Abstract
NASP (nuclear autoantigenic sperm protein) is a linker histone-binding protein found in all dividing cells that is regulated by the cell cycle (Richardson, R. T., Batova, I. N., Widgren, E. E., Zheng, L. X., Whitfield, M., Marzluff, W. F., and O'Rand, M. G. (2000) J. Biol. Chem. 275, 30378-30386), and in the nucleus linker histones not bound to DNA are bound to NASP (Alekseev, O. M., Bencic, D. C., Richardson R. T., Widgren E. E., and O'Rand, M. G. (2003) J. Biol. Chem. 278, 8846-8852). In mouse spermatogenic cells tNASP binds the testis-specific linker histone H1t. Utilizing a cross-linker, 3,3'-dithiobissulfosuccinimidyl propionate, and mass spectrometry, we have identified HSP90 as a testis/embryo form of NASP (tNASP)-binding partner. In vitro assays demonstrate that the association of tNASP with HSP90 stimulated the ATPase activity of HSP90 and increased the binding of H1t to tNASP. HSP90 and tNASP are present in both nuclear and cytoplasmic fractions of mouse spermatogenic cells; however, HSP90 bound to NASP only in the cytoplasm. In vitro nuclear import assays on permeabilized HeLa cells demonstrate that tNASP, in the absence of any other cytoplasmic factors, transports linker histones into the nucleus in an energy and nuclear localization signal-dependent manner. Consequently we hypothesize that in the cytoplasm linker histones are bound to a complex containing NASP and HSP90 whose ATPase activity is stimulated by binding NASP. NASP-H1 is subsequently released from the complex and translocates to the nucleus where the H1 is released for binding to the DNA.
Highlights
NASP is a linker histone-binding protein found in all dividing cells that is regulated by the cell cycle
Identification of testis/embryo form of NASP (tNASP)-binding Partners—To identify additional binding partners that may be complexed with tNASP in mouse germ cells, we used size exclusion chromatography and gel electrophoresis to isolate tNASP complexes. tNASP complexes were identified by SDS-PAGE and Western blot analysis with anti-NASP antibodies following size exclusion HPLC separation of a mouse testis lysate on a Bio-Sep SEC-S 3000 column (Fig. 2A)
In this study we have demonstrated that the histone-binding protein tNASP binds the testis-specific linker histone H1t and that the association of tNASP with HSP90 stimulated the ATPase activity of HSP90 and the binding of H1t to tNASP
Summary
H1, linker histone; tNASP, testis/embryo form of NASP; HSP90, heat shock protein 90; NLS, nuclear localization signal; H1t, testis form of H1; HPLC, high pressure liquid chromatography; DTSSP, 3,3Ј-dithiobissulfosuccinimidyl propionate; MALDI, matrix assisted laser desorption/ionization; TOF, time of flight; TPR, tetratricopeptide repeat. H1t is expressed during spermatogenesis, representing as much as 50% of the total H1 histones in pachytene spermatocytes [13,14,15], and is preceded by tNASP expression in preleptotene spermatocytes Their exact function during spermatogenesis is unknown, gene-targeting experiments [16] have shown that the absence of H1t and H1a in double null mice does not compromise spermatogenesis because compensation by H1c, H1d, and H1e most likely maintained normal spermatogenesis despite a reduced H1/nucleosome ratio [16]. Under these conditions the major H1 histone binding activity of tNASP could have shifted from H1t to H1c, H1d, and H1e in double null mice. TNASP can transport both H1t and somatic linker histones into nuclei
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