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Molecular and Cellular Biology, July 2008, p. 4331-4341, Vol. 28, No. 13
0270-7306/08/$08.00+0     doi:10.1128/MCB.00519-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Involvement of Importin-4 in the Transport of Transition Protein 2 into the Spermatid Nucleus {triangledown}

M. M. Pradeepa,1 S. Manjunatha,1 V. Sathish,1 Shipra Agrawal,1,{dagger} and M. R. S. Rao1,2*

Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore 560064,1 Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India2

Received 26 March 2007/ Returned for modification 11 May 2007/ Accepted 26 July 2007

Mammalian spermiogenesis is characterized by a unique chromatin-remodeling process in which histones are replaced by transition protein 1 (TP1), TP2, and TP4, which are further replaced by protamines. We showed previously that the import of TP2 into the haploid spermatid nucleus requires the components of cytosol and ATP. We have now carried out a detailed analysis to characterize the molecular components underlying the nuclear translocation of TP2. Real-time PCR analysis of the expression of different importins in testicular germ cells revealed that importin-4 and importin-β3 are significantly up-regulated in tetraploid and haploid germ cells. We carried out physical interaction studies as well as an in vitro nuclear transport assay using recombinant TP2 and the nuclear localization signal of TP2 (TP2NLS) fused to glutathione S-transferase in digitonin-permeabilized, haploid, round spermatids and identified importin-4 to be involved in the import of TP2. A three-dimensional model of the importin-4 protein was generated using the crystal structure of importin-β1 as the template. Molecular docking simulations of TP2NLS with the importin-4 structure led to the identification of a TP2NLS binding pocket spanning the three helices (helices 21 to 23) of importin-4, which was experimentally confirmed by in vitro interaction and import studies with different deletion mutants of importin-4. In contrast to TP2, TP1 import was accomplished through a passive diffusion process.

* Corresponding author. Mailing address: Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore 560064, India. Phone: 91-80-2208 2864. Fax: 91-80-2362 2762. E-mail: mrsrao{at}jncasr.ac.in

{triangledown} Published ahead of print on 6 August 2007.

{dagger} Present address: Institute of Bioinformatics and Applied Biotechnology, ITPL, Bangalore 560066, India.

Molecular and Cellular Biology, July 2008, p. 4331-4341, Vol. 28, No. 13
0270-7306/08/$08.00+0     doi:10.1128/MCB.00519-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Pradeepa, M. M., Nikhil, G., Hari Kishore, A., Bharath, G. N., Kundu, T. K., Rao, M. R. S. (2009). Acetylation of Transition Protein 2 (TP2) by KAT3B (p300) Alters Its DNA Condensation Property and Interaction with Putative Histone Chaperone NPM3. J. Biol. Chem. 284: 29956-29967 [Abstract] [Full Text]  
  • Poplonska, K., Kwiatkowska, M., Wojtczak, A., Polit, J. (2009). Immunogold Evidence Suggests That Endoplasmic Reticulum Is the Site of Protamine-Type Protein Synthesis and Participates in Translocation of These Proteins into the Nucleus During Chara vulgaris Spermiogenesis. Biol. Reprod. 80: 572-580 [Abstract] [Full Text]  


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