Characterization and potential function of a novel testis-specific nucleoporin BS-63.
A 1933 bp cDNA fragment, coding a truncated testis-specific novel nucleoporin, was isolated from a human testis lambdaZAPII 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.
Cai
Ying
Y
National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College, Dong Dan San Tiao, Beijing, China.
Gao
Ying
Y
Sheng
Qi
Q
Miao
Shiying
S
Cui
Xiuyun
X
Wang
Linfang
L
Zong
Shudong
S
Koide
Samuel S
SS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D028884:Karyopherins / Q000737:chemistry; D008297:Male; D051379:Mice; D008855:Microscopy, Electron, Scanning; D008969:Molecular Sequence Data; D009685:Nuclear Envelope / Q000737:chemistry / Q000378:metabolism; D022022:Nuclear Pore / Q000737:chemistry / Q000378:metabolism; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; D009928:Organ Specificity; D010766:Phosphorylation; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism; D021381:Protein Transport; D012689:Sequence Homology, Nucleic Acid; D013094:Spermatozoa / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure; D013737:Testis / Q000166:cytology / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D020931:ran GTP-Binding Protein / Q000737:chemistry
D018076:DNA, Complementary; D006023:Glycoproteins; D028884:Karyopherins; C092513:MLLT10 protein, human; C490477:Mllt10 protein, mouse; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; C119930:RGPD8 protein, human; D014157:Transcription Factors; D011493:Protein Kinase C; D020931:ran GTP-Binding Protein