Jullien D, Görlich D, Laemmli U K, Adachi Y
Institute of Cell & Molecular Biology, The University of Edinburgh, Edinburgh EH9 3JR, UK.
EMBO J. 1999 Aug 2;18(15):4348-58. doi: 10.1093/emboj/18.15.4348.
Replication protein A (RPA) is a eukaryotic single-stranded (ss) DNA-binding protein that is essential for general DNA metabolism. RPA consists of three subunits (70, 33 and 14 kDa). We have identified by two-hybrid screening a novel Xenopus protein called XRIPalpha that interacts with the ssDNA-binding domain of the largest subunit of RPA. XRIPalpha homologues are found in human and in Drosophila but not in yeast. XRIPalpha is complexed with RPA in Xenopus egg extracts together with another 90 kDa protein that was identified as importin beta. We have demonstrated that XRIPalpha, but not importin alpha, is required for nuclear import of RPA. Immunodepletion of XRIPalpha from the egg extracts blocks nuclear import of RPA but not that of nucleoplasmin, a classical import substrate. RPA import can be restored by addition of recombinant XRIPalpha. Conversely, depletion of importin alpha blocks import of nucleoplasmin but not that of RPA. GST-XRIPalpha pull-down assay shows that XRIPalpha interacts directly with recombinant importin beta as well as with RPA in vitro. Finally, RPA import can be reconstituted from the recombinant proteins. We propose that XRIPalpha plays the role of importin alpha in the RPA import scheme: XRIPalpha serves as an adaptor to link RPA to importin beta.
复制蛋白A（RPA）是一种真核单链（ss）DNA结合蛋白，对一般DNA代谢至关重要。RPA由三个亚基（70、33和14 kDa）组成。我们通过双杂交筛选鉴定出一种名为XRIPα的非洲爪蟾新蛋白，它与RPA最大亚基的ssDNA结合结构域相互作用。在人类和果蝇中发现了XRIPα同源物，但在酵母中未发现。在非洲爪蟾卵提取物中，XRIPα与RPA以及另一种被鉴定为输入蛋白β的90 kDa蛋白形成复合物。我们已经证明，RPA的核输入需要XRIPα，而不是输入蛋白α。从卵提取物中免疫去除XRIPα会阻断RPA的核输入，但不会阻断核质蛋白（一种经典的输入底物）的核输入。添加重组XRIPα可以恢复RPA的输入。相反，去除输入蛋白α会阻断核质蛋白的输入，但不会阻断RPA的输入。GST-XRIPα下拉试验表明，XRIPα在体外直接与重组输入蛋白β以及RPA相互作用。最后，可以用重组蛋白重建RPA的输入。我们提出，XRIPα在RPA输入机制中发挥输入蛋白α的作用：XRIPα作为衔接子将RPA与输入蛋白β连接起来。
