Characterization and potential function of a novel testis-specific nucleoporin BS-63
Characterization and potential function of a novel testis-specific nucleoporin BS-63
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DOI：
10.1002/mrd.1139
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发表时间：
2002-01-01
影响因子：
2.5
通讯作者：
Koide, SS
中科院分区：
文献类型：
--
作者：
Cai, Y;Gao, Y;Koide, SS
从人睾丸λ ZAP Ⅱ cDNA文库中分离到一个长度为1933 bp的cDNA片段，命名为BS-63，GenBank登录号为U64675。采用5 'RACE和PCR技术，获得了BS-63的全长cDNA，全长为5475bp。BS-63 cDNA含有一个由1765个密码子组成的开放阅读框架和XFXFG或GLGG重复序列基序。这些重复基序是核孔蛋白的结构特征。BS-63 cDNA与Nup358/Ran BP2有较高的同源性。将BS-63 cDNA的C-端片段克隆到大肠杆菌中，在大肠杆菌中表达。coli BL21（DE3）。用亲和层析和SDS-PAGE对重组蛋白进行纯化，并制备了多克隆抗体。在大鼠睾丸切片中，BS-63蛋白用免疫金透射电镜定位于精子细胞核孔部位，用胶体银免疫金扫描电镜定位于Triton X处理的精子核膜上。重组BS-63蛋白在体外可被PKC和p34（cdc2）磷酸化。利用酵母双杂交系统从小鼠睾丸cDNA文库中筛选与BS-63相互作用的蛋白质。使用1.6 kb的cDNA片段作为诱饵，鉴定了以下相互作用蛋白：Ran，转运蛋白（核浆转运蛋白β 2），与核质转运蛋白和aF10蛋白相关的两种蛋白。后一种蛋白质是一个假定的转录子，含有富含半胱氨酸的N-末端，一个NH4/PHD指，一个亮氨酸拉链结构域和一个富含谷氨酰胺的C-末端。它在小鼠睾丸中也高度表达，并且位于细胞核和细胞质中。通过表面等离子体共振和亲和沉淀结合Western blot验证BS-63与aF10（696 - 1001aa）的相互作用。aF10（696 - 1001aa）与大鼠睾丸生殖细胞提取物BS-63在体外相互作用。推测BS-63是睾丸特异性核孔蛋白，可能作为Ran和转运蛋白的对接位点和共转运蛋白。该复合物在精子发生过程中执行将aF10转运到生殖细胞核中的载体系统的任务。摩尔Reprod. dev. 61：126 - 134，2002. (C)2002 Wiley-Liss，Inc.
A 1933 bp cDNA fragment, coding a truncated testis-specific novel nucleoporin, was isolated from a human testis lambda ZAPII cDNA library, designated as BS-63 and assigned GenBank accession number: U64675. By applying the methods of rapid amplification of cDNA ends (5' RACE) and PCR, a full-length BS-63 cDNA composed of 5475 bp was obtained. BS-63 cDNA contained an open reading frame consisting of 1765 codons and XFXFG or GLFG repetitive sequence motifs. These repetitive motifs are structural characteristic of nucleoporins. BS-63 cDNA has high homology with Nup358/Ran BP2. A 1599 bp fragment, corresponding to the C-terminus of BS-63 cDNA, was prepared and expressed in E. coli BL21(DE3). The recombinant product was purified by affinity chromatography and SDS-PAGE and polyclonal antibodies raised. In rat testis section, the BS-63 protein was localized at the sites of nuclear pores in spermatids by immuno-gold transmission electron microscopy and on the nuclear membrane of Triton X-treated sperm by colloidal silver immuno-gold scanning electron microscopy. The recombinant BS-63 protein can be phosphorylated in vitro with PKC and p34(cdc2). A yeast two-hybrid system was used to screen a mouse testis cDNA library to identify proteins capable of interacting with BS-63. Using the 1.6 kb cDNA fragment as bait, the following interacting proteins were identified: Ran, transportin (karyopherin beta2), two proteins related to the nucleocytoplasmic transporter and aF10 protein. The latter protein is a putative transcriptor containing a cysteine-rich N-terminus, a LAP/PHD finger, a leucine zipper domain and a glutamine-rich C-terminus. Also it is highly expressed in murine testis and is located in the cell nucleus and cytoplasm. The interaction of BS-63 with aF10 (696-1001aa) was validated by surface plasmon resonance and by affinity precipitation combined with Western blot. aF10 (696-1001aa) interacted in vitro with BS-63 extracted from rat testis germ cells. It is hypothesized that BS-63 is a testis-specific nucleoporin and possibly acts as a docking site and a cotransporter of Ran and transportin. The complex performs the task of a carrier system in transporting aF10 into the nucleus of germ cells during spermiogenesis. Mol. Reprod. Dev. 61: 126-134, 2002. (C) 2002 Wiley-Liss, Inc.