Department of Biochemistry & Molecular Biology, Monash University, Clayton, Victoria, Australia.
Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan; Embryology Unit, Children's Medical Research Institute, Westmead, Australia.
Biochim Biophys Acta Mol Cell Res. 2017 Mar;1864(3):546-561. doi: 10.1016/j.bbamcr.2016.12.017. Epub 2016 Dec 16.
Importin 13 (Imp13) is a bidirectional nuclear transporter of proteins involved in a range of important cellular processes, with an N-terminally truncated inhibitory isoform (tImp13) specifically expressed in testis. To gain insight into tImp13 function, we performed a yeast-2-hybrid screen from a human testis cDNA library, identifying for the first time a suite of interactors with roles in diverse cellular process. We validated the interaction of tImp13 with Eukaryotic translation initiation factor 4γ2 (EIF4G2) and High mobility group containing protein 20A (HMG20A), benchmarking that with glucocorticoid receptor (GR), a known Imp13 interactor expressed in testis. Coimmunoprecipitation assays indicated association of both tImp13 and Imp13 with EIF4G2, HMG20A and GR. Quantitative confocal microscopic analysis revealed the ability of tImp13 to inhibit the nuclear localisation of EIF4G2, HMG20A and GR, as well as that of Imp13 to act as a nuclear exporter for both EIF4G2 and HMG20A, and as a nuclear importer for GR. The physiological relevance of these results was highlighted by the cytoplasmic localisation of EIF4G2, HMG20A and GR in pachytene spermatocytes/round spermatids in the murine testis where tImp13 is present at high levels, in contrast to the nuclear localisation of HMG20A and GR in spermatogonia, where tImp13 is largely absent. Interestingly, Imp13, EIF4G2, HMG20A and GR were found together in the acrosome vesicle of murine epididymal spermatozoa. Collectively, our findings show, for the first time, that tImp13 may have a functional role in the mature spermatozoa, in addition to that in the meiotic germ cells of the testis.
输入蛋白 13（Imp13）是一种双向核蛋白转运体，参与多种重要的细胞过程，其 N 端截短的抑制性同工型（tImp13）特异性表达于睾丸。为了深入了解 tImp13 的功能，我们从人睾丸 cDNA 文库中进行了酵母双杂交筛选，首次鉴定了一系列具有不同细胞过程作用的相互作用蛋白。我们验证了 tImp13 与真核翻译起始因子 4γ2（EIF4G2）和高迁移率族蛋白 20A（HMG20A）的相互作用，并将其与已知在睾丸中表达的 Imp13 相互作用物糖皮质激素受体（GR）进行了基准测试。免疫共沉淀实验表明 tImp13 和 Imp13 均可与 EIF4G2、HMG20A 和 GR 结合。定量共聚焦显微镜分析显示 tImp13 能够抑制 EIF4G2、HMG20A 和 GR 的核定位，Imp13 能够作为 EIF4G2 和 HMG20A 的核输出物，以及 GR 的核输入物。这些结果的生理相关性在小鼠睾丸中 pachytene 精母细胞/圆形精子中得到了强调，在 pachytene 精母细胞/圆形精子中 tImp13 水平较高，EIF4G2、HMG20A 和 GR 位于细胞质中，而在精原细胞中 HMG20A 和 GR 位于核内，tImp13 则大量缺失。有趣的是，在小鼠附睾精子的顶体小泡中发现了 Imp13、EIF4G2、HMG20A 和 GR。总的来说，我们的研究结果首次表明，tImp13 除了在睾丸的减数分裂生殖细胞中具有功能外，还可能在成熟精子中具有功能。
