2 and EGFP-X11L are shuttled between the cytoplasm and nucleus, the former more effectively than the latter. We identified a nuclear export signal (NES) in the N-terminus of X11L2, mutation of which induces nuclear accumulation of EGFP-X11L2 in the absence of LMB. X11L2 fused to the Gal4 DNA binding domain (DBD) showed transcriptional activity, suggesting that X11L2 could function as a transcriptional activator if tethered near a promoter. Interestingly, attenuation of the nucleo-cytoplasmic shuttling of GAL4-DBD-X11L2 by mutating the NES or attaching the SV40 nuclear localization signal significantly decreased the apparent transcriptional activity. Our observations suggest that X11L2 functions in the nucleus by a mechanism distinct from conventional transactivators.
X11/MINT家族蛋白是参与多蛋白复合物形成以及膜蛋白（如β-淀粉样前体蛋白）运输和代谢的衔接支架蛋白。我们发现，小鼠脑匀浆的核组分中可回收相当一部分的X11L和X11L2。在N2a神经母细胞瘤细胞的细胞核中未检测到EGFP-X11s；然而，施用 leptomycin B（LMB）会诱导EGFP-X11L和EGFP-X11L2大量在细胞核中积累，而EGFP-X11几乎没有积累。光漂白荧光损失（FLIP）分析表明，EGFP-X11L2和EGFP-X11L在细胞质和细胞核之间穿梭，前者比后者更有效。我们在X11L2的N端鉴定出一个核输出信号（NES），其突变会在没有LMB的情况下诱导EGFP-X11L2在细胞核中积累。与Gal4 DNA结合结构域（DBD）融合的X11L2表现出转录活性，这表明如果X11L2与启动子附近相连，它可以作为转录激活因子发挥作用。有趣的是，通过突变NES或连接SV40核定位信号来减弱GAL4-DBD-X11L2的核质穿梭，会显著降低其表观转录活性。我们的观察结果表明，X11L2在细胞核中的功能机制不同于传统的转录激活因子。
