The Relative Levels of Translin-associated Factor X (TRAX) and Testis Brain RNA-binding Protein Determine Their Nucleocytoplasmic Distribution in Male Germ Cells
2004; Elsevier BV; Volume: 279; Issue: 30 Linguagem: Inglês
10.1074/jbc.m401442200
ISSN1083-351X
AutoresYoon Shin Cho, Vargheese M. Chennathukuzhi, Mary Ann Handel, John J. Eppig, Norman B. Hecht,
Tópico(s)Sperm and Testicular Function
ResumoTestis brain RNA-binding protein (TB-RBP), the mouse orthologue of human translin, is an RNA and single-stranded DNA-binding protein abundant in testis and brain. Translin-associated factor X (TRAX) was identified as a protein that interacts with TB-RBP and is dependent upon TB-RBP for stabilization. Using immunohistochemistry to investigate the subcellular locations of TB-RBP and TRAX during spermatogenesis, both proteins localize in nuclei in meiotic pachytene spermatocytes and in the cytoplasm of subsequent meiotic and post-meiotic cells. An identical subcellular distribution is seen in female germ cells. Western blot analysis of germ cell protein extracts reveals an increased ratio of TRAX to TB-RBP in meiotic pachytene spermatocytes compared with the post-meiotic round and elongated spermatids. Using COS-1 cells and mouse embryonic fibroblasts derived from TB-RBP null mice as model systems to examine the shuttling of TB-RBP and TRAX, we demonstrate that TRAX contains a functional nuclear localization signal and TB-RBP contains a functional nuclear export signal. Coexpression of both proteins in COS-1 cells and TB-RBP-deficient mouse embryonic fibroblasts reveals that the ratio of TRAX to TB-RBP determines their subcellular locations, i.e. increased TRAX to TB-RBP ratios lead to nuclear localizations, whereas TRAX remains in the cytoplasm when TB-RBP levels are elevated. These subcellular distributions require interaction between TB-RBP and TRAX. We propose that the subcellular locations of TB-RBP and TRAX in male germ cells are modulated by the relative ratios of TRAX and TB-RBP. Testis brain RNA-binding protein (TB-RBP), the mouse orthologue of human translin, is an RNA and single-stranded DNA-binding protein abundant in testis and brain. Translin-associated factor X (TRAX) was identified as a protein that interacts with TB-RBP and is dependent upon TB-RBP for stabilization. Using immunohistochemistry to investigate the subcellular locations of TB-RBP and TRAX during spermatogenesis, both proteins localize in nuclei in meiotic pachytene spermatocytes and in the cytoplasm of subsequent meiotic and post-meiotic cells. An identical subcellular distribution is seen in female germ cells. Western blot analysis of germ cell protein extracts reveals an increased ratio of TRAX to TB-RBP in meiotic pachytene spermatocytes compared with the post-meiotic round and elongated spermatids. Using COS-1 cells and mouse embryonic fibroblasts derived from TB-RBP null mice as model systems to examine the shuttling of TB-RBP and TRAX, we demonstrate that TRAX contains a functional nuclear localization signal and TB-RBP contains a functional nuclear export signal. Coexpression of both proteins in COS-1 cells and TB-RBP-deficient mouse embryonic fibroblasts reveals that the ratio of TRAX to TB-RBP determines their subcellular locations, i.e. increased TRAX to TB-RBP ratios lead to nuclear localizations, whereas TRAX remains in the cytoplasm when TB-RBP levels are elevated. These subcellular distributions require interaction between TB-RBP and TRAX. We propose that the subcellular locations of TB-RBP and TRAX in male germ cells are modulated by the relative ratios of TRAX and TB-RBP. Spermatogenesis is the dynamic developmental process in the testis where the male gamete differentiates into spermatozoa. Following spermatogonial proliferation and differentiation, recombination occurs during meiosis and haploid daughter cells are produced from the two meiotic divisions. This is followed by spermiogenesis where the haploid cells mature into spermatozoa. Throughout spermatogenesis, many testis-specific and testis-enriched mRNAs are temporally and spatially expressed in a tightly controlled manner (1Braun R.E. Int. J. Androl. 2000; 23: 92-94Google Scholar, 2Eddy E.M. Recent Prog. Horm. Res. 2002; 57: 103-128Google Scholar, 3Hecht N.B. BioEssays. 1998; 20: 555-561Google Scholar). A number of testicular nucleic acid-binding proteins believed to be involved in post-transcriptional regulation have been identified (1Braun R.E. Int. J. Androl. 2000; 23: 92-94Google Scholar, 3Hecht N.B. BioEssays. 1998; 20: 555-561Google Scholar, 4Ma K. Inglis J.D. Sharkey A. Bickmore W.A. Hill R.E. Prosser E.J. Speed R.M. Thomson E.J. Jobling M. Taylor K. Wolfe J. Cooke H.J. Hargreave T.B. Chandley A.C. Cell. 1993; 75: 1287-1295Google Scholar, 5Reijo R. Lee T.-Y. Salo P. Alagappan R. Brown L.G. Rosenberg M. Rozen S. Jaffe T. Straus D. Hovatta O. de la Chapelle A. Silber S. Page D.C. Nat. Genet. 1995; 10: 383-393Google Scholar, 6Venables J.P. Vernet C. Chew S.L. Elliott D.J. Cowmeadow R.B. Wu J. Cooke H.J. Artzt K. Eperon I.C. Hum. Mol. Genet. 1999; 8: 959-969Google Scholar).One of the nucleic acid-binding proteins, testis brain RNA-binding protein (TB-RBP), 1The abbreviations used are: TB-RBP, testis brain RNA-binding protein; NES, nuclear export signal; TRAX, translin-associated factor X; NLS, nuclear localization signal; MEF, mouse embryonic fibroblast; LZ, leucine zipper; GFP, green fluorescent protein; IRES, internal ribosome entry site; LMB, leptomycin B. 1The abbreviations used are: TB-RBP, testis brain RNA-binding protein; NES, nuclear export signal; TRAX, translin-associated factor X; NLS, nuclear localization signal; MEF, mouse embryonic fibroblast; LZ, leucine zipper; GFP, green fluorescent protein; IRES, internal ribosome entry site; LMB, leptomycin B. the mouse orthologue of human translin (7Aoki K. Suzuki K. Sugano T. Tasaka T. Nakahara K. Kuge O. Omori A. Kasai M. Nat. Genet. 1995; 10: 167-174Google Scholar, 8Aoki K. Suzuki K. Ishida R. Kasai M. FEBS Lett. 1999; 443: 363-366Google Scholar, 9Kasai M. Matsuzaki T. Katayanagi K. Omori A. Maziarz R.T. Strominger J.L. Aoki K. Suzuki K. J. Biol. Chem. 1997; 272: 11402-11407Google Scholar), has emerged as a highly versatile molecule. Translin, believed associated with chromosomal translocations, is in the nucleus in human lymphoid cell lines where active nuclear transport has been proposed to be involved in processes such as Ig/TCR rearrangements (7Aoki K. Suzuki K. Sugano T. Tasaka T. Nakahara K. Kuge O. Omori A. Kasai M. Nat. Genet. 1995; 10: 167-174Google Scholar). DNA damage has also been proposed to stimulate transport of translin into nuclei (9Kasai M. Matsuzaki T. Katayanagi K. Omori A. Maziarz R.T. Strominger J.L. Aoki K. Suzuki K. J. Biol. Chem. 1997; 272: 11402-11407Google Scholar). TB-RBP is a 28-kDa protein with several domains including a putative nuclear export signal (NES), a leucine zipper domain, and two basic domains in its N terminus (10Chennathukuzhi V. Kurihara Y. Bray J.D. Hecht N.B. J. Biol. Chem. 2001; 276: 13256-13263Google Scholar). TB-RBP functions as both a RNA-binding protein and a single-stranded DNA-binding protein in the testis and brain. As an RNA-binding protein, it links specific mRNAs to microtubules (11Han J.R. Yiu G.K. Hecht N.B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9550-9554Google Scholar, 12Wu X.-Q. Hecht N.B. Biol. Reprod. 2000; 62: 720-725Google Scholar), mediates intracellular and intercellular mRNA transport, and regulates timing of translation of specific mRNAs in male germ cells (13Morales C.R. Wu X.-Q. Hecht N.B. Dev. Biol. 1998; 201: 113-123Google Scholar, 14Morales C.R. Lefrancois S. Chennathukuzhi V. El-Alfy M. Wu X.-Q. Yang J. Gerton G.L. Hecht N.B. Dev. Biol. 2002; 246: 480-494Google Scholar). As a DNA-binding protein, TB-RBP/translin has been proposed to bind to breakpoint junctions of chromosomal translocations (7Aoki K. Suzuki K. Sugano T. Tasaka T. Nakahara K. Kuge O. Omori A. Kasai M. Nat. Genet. 1995; 10: 167-174Google Scholar, 8Aoki K. Suzuki K. Ishida R. Kasai M. FEBS Lett. 1999; 443: 363-366Google Scholar, 9Kasai M. Matsuzaki T. Katayanagi K. Omori A. Maziarz R.T. Strominger J.L. Aoki K. Suzuki K. J. Biol. Chem. 1997; 272: 11402-11407Google Scholar, 15Lee S.P. Fuior E. Lewis M.S. Han M.K. Biochemistry. 2001; 40: 14081-14088Google Scholar, 16Sengupta K. Rao B.J. Biochemistry. 2002; 41: 15315-15326Google Scholar, 17VanLoock M.S. Yu X. Kasai M. Egelman E.H. J. Struct. Biol. 2001; 135: 58-66Google Scholar) and regulate cell proliferation rates (18Chennathukuzhi V. Stein J.M. Abel T. Donlon S. Yang S. Miller J.P. Allman D.M. Seykora J. Simmons R.A. Hecht N.B. Mol. Cell. Biol. 2003; 23: 6419-6434Google Scholar, 19Ishida R. Okado H. Sato H. Shionoiri C. Aoki K. Kasai M. FEBS Lett. 2002; 525: 105-110Google Scholar). The official nomenclature for the mouse TB-RBP gene is Tsn.Translin-associated factor X (TRAX) has been identified by yeast two-hybrid assays (20Aoki K. Ishida R. Kasai M. FEBS Lett. 1997; 401: 109-112Google Scholar) and by immunoprecipitation (21Wu X.-Q. Lefrancois S. Morales C. Hecht N.B. Biochemistry. 1999; 38: 11261-11270Google Scholar) as a protein that interacts with TB-RBP. TRAX is a 33-kDa protein that shows high amino acid sequence homology to TB-RBP (20Aoki K. Ishida R. Kasai M. FEBS Lett. 1997; 401: 109-112Google Scholar). Like TB-RBP, TRAX is also highly expressed in testis and brain, but does not bind nucleic acids directly (10Chennathukuzhi V. Kurihara Y. Bray J.D. Hecht N.B. J. Biol. Chem. 2001; 276: 13256-13263Google Scholar). TRAX contains a putative bipartite nuclear localization signal (NLS) and a leucine zipper domain (10Chennathukuzhi V. Kurihara Y. Bray J.D. Hecht N.B. J. Biol. Chem. 2001; 276: 13256-13263Google Scholar, 20Aoki K. Ishida R. Kasai M. FEBS Lett. 1997; 401: 109-112Google Scholar). The leucine zipper domain of TRAX is essential for interactions with not only TB-RBP but also with C1D, an activator of the DNA-dependent protein kinase involved in the repair of DNA-double strand breaks and V(D)J recombination (22Erdemir T. Bilican B. Cagatay T. Goding C.R. Yavuzer U. Mol. Microbiol. 2002; 46: 947-957Google Scholar, 23Erdemir T. Bilican B. Oncel D. Goding C.R. Yavuzer U. J. Cell Sci. 2002; 115: 207-216Google Scholar). Although the function of TRAX is unknown, it has been postulated that TRAX complexed with translin is involved in dendritic RNA processing (24Finkenstadt P.M. Kang W.S. Jeon M. Taira E. Tang W. Baraban J.M. J. Neurochem. 2000; 75: 1754-1762Google Scholar) and in DNA double-strand break repair as an interacting partner with C1D (23Erdemir T. Bilican B. Oncel D. Goding C.R. Yavuzer U. J. Cell Sci. 2002; 115: 207-216Google Scholar). All or half of the TRAX protein is absent in null and heterozygous mice lacking both or one allele of TB-RBP, suggesting a close relationship between TB-RBP and TRAX (18Chennathukuzhi V. Stein J.M. Abel T. Donlon S. Yang S. Miller J.P. Allman D.M. Seykora J. Simmons R.A. Hecht N.B. Mol. Cell. Biol. 2003; 23: 6419-6434Google Scholar). Transfecting TRAX and TB-RBP cDNAs into embryonic fibroblasts derived from TB-RBP null mice has confirmed that TB-RBP is required to stabilize TRAX protein (25Yang S. Cho Y.S. Chennathukuzhi V.M. Underkoffler L.A. Loomes K. Hecht N.B. J. Biol. Chem. 2004; 279: 12605-12614Google Scholar). In the absence of TB-RBP, TRAX is ubiquitinated and degraded (25Yang S. Cho Y.S. Chennathukuzhi V.M. Underkoffler L.A. Loomes K. Hecht N.B. J. Biol. Chem. 2004; 279: 12605-12614Google Scholar). The official nomenclature for the mouse TRAX gene is Tsnax.The location of a protein is often indicative of its function (26Gasca S. Canizares J. de Santa Barbara P. Mejean C. Poulat F. Berta P. Boizet-Bonhoure B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11199-11204Google Scholar, 27Moore J.D. Kirk J.A. Hunt T. Science. 2003; 300: 987-990Google Scholar). RNA-binding proteins that participate in post-transcriptional regulation of mRNA frequently exhibit a dynamic subcellular distribution between the nucleus and the cytoplasm (28Shyu A.-B. Wilkinson M.F. Cell. 2000; 102: 135-138Google Scholar, 29Vera Y. Dai T. Sinha Hikim A.P. Lue Y. Salido E.C. Swerdloff R.S. Yen H. J. Androl. 2002; 23: 622-628Google Scholar). Many nucleocytoplasmic shuttling proteins contain both a nuclear localization signal and a nuclear export signal, domains that are recognized by specific receptors and adaptors in nuclear pore complexes (30Gama-Carvalho M. Carmo-Fonseca M. FEBS Lett. 2001; 498: 157-163Google Scholar, 31Macara I.G. Mol. Biol. Rev. 2001; 65: 1-25Google Scholar). Translin/TB-RBP contains a leucine-rich nuclear export signal that may be dependent on the CRM1 cellular export receptor for subcellular movement (10Chennathukuzhi V. Kurihara Y. Bray J.D. Hecht N.B. J. Biol. Chem. 2001; 276: 13256-13263Google Scholar, 32Henderson B.R. Eleftheriou A. Exp. Cell Res. 2000; 256: 213-224Google Scholar, 33Stade K. Ford C.S. Guthrie C. Weis K. Cell. 1997; 90: 1041-1050Google Scholar), whereas TRAX contains putative bipartite nuclear sequences suggesting an involvement of TRAX in the nuclear transport of translin/TB-RBP (20Aoki K. Ishida R. Kasai M. FEBS Lett. 1997; 401: 109-112Google Scholar).To gain insight into the cellular movements of TB-RBP and TRAX during spermatogenesis, we have investigated their subcellular localizations in mouse testes and have used COS-1 cells and mouse embryonic fibroblasts (MEF) from TB-RBP null mice as model systems to investigate protein shuttling. Immunohistochemistry demonstrates that both proteins are in the nuclei of pachytene spermatocytes and in the cytoplasm of diplotene/diakinesis spermatocytes and the post-meiotic spermatids. An identical subcellular distribution is seen in female germ cells. Transfecting COS-1 cells and TB-RBP-deficient MEFs, we find that the NLS of TRAX and the NES of TB-RBP are functional and provide a means for the two proteins to shuttle between the nucleus and cytoplasm as a complex. Moreover, the ratio of TB-RBP to TRAX determines the "steady state" subcellular locations of the two proteins.MATERIALS AND METHODSImmunohistochemistry—Testes from adult male mice and ovaries from pregnant females at days 16 and 18 post-coitum were fixed and processed by the Histological Core Facility of the Children's Hospital of Pennsylvania and the Jackson Laboratory. Immunohistochemistry was performed as previously described (13Morales C.R. Wu X.-Q. Hecht N.B. Dev. Biol. 1998; 201: 113-123Google Scholar, 34Gu W. Wu X.-Q. Meng X.H. Morales C.R. El-Alfy M. Hecht N.B. Mol. Reprod. Dev. 1998; 49: 219-228Google Scholar).Preparation of Testis and Germ Cell Extracts—Total testis extracts were prepared from adult and 17-day-old male CD-1 mice (Charles River Laboratories, Wilmington, MA) using a modification of the procedure of Wu et al. (35Wu X.-Q. Gu W. Meng X. Hecht N.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5640-5645Google Scholar). Testes were decapsulated, washed with phosphate-buffered saline buffer, and resuspended in 300 μl of RIPA buffer (10 mm sodium phosphate, pH 7.2, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 150 mm NaCl) containing 0.5 mm phenylmethylsulfonyl fluoride, 0.5 μg/ml leupeptin, 0.7 μg/ml pepstatin, and 2 μg/ml aprotinin. Testes were homogenized in a Teflon glass homogenizer on ice until most of cells were lysed and homogenates were centrifuged at 14,000 rpm at 4 °C for 15 min. The supernatants were stored at –80 °C until use. Testicular germ cells were separated by STA-PUT centrifugation as previously described (36Travis A.J. Foster J.A. Rosenbaum N.A. Visconti P.E. Gerton G.L. Kopf G.S. Moss S.B. Mol. Biol. Cell. 1998; 9: 263-276Google Scholar).Western Blot Analyses—For protein analyses of mouse testes, aliquots from total testis (10 μg) or germ cell extracts (70 μg) were separated by 10% SDS-PAGE. Whole cell lysates from COS-1 cells and TB-RBP-deficient MEFs were prepared in RIPA buffer as previously described (37Munshi N. Groopman J.E. Gill P.S. Ganju R.K. J. Immunol. 2000; 164: 1169-1174Google Scholar). Protein concentrations were determined with a BCA Protein Assay Kit (Pierce). For Western blotting, proteins separated by SDS-PAGE were blotted onto polyvinylidene difluoride membranes (Millipore, Bedford, MA), and anti-TB-RBP antibody (1:10,000 dilution) and anti-TRAX antibody (1:1,000 dilution) were used as primary antibodies. TB-RBP and TRAX were visualized with horseradish peroxidase-conjugated protein A (1:3,000 dilution) and the enhanced chemiluminescent (ECL) detection kit (Amersham Biosciences).Plasmid Construction—The open reading frame of wild type TB-RBP was amplified by PCR and subcloned to generate the expression plasmid pEGFP-TB-RBP (10Chennathukuzhi V. Kurihara Y. Bray J.D. Hecht N.B. J. Biol. Chem. 2001; 276: 13256-13263Google Scholar). Wild type mouse TRAX cDNA was obtained by RT-PCR from testis RNA and inserted into pEGFP-C2 (Clontech, Palo Alto, CA) to generate the expression plasmid pEGFP-TRAX. To create mutated forms of TB-RBP and TRAX expression plasmids as well as coexpression plasmids, wild type or mutated forms of TB-RBP and TRAX were amplified by PCR and inserted into plasmids, pEGFP-C2 or pIRES (Clontech) using restriction enzyme sites introduced with PCR primers. For the NES deletion form of TB-RBP, the plasmid was constructed by insertion of two PCR products (one for amino acids 1–145 and the other for 158–228) into the multiple cloning sites of pEGFP-C2, which resulted in replacing the entire NES with two amino acids, glycine and threonine. To make the leucine zipper (LZ) deletion form of TRAX, the PCR-mediated mutagenesis method (38Yang S. Sun Y. Zhang H. J. Biol. Chem. 2001; 276: 4889-4893Google Scholar) was applied using two sets of primers, 5′-GCCATGAACGGCAAAGAAGGACCA-3′ and 5′-CATGTCTTCCCCCGATATCTCCTCCATA-3′ for the amplification of the upstream open reading frame from the LZ and 5′-GATATGGAGGAGATATCGGGGGAAGACATGC-3′ and 5′-GCCTTAAGAAATGCTCTCTTCCTGATC-3′ for the downstream open reading frame from the LZ. Both amplified PCR products lack the LZ but contain an overlapping sequence (about 30 bp) by which two PCR products can be partially annealed and used as the template for the second round of PCR to generate the LZ deletion form of TRAX. All constructs were sequenced before use. The PCR primers and templates used for the creation of each plasmid are described in Table I.Table IThe PCR primers and templates used for the creation of expression plasmidsPlasmidForward primerReverse primerTemplatepEGFP-TRAX5′-CACGAATTCEATGAACGGCAAAGAAGGA-3′5′-CACGGATCCBTTAAGAAATGCTCTCTTC-3′RT product from mouse testis RNApEGFP-TRAX(—NLS)5′-CACGAATTCEGATGCCAGTTTGTCTTCG-3′5′-CACGGATCCBTTAAGAAATGCTCTCTTC-3′pEGFP-TRAXpEGFP-TB-RBP5′-CACGAATTCEATGTCTGTGAGCGAGATC-3′5′-CTGCCCGGGSmCTATTTTTCACCACAAGCCGCTGC-3′10pEGFP-(—NES)TB-RBP (for N terminus (1-145) amplification)5′-CACGAATTCEATGTCTGTGAGCGAGATC-3′5′-CTCGGTACCKAATTAAAACTCCTGA-3′pEGFP-TB-RBPpEGFP-(—NES)TB-RBP (for C terminus (158-228) amplification)5′-CTCGGTACCKAGTGTCACTGCTGGAGAC-3′5′-CTGCCCGGGSmCTATTTTTCACCACAAGCCGCTGC-3′pEGFP-TB-RBPpTRAX-IRES-EGFP-TB-RBP (for TRAX amplification)5′-CTCTCTAGAXGCTAGCNGCCACCATGAACGGCAAAGAAGGACCA-3′5′-CTCGTCGACSACGCGTMTTAAGAAATGCTCTCTTCCTG-3′pEGFP-TRAXpTRAX-IRES-EGFP-TB-RBP (for GFP-TB-RBP amplification)5′-CACTCTAGAXGCTAGCNGCTACCGGTCGCCACCATGG-3′5′-CTCGTCGACSACGCGTM CTATTTTTCACCACAAGCCGCTGC-3′pEGFP-TB-RBPpTB-RBP-IRES-EGFP-TRAX (for TB-RBP amplification)5′-CTCTCTAGAXGCTAGCN GCCACCATGTCTGTGAGCGAGATCTTC-3′5′-CTCGTCGACSACGCGTM CTATTTTTCACCACAAGCCGCTGC-3′pEGFP-TB-RBPpTB-RBP-IRES-EGFP-TRAX (for GFP-TRAX amplification)5′-CACTCTAGAXGCTAGCNGCTACCGGTCGCCACCATGG-3′5′-CTCGTCGACSACGCGTMTTAAGAAATGCTCTCTTCCTG-3′pEGFP-TRAXpEGFP-TB-RBP-(145)5′-CACGAATTCEATGTCTGTGAGCGAGATC-3′5′-CTCGGTACCKCTAAATTAAAACTCCTGAGAGATA-3′pEGFP-TB-RBPpEGFP-TB-RBP-(157)5′-CACGAATTCEATGTCTGTGAGCGAGATC-3′5′-CTCGGTACCKCTAGTTGACAGACAGCCTCGACAG-3′pEGFP-TB-RBPpEGFP-TB-RBP-(158-228)5′-CTCGGTACCKAGTGTCACTGCTGGAGAC-3′5′-CTGCCCGGGSmCTATTTTTCACCACAAGCCGCTGC-3′pEGFP-TB-RBPpTRAX(—NLS)-IRES-EGFP-TB-RBP (for TRAX(—NLS) amplification5′-CTCGCTAGCNGATGCCAGTTTGTCTTCGCCG-3′5′-CTCGTCGACSACGCGTM TTAAGAAATGCTCTCTTCCTG-3′pEGFP-TRAXpTRAX(ΔLZ)-IRES-EGFP-TB-RBP (for TRAX(ΔLZ) amplification)5′-CTCTCTAGAXGCTAGCN GCCACCATGAACGGCAAAGAAGGACCA-3′5′-CTCGTCGACSACGCGTM TTAAGAAATGCTCTCTTCCTG-3′pEGFP-TRAX(ΔLZ)pTB-RBP-(157)-IRES-EGFP-TRAX (for TB-RBP-(157) amplification)5′-CTCTCTAGAXGCTAGCN GCCACCATGTCTGTGAGCGAGATCTTC-3′5′-CTCACGCGTMCTAGTTGACAGACAGCCTCGACAG-3′pEGFP-TB-RBP Open table in a new tab Cell Culture, Transfections, and Microscopy—COS-1 cells and TB-RBP-deficient MEFs were maintained in 10-cm tissue culture dishes containing Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and streptomycin. For transient transfections of COS-1 cells and TB-RBP-deficient MEFs, FuGENE 6 reagent (Roche Applied Science) was used according to the manufacturer's protocol. When leptomycin B (LMB) (3 ng/ml) was used, it was added to the transfected COS-1 cell cultures 4 h before fixation. For fluorescence microscopy, cells grown on two-well chamber slides (Lab-Tek, Champbell, CA) were fixed using methanol and mounted using 95% glycerol in phosphate-buffered saline. Green fluorescent (GFP) fusion proteins in the transfected COS-1 cells and TB-RBP-deficient MEFs were detected by fluorescence microscopy and their subcellular locations were confirmed following 4′,6-diamidino-2-phenylindole counterstaining. Similar subcellular distributions were obtained with other fixation methods (4% paraformaldehyde).RESULTSTB-RBP and TRAX Are in Nuclei of Meiotic Pachytene Spermatocytes and in the Cytoplasm of Subsequent Germ Cells—To help define the cellular roles of TB-RBP and TRAX during spermatogenesis, we have investigated by immunostaining the subcellular steady state locations of both proteins in mouse testes using affinity purified antibodies to TB-RBP and TRAX (Fig. 1A). As previously shown (13Morales C.R. Wu X.-Q. Hecht N.B. Dev. Biol. 1998; 201: 113-123Google Scholar, 18Chennathukuzhi V. Stein J.M. Abel T. Donlon S. Yang S. Miller J.P. Allman D.M. Seykora J. Simmons R.A. Hecht N.B. Mol. Cell. Biol. 2003; 23: 6419-6434Google Scholar), TB-RBP is localized in the nuclei of pachytene spermatocytes and in the cytoplasm of subsequent cell types (Fig. 1A, a and b). A more detailed analysis of the stages of meiotic prophase after pachytene reveals TB-RBP rapidly appears in the cytoplasm in diplotene/diakinesis cells where it remains in the post-meiotic round spermatids (Fig. 1A, c). TRAX shows a similar pattern of nuclear localization in pachytene spermatocytes and cytoplasmic localization in later stage male germ cells (Fig. 1A, d and f).A similar pattern of subcellular localization of TB-RBP is seen in female germ cells. At the time mouse oocytes are in pachytene (day 16 post-coitum) (Fig. 1B, a), TB-RBP is in nuclei, whereas at day 18 post-coitum (Fig. 1B, b) when oocytes are arrested in diplotene/diakinesis, TB-RBP is in the cytoplasm. These data suggest that TB-RBP and TRAX have nuclear functions in pachytene-stage meiotic germ cells and cytoplasmic functions in subsequent meiotic and post-meiotic germ cells. The change in subcellular location of both proteins in the testis germ cells as the germ cells differentiate raises the possibility of a coordinated shuttling of proteins between the nucleus and cytoplasm in mouse germ cells.The TRAX to TB-RBP Ratio Is Increased in Pachytene Spermatocytes—Extracts from testes of 17-day-old mice and from adult mice were prepared for Western blotting and analyzed for TB-RBP and TRAX using anti-TB-RBP and anti-TRAX (Fig. 2A). The testes of 17-day-old mice contain pachytene spermatocytes, but lack post-meiotic germ cells. The ratio of TRAX to TB-RBP was increased in testes of 17-day-old mice compared with the testes of adult mice (Fig. 2A).Fig. 2Western blot of subcellular fractions and cell types from mouse testes. A, TB-RBP and TRAX in the total testis extract from 17-day-old (17d) and adult (A) mice. Total protein (10 μg) from total testis extract was separated by a 10% SDS-PAGE gel. B, TB-RBP and TRAX in enriched populations of male germ cells. PS, pachytene spermatocytes; RS, round spermatids; ES, elongated spermatids; and MGC, mixed germ cells. Total protein (70 μg) from each cell type was separated by a 10% SDS-PAGE gel. C, quantitation of relative amounts of TRAX and TB-RBP in B was performed by densitometry using ImageQuant software. Similar quantitations were obtained with additional blots.View Large Image Figure ViewerDownload (PPT)To more precisely compare the ratio of TRAX to TB-RBP in germ cells where the two proteins are predominately either in nuclei or cytoplasm, we quantitated the level of TB-RBP and TRAX in Western blots of enriched populations of pachytene spermatocytes, round spermatids, elongated spermatids, and mixed germ cells (Fig. 2, B and C). Although similar amounts of TB-RBP are seen in all the cell types, pachytene spermatocytes contain a higher relative amount of TRAX compared with TB-RBP than the other germ cell types examined. The post-meiotic cell types (round and elongated spermatids) maintain similar levels of TB-RBP, but lower amounts of TRAX relative to TB-RBP.TRAX Is in Nuclei and TB-RBP in the Cytoplasm in Singly Transfected COS-1 Cells—To determine whether the subcellular localization of TB-RBP could be influenced by TRAX, COS-1 cells were used as a model system. Plasmid constructs expressing TB-RBP and TRAX fused to GFP were individually transfected into COS-1 cells. In transfected COS-1 cells, TRAX is predominantly nuclear (about 91%) (Fig. 3A) with the remaining transfected cells showing both nuclear and cytoplasmic localization of GFP-TRAX (Table II). This is in agreement with previous findings (23Erdemir T. Bilican B. Oncel D. Goding C.R. Yavuzer U. J. Cell Sci. 2002; 115: 207-216Google Scholar).Fig. 3Subcellular localization of GFP-TRAX and GFP-TB-RBP in COS-1 cells. COS-1 cells transfected with pEGFP-TRAX (A), pEGFP-TRAX(–NLS) (C), and pEGFP-TB-RBP (E) are shown in the left panels. Corresponding 4′,6-diamidino-2-phenylindole stainings for each transfection are shown in the right panels (B, D, and F).View Large Image Figure ViewerDownload (PPT)Table IISubcellular localization of GFP-TB-RBP and GFP-TRAX in the presence or absence of LMBPlasmidLMBNN + CCNo. of cells counted%pEGFP-TRAX-90.99.10328pEGFP-TRAX(—NLS)-06.094.0302pEGFP-TB-RBP-04.395.7601+0.45.694.0503pTRAX-IRES-EGFP-TB-RBP-36.916.546.5157+86.710.62.7472pTRAX(-NLS)-IRES-EGFP-TB-RBP-03.496.6379pTRAX(ΔLZ)-IRES-EGFP-TB-RBP-04.695.4438pTB-RBP-IRES-EGFP-TRAX-1.41.796.9354+4.315.180.6186pTB-RBP-(157)-IRES-EGFP-TRAX-94.54.11.4290 Open table in a new tab To determine whether the bipartite NLS at the N terminus of TRAX (amino acids 11–27) is functional, we fused GFP to a construct lacking the N-terminal 27 amino acids of TRAX (TRAX-NLS). After transfection into COS-1 cells, TRAX-NLS remained in the cytoplasm (about 94%), suggesting that the NLS is needed for TRAX to move to the nucleus (Fig. 3C, Table II).In contrast to GFP-TRAX, GFP-TB-RBP is mostly in the cytoplasm (about 96%) (Fig. 3E, Table II), consistent with previous observations that TB-RBP contains a functional leucine-rich NES (10Chennathukuzhi V. Kurihara Y. Bray J.D. Hecht N.B. J. Biol. Chem. 2001; 276: 13256-13263Google Scholar). To determine whether the predominantly cytoplasmic localization of GFP-TB-RBP in the transfected COS-1 cells resulted from the export of TB-RBP from nuclei, LMB, the inhibitor for the cellular export receptor CRM1, was added to the COS-1 cell cultures 4 h before fixation. Leptomycin B treatment did not change the subcellular distribution of GFP-TB-RBP in the transfected COS-1 cells (Table II), suggesting that TB-RBP had not shuttled out of the nucleus. Thus, the cytoplasmic localization of GFP-TB-RBP could result from insufficient levels of TRAX for the GFP-TB-RBP in the transfected COS-1 cells, preventing the formation of GFP-TB-RBP complexes that could move into the nucleus.TB-RBP Translocation to the Nucleus Is Dependent upon TRAX—Because TB-RBP and TRAX interact forming oligomeric complexes via their leucine zipper domains (21Wu X.-Q. Lefrancois S. Morales C. Hecht N.B. Biochemistry. 1999; 38: 11261-11270Google Scholar, 23Erdemir T. Bilican B. Oncel D. Goding C.R. Yavuzer U. J. Cell Sci. 2002; 115: 207-216Google Scholar) and TRAX is dependent upon TB-RBP for its cellular stabilization (25Yang S. Cho Y.S. Chennathukuzhi V.M. Underkoffler L.A. Loomes K. Hecht N.B. J. Biol. Chem. 2004; 279: 12605-12614Google Scholar), we reasoned that TB-RBP could translocate to the nucleus as a complex dependent upon the NLS of TRAX. To test whether GFP-TB-RBP could be transported into nuclei by increasing the amount of TRAX in the transfected COS-1 cells, we utilized a coexpression plasmid, pTRAX-IRES-EGFP-TB-RBP, which allows the expression of two consecutive open reading frames from one mRNA. In plasmid pTRAX-IRES-EGFP-TB-RBP, the internal ribosome entry site (IRES) is located between two open reading frames of TRAX and GFP-TB-RBP (Fig. 4A, a). Because of a partially disabled IRES sequence, the rate of translation initiation of the second open reading frame is reduced to about 20% of the first open reading frame. Therefore, in the plasmid pTRAX-IRES-EGFP-TB-RBP we obtained attenuated expression of the second gene, GFP-TB-RBP, compared with TRAX (Fig. 4B, lane 5). When relatively high levels of TRAX are produced, about 37% of transfected COS-1 cells show nuclear localization of GFP-TB-RBP, 46.5% cytoplasmic, and 16.5% show both nuclear and cytoplasmic localization (Fig. 4C, a, Table II), suggesting that TRAX enhances the translocation of TB-RBP into the nucleus. Furthermore, when these transfected cells were incubated with leptomycin B, the percentage of cells showing nuclear localization of GFP-TB-RBP increased to about 87% (Fig. 4C, c, Table II). This indicates that export of GFP-TB-RBP from the nucleus is the reason about half of the cell
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