Separate nuclear import pathways converge on the nucleoporin Nup153 and can be dissected with dominant-negative inhibitors

Background: Proteins generally enter or exit the nucleus as cargo of one of a small family of import and export receptors. These receptors bear distant homology to importin β, a subunit of the receptor for proteins with classical nuclear localisation sequences (NLSs). To understand the mechanism of nuclear transport, the next question involves identifying the nuclear pore proteins that interact with the different transport receptors as they dock at the pore and translocate through it.Results: Two pathways of nuclear import were found to intersect at a single nucleoporin, Nup153, localized on the intranuclear side of the nuclear pore. Nup153 contains separate binding sites for importin α/β, which mediates classical NLS import, and for transportin, which mediates import of different nuclear proteins. Strikingly, a Nup153 fragment containing the importin β binding site acted as a dominant-negative inhibitor of NLS import, with no effect on transportin-mediated import. Conversely, a Nup153 fragment containing the transportin binding site acted as a strong dominant-negative inhibitor of transportin import, with no effect on classical NLS import. The interaction of transportin with Nup153 could be disrupted by a non-hydrolyzable form of GTP or by a GTPase-deficient mutant of Ran, and was not observed if transportin carried cargo. Neither Nup153 fragment affected binding of the export receptor Crm1 at the nuclear rim.Conclusions: Two nuclear import pathways, mediated by importin β and transportin, converge on a single nucleoporin, Nup153. Dominant-negative fragments of Nup153 can now be used to distinguish different nuclear import pathways and, potentially, to dissect nuclear export.     

Different structural and kinetic requirements for the interaction of Ran with the Ran-binding domains from RanBP2 and importin-beta

The cytoplasmic disassembly of Ran.GTP.importin and Ran.GTP.exportin. cargo complexes is an essential step in the corresponding nuclear import and export cycles. It has previously been shown that such disassembly can be mediated by RanBP1 in the presence of RanGAP. The nuclear pore complex protein RanBP2 (Nup358) contains four Ran-binding domains (RanBDi) that might function like RanBP1. We used biophysical assays based on fluorescence-labeled probes and on surface plasmon resonance to investigate the dynamic interplay of Ran in its GDP- and GTP-complexed states with RanBDis and with importin-beta. We show that RanBP1 and the four RanBDis from RanBP2 have comparable affinities for Ran.GTP (10(8)-10(9) M(-1)). Deletion of Ran's C-terminal (211)DEDDDL(216) sequence weakens the interaction of Ran.GTP with RanBPis approximately 2000-fold, but accelerates the association of Ran.GTP with importin-beta 10-fold. Importin-beta binds Ran.GTP with a moderate rate, but attains a high affinity for Ran (K(D) = 140 pM) via an extremely low dissociation rate of 10(-5) s(-)(1). Association with Ran is accelerated 3-fold in the presence of RanBP1, which presumably prevents steric hindrance caused by the Ran C-terminus. In addition, we show that the RanBDis of RanBP2 are full equivalents of RanBP1 in that they also costimulate RanGAP-catalyzed GTP hydrolysis in Ran and relieve the GTPase block in a Ran.GTP.transportin complex. Our data suggest that the C-terminus of Ran functions like a loose tether in Ran.GTP complexes of importins or exportins that exit the nucleus. This flag is then recognized by the multiple RanBDis at or near the nuclear pore complex, allowing efficient disassembly of these Ran.GTP complexes.     

Molecular cloning and chromosomal mapping of the human gene for the testis-specific catalytic subunit of calmodulin-dependent protein phosphatase (calcineurin A).

A cDNA for an alternatively spliced variant of the testis-specific catalytic subunit of calmodulin dependent protein phosphatase (CaM-PrP) was cloned from a human testis library. The nucleotide sequence of 2134 base pairs (bp) encodes a protein of 502 amino acids (Mr approximately 57,132) and pI 7.0. The cDNA sequence differs from the murine form of this gene by a 30 bp deletion in the coding region, the position of which matches those in the two other genes for the catalytic subunit. These data indicate that this alternative splicing event arose prior to the divergence of the three genes. The deduced sequence of the human protein is only 88% identical to the homologous murine form, in striking contrast to the other two CaM-PrP catalytic subunits which are highly conserved between mouse and human (approximately 99%); this indicates a more rapid rate of evolution for the testis-specific gene. Analysis of Southern blots containing DNA from human-hamster somatic cell hybrids show that the gene is on human chromosome 8.     

一个新的睾丸特异表达基因的克隆及功能的初步分析

运用“数据库消减杂交”(digital differential display)方法来筛选人类睾丸特异表达新基因,获得了有差异显示的代表新基因的克隆重叠群。挑选其中一个克隆重叠群HS．326528进行多组织RT—PCR,初步获得该重叠群在人睾丸中有高表达。从该重叠群的IMAGE出发,采用生物信息学的方法快速克隆了一个人类新基因的全长cDNA序列,其全长1044bp,开放阅读框为214～529bp,定位于15q26．2,编码由105个氨基酸组成、分子量为11．7kD、等电点为10．09的一个碱性蛋白,该蛋白与已知蛋白无明显的同源性,克隆实验证明该基因的阅读框完全正确,RT—PCR和Northern blot显示该基因在人类睾丸中特异表达,实时PCR结果表明：该基因在成人睾丸中高表达,在精子中有中度表达,在胚胎睾丸中低表达,推测该基因与精子的生成有关,命名为SRG8(homo sapiens spermatogenesis—related gene 8)(GenBank登录号：AY489187),该基因编码的蛋白定位于细胞核。流式结果分析表明,SRG8基因能够促使HeLa细胞由S期向G2期的转变,从而加速细胞的分裂。这些结果表明SRG8基因可能在睾丸的发育及精子的形成过程中起重要的作用。     

Cloning,expression, and genomic structure of a novel human Rap2 interacting gene (RPIP9)

During a large-scale screen of a human fetal brain cDNA library, a full-length cDNA encoding a novel Rap2 interacting protein was isolated and sequenced. The cDNA is 3397 bp long and has a predicted open reading frame encoding a protein of 329 aa. The predicted protein shows high homology to mouse and human RPIP8, and has a RUN domain near its C-terminus. The gene was mapped to human chromosome 7q21–7q22 and has 9 exons and 8 introns. The expression pattern was also detected by cycle-limited reverse polymerase chain reaction (RT-PCR).     

Cloning and characterization of a testis-specific thymosin beta 10 cDNA. Expression in post-meiotic male germ cells.

Thymosin beta 10 is one of a small family of proteins closely related in sequence to thymosin beta 4, recently identified as an actin-sequestering protein. A single molecular weight species of thymosin beta 10 mRNA is present in a number of rat tissues. In adult rat testis, an additional thymosin beta 10 mRNA of higher molecular weight was identified. Nucleotide sequencing of cDNA clones complementary to the testis-specific thymosin mRNA indicated that this mRNA differed from the ubiquitous thymosin beta 10 mRNA only in its 5'-untranslated region, beginning 14 nucleotides upstream of the translation initiation codon. These results, together with primer extension experiments, suggest that the two thymosin beta 10 mRNAs are transcribed from the same gene through a combination of differential promoter utilization and alternative splicing. The novel thymosin beta 10 mRNA could be detected only in RNA isolated from sexually mature rat testis. Both mRNAs were present in pachytene spermatocytes; only the testis-specific mRNA was detected in postmeiotic haploid spermatids. Immunoblot analysis using specific antibodies showed that the thymosin beta 10 protein synthesized in adult testis was identical in size to that synthesized in brain. Immunohistochemical analysis showed that the protein was present in differentiating spermatids, suggesting that the testis-specific thymosin beta 10 mRNA is translated in haploid male germ cells.     

Expression of a novel HsMCAK mRNA splice variant,tsMCAK gene,in human testis

Identification of specifically expressed genes in the adult or fetal testis is very important for the study of genes related to the development and function of the testis. In this study, a human adult testis cDNA microarray was constructed and hybridized with 33P-labeled human adult and embryo testis cDNA probes, respectively. After differential display analyzing, a number of new genes related to the development of testis and spermatogenesis had been identified. One of these new genes is tsMCAK. tsMCAK was expressed 2.62 folds more in human adult testis than fetal testis. The full length of tsMCAK is 2401 bp and contains a 2013 bp open reading frame, encoding a 671-amino-acid protein. Sequence analysis showed that it has a central kinesin motor domain and is homologous to HsMCAK gene of the somatic cells. Blasting human genome database localized tsMCAK to human chromosome 1P34 and further investigation showed that it is a splice variant of HsMCAK. The tissue distribution of tsMCAK was determined by RT-PCR and it is expressed highly and specifically in the testis. Southern blot studies of its expression in patients with infertility indicated its specific expression in spermatogenic cells and its correlation with male infertility. The above results suggested that tsMCAK is a candidate gene for the testis-specific KRPs and its specific expression in the testis was correlated with spermatogenesis and may be correlated with male infertility.     

The nucleotide sequence of boar transition protein 2 (TNP2) cDNA and haploid expression of the gene during spermatogenesis

A cDNA clone coding for boar transition protein 2 (TNP 2) was isolated from a randomly primed cDNA library of boar testis. Sequence analysis revealed an open reading frame of 414 bp (corresponding to 138 amino acids), 33 bp of the 5' untranslated and about 300 bp of the 3' untranslated region. As compared to TNP 2 of mouse and rat, similarity with TNP 2 of the boar is approximately 70% at the nucleotide level and only about 40% on the basis of amino acid sequence. The similarity between boar and bull TNP2 is 77% and 64%, respectively. Northern blot experiments with RNA of different boar tissues and in situ hybridization on mature boar testis sections revealed testis-specific expression of the TNP 2 gene which is restricted to haploid germ cells. Hybridization experiments of boar TNP2 cDNA with testicular RNA of boar, bull, rat and mouse revealed decreasing intensities of the hybridization signals. With human testicular RNA no hybridization could be obtained.     

一种与Calpastatin具有部分同源结构的人精子膜蛋白的发现与研究

在以前工作中我们从人精子中分离纯化出一种与生育有关的糖蛋白,命名为ＢＳ－１７。本文用其多克隆抗血清从人睾丸λｇｔ１１ｃＤＮＡ表达库中克隆了编码ＢＳ－１７的ｃＤＮＡ片段。序列分析表明ＢＳ－１７ｃＤＮＡ片段长７９１ｂｐ,开放阅读框架５５８ｂｐ,可编码１８６个氨基酸。经数据库检索,该ｃＤＮＡ片段与人Ｃａｌｐａｓｔａｔｉｎ（Ｃａ￣（２＋）依赖的半胱氨酸蛋白酶ｃａｌｐａｉｎ抑制剂）基因３’端顺序具有９９．７％的同源,与Ｃａｌｐａｓｔａｔｉｎ蛋白质羧基末端同源性为９９．５％。用ｃＲＮＡ进行组织原位杂交结果表明,ＢＳ－１７基因表达于人精子减数分裂后期单倍精细胞阶段。     

Cloning and partial characterization of the cDNA encoding the fox sperm protein FSA-Acr.1 with similarities to the SP-10 antigen

We have isolated and characterized a cDNA, cFSA-Acr.1, encoding a testis-specific fox sperm antigen. The antigen is located on the inner acrosomal compartment, and is expressed during spermatogenesis on the developing acrosome of round and elongating spermatids. Database searches with the deduced amino acid sequence of cFSA-Acr.1 revealed that the clone has high homology to both human and baboon sperm protein SP-10, and the mouse sperm protein, MSA-63. The region of highest homology is within the carboxyl terminus. In the middle of the open reading frame, the fox sequence shows unique sequences absent from both the human, baboon SP-10, and mouse MSA-63 sequences. In addition to cFSA-Acr.1, two other clones were also isolated from the same fox testis cDNA library, and sequence analysis shows that they may represent alternatively spliced mRNAs coding for other FSA-Acr proteins. © 1995 Wiley-Liss, Inc.     

A testis-specific gene encoding a nuclear high-mobility-group box protein located in elongating spermatids.

A cDNA encoding a DNA-binding protein has been isolated by screening a mouse testicular expression cDNA library with a concatemer of a 12-bp putative protein-binding element present in the promoter of the testis-specific gene PGK-2. Sequence analysis of the isolated cDNA indicated the presence of an open reading frame that encodes a protein with two conserved DNA-binding motifs known as the high-mobility-group (HMG) boxes. Northern (RNA) blot analysis demonstrated that expression of the gene is restricted to the postpuberal testis. The DNA-binding activity and sequence specificity of the recombinant HMG protein were confirmed by DNA mobility shift assay using the initial concatemer of the PGK-2 promoter element as a probe as well as the wild-type or mutated versions of the 12-bp element within its natural sequence context. Immunocytochemical staining of adult testis sections with polyclonal antisera recognizing this recombinant HMG protein demonstrated that it is located predominantly in the nuclei of elongated spermatids at steps 9 and 10. These results suggest that this novel HMG box protein gene may be involved in the regulation of gene expression of the haploid male genome. The gene from which the cDNA was derived has been termed testis-specific HMG (tsHMG).