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Abstract

Characterization and potential function of a novel testis-specific nucleoporin BS-63 (HTML) 
Cai,Ying; Gao,Ying; Sheng,Qi; Miao,Shiying; Cui,Xiuyun; Wang,Linfang; Zong,Shudong; Koide,Samuel S.
Molecular Reproduction and Development 61(1): 126-134
Publication date: 2002

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A 1933 bp cDNA fragment, coding a truncated testis-specific novel nucleoporin, was isolated from a human testis 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. 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.

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