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Characterization and potential function of a novel testis‐specific nucleoporin BS‐63
Author(s) -
Cai Ying,
Gao Ying,
Sheng Qi,
Miao Shiying,
Cui Xiuyun,
Wang Linfang,
Zong Shudong,
Koide Samuel S.
Publication year - 2002
Publication title -
molecular reproduction and development
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.745
H-Index - 105
eISSN - 1098-2795
pISSN - 1040-452X
DOI - 10.1002/mrd.1139
Subject(s) - complementary dna , biology , microbiology and biotechnology , cdna library , rapid amplification of cdna ends , nucleoporin , nuclear pore , nuclear protein , biochemistry , molecular cloning , gene , cytoplasm , transcription factor
A 1933 bp cDNA fragment, coding a truncated testis‐specific novel nucleoporin, was isolated from a human testis λZAPII 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. © 2002 Wiley‐Liss, Inc.