Pradeepa, MM and Manjunatha, S and Sathish, V and Agrawal, Shipra and Rao, MRS (2008) Involvement of Importin-4 in the Transport of Transition Protein 2 into the Spermatid Nucleus. In: Molecular and Cellular Biology, 28 (13). pp. 4331-4341.
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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 arried 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- \beta 3 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 ignal 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- \beta 1 as the template. Molecular docking simulations of $TP2_{NLS}$ with the importin-4 structure led to the identification of a $TP2_{NLS}$ 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.
| Item Type: | Journal Article |
|---|---|
| Publication: | Molecular and Cellular Biology |
| Publisher: | American Society for Microbiology |
| Additional Information: | Copyright of this article belongs to American Society for Microbiology. |
| Department/Centre: | Division of Biological Sciences > Biochemistry |
| Date Deposited: | 21 Jul 2008 |
| Last Modified: | 27 Aug 2008 13:37 |
| URI: | http://eprints.iisc.ac.in/id/eprint/15181 |
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