Skip to main content
SpringerLink
Log in

Cloning, expression and characterization of the putative nuclear transport factor 2 (NTF2) gene from moss Conocephalum conicum(L.) Dum

  • Published:
Molecular Biology Reports Aims and scope Submit manuscript

Abstract

Biomacromolecules import into the nucleus is a complex progress which requires the participation of several cytosolic factors, and nuclear transport factor 2 (NTF2) is one of essential components in nuclear trafficking. Its main role is to transport RanGDP from cytoplasm to nucleus by interacting with FxFG nucleoporin repeats. In the study a putative new gene, designated as CcNTF2, was obtained from the moss (Conocephalum conicum) cDNA library we have constructed. The full-length cDNA sequence is 913 bp in size contains a 372 bp open reading frame (ORF) flanked by a 195 bp 5′-untranslated sequence and a long 346 bp 3′-non-coding region, encoding 123 amino acids of 13,575.3 Da. Part of the genomic sequence was also cloned and sequenced, which is 1,602 bp long and possesses two exons and one intron. Alignment analysis showed that the CcNTF2 protein is high conserved among plant NTF2 and shares 81% similarity with the ones from Arabidopsis thaliana and Brassica rapa. The expression of wild-type CcNTF2 was detected by immunoblotting of extraction of C. conicum and it indicated the putative protein is integral. Through functional expression of CcNTF2-green fluorescent protein (GFP) in tobacco, it was demonstrated that CcNTF2 can accumulate at the nuclear rim. Site-directed mutagenesis analysis confirmed CcNTF2 P71K has influence on the protein import into nucleus. In addition, overexpression of CcNTF2 P71K was observed to be deleterious for the plant cell. It is the first illumination of NTF2 in moss, and our study established the primary foundation for further research on moss NTF2.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Quimby BB, Leung SW, Bayliss R, Harreman MT, Thirumala G, Stewart M, Corbett AH (2001) Functional analysis of the hydrophobic patch on nuclear transport factor 2 involved in interactions with the nuclear pore in vivo. J Biol Chem 276:38820–38829

    Article  CAS  PubMed  Google Scholar 

  2. Rout MP, Aitchison JD, Suprapto A, Hjertaas K, Zhao Y, Chait BT (2000) The yeast nuclear pore complex: composition, architecture, and transport mechanism. J Cell Biol 148:635–652

    Article  CAS  PubMed  Google Scholar 

  3. Rout MP, Wente SR (1994) Pores for thought: nuclear pore complex proteins. Trends Cell Biol 4:357–365

    Article  CAS  PubMed  Google Scholar 

  4. Macara IG (2001) Transport into and out of the nucleus. Microbiol Mol Biol Rev 65:570–594

    Article  CAS  PubMed  Google Scholar 

  5. Moore MS (1998) Ran and nuclear transport. J Biol Chem 273:22857–22860

    Article  CAS  PubMed  Google Scholar 

  6. Avis JM, Clarke PR (1996) Ran, a GTPase involved in nuclear processes: its regulators and effectors. J Cell Sci 109:2423–2427

    CAS  PubMed  Google Scholar 

  7. Matunis MJ, Coutavas E, Blobel G (1996) A novel ubiquitin-like modification modulates the partitioning of the Ran-GTPase-activating protein RanGAP1 between the cytosol and the nuclear pore complex. J Cell Biol 135:1457–1470

    Article  CAS  PubMed  Google Scholar 

  8. Gorlich D, Mattaj IW (1996) Nucleocytoplasmic transport. Science 271:1513–1518

    Article  CAS  PubMed  Google Scholar 

  9. Kubitscheck U, Grünwald D, Hoekstra A, Rohleder D, Kues T, Siebrasse JP, Peters R (2005) Nuclear transport of single molecules:dwell times at the nuclear pore complex. J Cell Biol 168(2):233–243

    Article  PubMed  Google Scholar 

  10. Moore MS, Blobel G (1994) Purification of a Ran-interacting protein that is required for protein import into the nucleus. Proc Natl Acad Sci 91:10212–10216

    Article  CAS  PubMed  Google Scholar 

  11. Clarkson WD, Kent HM, Stewart M (1996) Separate binding sites on nuclear transport factor 2 (NTF2) for GDP-Ran and the phenylalanine-rich repeat regions of nucleoporins p62 and Nsp1p. J Mol Biol 263:517–524

    Article  CAS  PubMed  Google Scholar 

  12. Paschal BM, Delphin C, Gerace L (1996) Nucleotide-specific interaction of Ran/TC4 with nuclear transport factors NTF2 and p97. Proc Natl Acad Sci 93:7679–7683

    Article  CAS  PubMed  Google Scholar 

  13. Stewart M, Kent HM, McCoy AJ (1998) Structural basis for molecular recognition between nuclear transport factor 2 (NTF2) and the GDP-bound form of the ras-family GTPase Ran. J Mol Biol 227:635–646

    Article  Google Scholar 

  14. Bayliss R, Leung SW, Baker RP, Quimby BB, Corbett AH, Stewart M (2002) Structural basis for the interaction between NTF2 and nucleoporin FxFG repeats. EMBO J 21(12):2843–2853

    Article  CAS  PubMed  Google Scholar 

  15. Bayliss R, Ribbeck K, Akin D, Kent HM, Feldherr CM, Görlich D, Stewart M (1999) Interaction between NTF2 and xFxFG-containing nucleoporins is required to mediate nuclear import of RanGDP. J Mol Biol 293:579–593

    Article  CAS  PubMed  Google Scholar 

  16. Chaillan-Huntington C, Braslavsky CV, Kuhlmann J, Stewart M (2000) Dissecting the interactions between NTF2, RanGDP, and the nucleoporin XFXFG repeats. J Biol Chem 275:5874–5879

    Article  CAS  PubMed  Google Scholar 

  17. Impe KV, Hubert T, Corte VD, Vanloo B, Boucherie C, Vandekerckhove J, Gettemans J (2008) A new role for nuclear transport factor 2 and Ran: nuclear import of CapG. Traffic 9(5):695–707

    Article  PubMed  Google Scholar 

  18. Rensing SA, Lang D, Zimmer AD, Terry A et al (2008) The physcomitrella genome reveals evolutionary insights into the conquest of land by plants. Science 319(5859):64–69

    Article  CAS  PubMed  Google Scholar 

  19. Fukayama H, Hatch MD, Tamai T, Tsuchida H, Sudoh S, Furbank RT, Miyao M (2003) Activity regulation and physiological impacts of maize C4-specific phosphoenolpyruvate carboxylase overproduced in transgenic rice plants. Photosynth Res 77:227–239

    Article  CAS  PubMed  Google Scholar 

  20. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning; a laboratory manual, 2nd edn. Cold Spring Harbor Press, Cold Spring Harbor, pp 880–898

    Google Scholar 

  21. Lescot M, Déhais P, Moreau Y, De Moor B, Rouzé P, Rombauts S (2002) PlantCARE: a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res 30(1):325–327

    Article  CAS  PubMed  Google Scholar 

  22. Bannai H, Tamada Y, Maruyama O, Nakai K, Miyano S (2002) Extensive feature detection of N-terminal protein sorting signals. Bioinformatics 18(2):298–305

    Article  CAS  PubMed  Google Scholar 

  23. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) ClustalW2 and ClustalX version 2. Bioinformatics 23(21):2947–2948

    Article  CAS  PubMed  Google Scholar 

  24. Kozak M (1987) An analysis of 5′-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res 15(20):8125–8148

    Article  CAS  PubMed  Google Scholar 

  25. De Angioletti M, Lacerra G, Sabato V, Carestia C (2004) β + 45 G → C: a novel silent β-thalassaemia mutation, the first in the Kozak sequence. Br J Haematol 124(2):224–231

    Article  PubMed  Google Scholar 

  26. Daraselia ND, Tarchevskaya S, Narita JO (1996) The promoter for tomato 3-hydroxy-3-methylglutaryl coenzyme A reductase gene 2 has unusual regulatory elements that direct high-level expression. Plant Physiol 112(2):727–733

    Article  CAS  PubMed  Google Scholar 

  27. Straub PF, Shen Q, Ho DTH (1994) Structure and promoter analysis of an ABA- and stress-regulated barley gene, HVA1. Plant Mol Biol 26(2):617–630

    Article  CAS  PubMed  Google Scholar 

  28. Smith A, Brownawell A, Macara IG (1998) Nuclear import of Ran is mediated by the transport factor NTF2. Curr Biol 8:1403–1406

    Article  CAS  PubMed  Google Scholar 

  29. Haizel T, Merkle T, Pay A, Fejes E, Nagy F (1997) Characterization of proteins that interact with the GTP-bound form of the regulatory GTPase Ran in Arabidopsis. Plant J 11:93–103

    Article  CAS  PubMed  Google Scholar 

  30. Pay A, Resch K, Frohnmeyer H, Fejes E, Nagy F, Nick P (2002) Plant RanGAPs are localized at the nuclear envelope in interphase and associated with microtubules in mitotic cells. Plant J 30:699–709

    Article  CAS  PubMed  Google Scholar 

  31. Zhao Q, Leung S, Corbett AH, Meier I (2006) Identification and characterization of the Arabidopsis orthologs of nuclear transport factor 2, the nuclear import factor of Ran. Plant Physiol 140:869–878

    Article  CAS  PubMed  Google Scholar 

  32. Mascarenhas D, Mettler IJ, Pierce DA, Lowe HW (1990) Intron-mediated enhancement of heterologous gene expression in maize. Plant Mol Biol 15(6):913–920

    Article  CAS  PubMed  Google Scholar 

  33. Jonsson JJ, Foresman MD, Wilson N, Mcivor RS (1990) Intron requirement for expression of the human purine nucleoside phosphorylase gene. Nucleic Acids Res 20(12):3191–3198

    Article  Google Scholar 

  34. Palmiter RD, Sandgren EP, Avarbock MR, Allen DD, Brinster RL (1991) Heterologous introns can enhance expression of transgenes in mice. Proc Natl Acad Sci 88(2):478–482

    Article  CAS  PubMed  Google Scholar 

  35. Rose AB, Last RL (2003) Introns act post-transcriptionally to increase expression of the Arabidopsis thaliana tryptophan pathway gene PAT1. Plant J 11(3):455–464

    Article  Google Scholar 

  36. Jeon JS, Lee S, Jung KH, Jun SH, Kim C, An G (2000) Tissue-preferential expression of a rice α-tubulin gene, OsTubA1, mediated by the first intron. Plant Physiol 123(3):1005–1014

    Article  CAS  PubMed  Google Scholar 

  37. Morello L, Bardini M, Sala F, Breviario D (2002) A long leader intron of the Ostub16 rice β-tubulin gene is required for high-level gene expression and can autonomously promote transcription both in vivo and in vitro. Plant J 29(1):33–44

    Article  CAS  PubMed  Google Scholar 

  38. Ho SH, So GMK, Chow KL (2001) Postembryonic expression of Caenorhabditis elegans mab-21 and its requirement in sensory ray differentiation. Dev Dynam 221(4):422–430

    Article  CAS  Google Scholar 

  39. Corbett AH, Silver PA (1996) The NTF2 gene encodes an essential, highly conserved protein that functions in nuclear transport in vivo. J Biol Chem 271:18477–18484

    Article  CAS  PubMed  Google Scholar 

  40. Ribbeck K, Lipowsky G, Kent HM, Stewart M, Gorlich D (1998) NTF2 mediates nuclear import of Ran. EMBO J 17:6587–6598

    Article  CAS  PubMed  Google Scholar 

  41. Bayliss R, Littlewood TD, Stewart M (2000) Structural basis for the interaction between FxFG nucleoporin repeats and importin-β in nuclear trafficking. Cell 102:99–108

    Article  CAS  PubMed  Google Scholar 

  42. Miao LL, Schulten K (2010) Transport-related structures and processes of the nuclear pore complex studied through molecular dynamics. Structure 17(3):449–459

    Article  Google Scholar 

  43. Tachibana T, Hieda M, Sekimoto T, Yoneda Y (1996) Exogenously injected nuclear import factor p10/NTF2 inhibits signal-mediated nuclear import and export of proteins in living cells. FEBS Lett 397:177–182

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Key laboratory for Molecular Biology and Biopharmaceuticals, Mianyang Normal University, Mianyang, 621000, Sichuan, China

    Chunxiang Bian, Qiping Ruan, Hongchun Ji, Lichun Jiang & Lijuan Yuan

  2. College of Life Science, China West Normal University, Nanchong, 637002, Sichuan, China

    Chunxiang Bian, Zhengsong Peng, Hongchun Ji & Lijuan Yuan

  3. College of Life Science and Bioengineering, Beijing University of Technology, Beijing, China, 100124

    Jintao Li

Authors
  1. Chunxiang Bian
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Qiping Ruan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Zhengsong Peng
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hongchun Ji
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Lichun Jiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jintao Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Lijuan Yuan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Qiping Ruan.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Bian, C., Ruan, Q., Peng, Z. et al. Cloning, expression and characterization of the putative nuclear transport factor 2 (NTF2) gene from moss Conocephalum conicum(L.) Dum. Mol Biol Rep 38, 2023–2032 (2011). https://doi.org/10.1007/s11033-010-0325-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11033-010-0325-6

Keywords

Search

Navigation