Role for Nucleolin/Nsr1 in the Cellular Localization of Topoisomerase I (2000)

Nucleolin functions in ribosome biogenesis and contains an acidic N terminus that binds nuclear localization sequences. In previous work we showed that human nucleolin associates with the N-terminal region of human topoisomerase I (Top1). We have now mapped the topoisomerase I interaction domain of nucleolin to the N-terminal 225 amino acids. We also show that theSaccharomyces cerevisiae nucleolin ortholog, Nsr1p, physically interacts with yeast topoisomerase I, yTop1p. Studies of isogenic NSR1 + andΔnsr1 strains indicate that NSR1is important in determining the cellular localization of yTop1p. Moreover, deletion of NSR1 reduces sensitivity to camptothecin, an antineoplastic topoisomerase I inhibitor. By contrast,Δnsr1 cells are hypersensitive to the topoisomerase II-targeting drug amsacrine. These findings indicate that nucleolin/Nsr1 is involved in the cellular localization of Top1 and that this localization may be important in determining sensitivity to drugs that target topoisomerases. Nucleolin functions in ribosome biogenesis and contains an acidic N terminus that binds nuclear localization sequences. In previous work we showed that human nucleolin associates with the N-terminal region of human topoisomerase I (Top1). We have now mapped the topoisomerase I interaction domain of nucleolin to the N-terminal 225 amino acids. We also show that theSaccharomyces cerevisiae nucleolin ortholog, Nsr1p, physically interacts with yeast topoisomerase I, yTop1p. Studies of isogenic NSR1 + andΔnsr1 strains indicate that NSR1is important in determining the cellular localization of yTop1p. Moreover, deletion of NSR1 reduces sensitivity to camptothecin, an antineoplastic topoisomerase I inhibitor. By contrast,Δnsr1 cells are hypersensitive to the topoisomerase II-targeting drug amsacrine. These findings indicate that nucleolin/Nsr1 is involved in the cellular localization of Top1 and that this localization may be important in determining sensitivity to drugs that target topoisomerases. topoisomerase I camptothecin RNA-binding domain glycine-arginine-rich nuclear localization sequence polymerase chain reaction glutathione S-transferase green fluorescent protein polyacrylamide gel electrophoresis amsacrine dithiothreitol phenylmethylsulfonyl fluoride 4′,6-diamidino- 2-phenylindole Human Top11 is a nuclear protein involved in the regulation of DNA structure and is the target of an important new class of antineoplastic drugs, the camptothecins (1Chen A.Y. Liu L.F. Annu. Rev. Pharmacol. Toxicol. 1994; 34: 191-218Crossref PubMed Scopus (780) Google Scholar, 2Wang J.C. Annu. Rev. Biochem. 1996; 65: 635-692Crossref PubMed Scopus (2076) Google Scholar). Studies in yeast provide convincing evidence that Top1 is the sole cellular target for camptothecins, with this knowledge facilitating attempts to understand the mechanisms by which the drug destroys cells (3Nitiss J. Wang J.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7501-7505Crossref PubMed Scopus (446) Google Scholar, 4Eng W.K. Faucette L. Johnson R.K. Sternglanz R. Mol. Pharmacol. 1988; 34: 755-760PubMed Google Scholar, 5Bjornsti M. Benedetti P. Vigilanti G. Wang J. Cancer Res. 1989; 49: 6318-6323PubMed Google Scholar). Top1 is a monomeric protein that relaxes supercoiled DNA by creating a transient single-strand nick, with this nick involving the covalent attachment of Top1 to the DNA phosphate backbone via a phosphotyrosine bond. Recent structural studies of Top1 and the Top1-DNA covalent complex led to molecular models of the mechanism by which camptothecins inhibit the catalytic activity of Top1 (6Redinbo M.R. Stewart L. Kuhn P. Champoux J.J. Hol W.G.J. Science. 1998; 279: 1504-1513Crossref PubMed Scopus (776) Google Scholar, 7Stewart L. Redinbo M.R. Qiu X. Hol W.G.J. Champoux J.J. Science. 1998; 279: 1534-1541Crossref PubMed Scopus (619) Google Scholar, 8Fan Y. Weinstein J.N. Kohn K.W. Shi L.M. Pommier Y. J. Med. Chem. 1998; 41: 2216-2226Crossref PubMed Scopus (185) Google Scholar). Nevertheless, formation of CPT-Top1-DNA ternary complexes is insufficient to explain the cytotoxic effects of CPT (9Goldwasser F. Bae I. Valenti M. Torres K. Pommier Y. Cancer Res. 1995; 55: 2116-2121PubMed Google Scholar, 10Beidler D.R. Chang J.Y. Zhou B.S. Cheng Y.C. Cancer Res. 1996; 56: 345-353PubMed Google Scholar, 11Sorensen M. Sehested M. Christensen I.J. Larsen J.K. Jensen P.B. Br. J. Cancer. 1998; 77: 2152-2161Crossref PubMed Scopus (8) Google Scholar). Current models invoke interactions between ternary complexes and replication or transcriptional machinery as being important in conversion of ternary complexes to lethal forms of DNA damage (12Hsiang Y. Lihou M. Liu L. Cancer Res. 1989; 49: 5077-5082PubMed Google Scholar, 13Wu J. Liu L.F. Nucleic Acids Res. 1997; 25: 4181-4186Crossref PubMed Scopus (133) Google Scholar). However, it is not known whether physical interactions between Top1 and other proteins (including proteins involved in replication or transcription) are important in the cytotoxic effects of CPT. Recently, an interaction between Top1 and the SV40 T antigen helicase was shown to modulate formation of Top1-CPT-DNA ternary complexes, suggesting that similar interactions between Top1 and cellular helicases may mediate the cytotoxicity of CPT (14Pommier Y. Kohlhagen G. Wu C. Simmons D.T. Biochemistry. 1998; 37: 3818-3823Crossref PubMed Scopus (31) Google Scholar). In addition, Top1 rapidly redistributes from the nucleolus to the nucleus or cytoplasm and is degraded after cellular exposure to CPT (15Beidler D.R. Cheng Y. Mol. Pharmacol. 1995; 47: 907-914PubMed Google Scholar, 16Desai S.D. Liu L.F. Vazquez-Abad D. D'Arpa P. J. Biol. Chem. 1997; 272: 24159-24164Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 17Danks M.K. Garrett K.E. Marion R.C. Whipple D.O. Cancer Res. 1996; 56: 1664-1673PubMed Google Scholar, 18Buckwalter C.A. Lin A.H. Tanizawa A. Pommier Y.G. Cheng Y.-C. Kaufmann S.H. Cancer Res. 1996; 56: 1674-1681PubMed Google Scholar, 19Wadkins R.M. Danks M.K. Horowitz L. Baker S.D. Exp Cell Res. 1998; 241: 332-339Crossref PubMed Scopus (13) Google Scholar). These alterations may confer transient cellular resistance to CPT and could be mediated by interactions between Top1 and other proteins. Top1 is known to physically interact with the following proteins: HMG17 (20Javaherian K. Liu L.F. Nucleic Acids Res. 1983; 11: 461-472Crossref PubMed Scopus (51) Google Scholar), casein kinase II (21Kordiyak G.J. Jakes S. Ingebritsen T.S. Benbow R.M. Biochemistry. 1994; 33: 13484-13491Crossref PubMed Scopus (31) Google Scholar), RNA polymerase I (22Rose K.M. Szopa J. Han F. Cheng Y.-C. Richter A. Scheer U. Chromosoma (Berl.). 1988; 96: 411-416Crossref PubMed Scopus (93) Google Scholar), nucleolin (23Bharti A.K. Olson M.O.J. Kufe D.W. Rubin E.H. J. Biol. Chem. 1996; 271: 1993-1997Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar), SV40 T antigen (24Simmons D.T. Melendy T. Usher D. Stillman B. Virology. 1996; 222: 365-374Crossref PubMed Scopus (76) Google Scholar, 25Haluska P. Saleem A. Edwards T.K. Rubin E.H. Nucleic Acids Res. 1998; 26: 1841-1847Crossref PubMed Scopus (36) Google Scholar), p53 (26Gobert C. Bracco L. Rossi F. Olivier M. Tazi J. Lavelle F. Larsen A.K. Riou J.F. Biochemistry. 1996; 35: 5778-5786Crossref PubMed Scopus (105) Google Scholar, 27Albor A. Kaku S. Kulesz-Martin M. Cancer Res. 1998; 58: 2091-2094PubMed Google Scholar), the TATA-binding protein (28Merino A. Madden K.R. Lane W.S. Champoux J.J. Reinberg D. Nature. 1993; 365: 227-232Crossref PubMed Scopus (323) Google Scholar), the splicing factors SF2/ASF (29Rossi F. Labourier E. Forne T. Divita G. Derancourt J. Riou J.F. Antoine E. Cathala G. Brunel C. Tazi J. Nature. 1996; 381: 80-82Crossref PubMed Scopus (287) Google Scholar) and PSF/p54nrb (30Straub T. Grue P. Uhse A. Lisby M. Knudsen B.R. Tange T. Westergaard O. Boege F. J. Biol. Chem. 1998; 273: 26261-26264Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), and a novel RING finger protein named topors (31Haluska P.H. Saleem A. Rasheed Z. Ahmed F. Su E.W. Liu L.F. Rubin E.H. Nucleic Acids Res. 1999; 27: 2538-2544Crossref PubMed Scopus (74) Google Scholar). Recently, two yTop1p-binding proteins were also identified (Tof1p and Tof2p) (32Park H. Sternglanz R. Yeast. 1999; 15: 35-41Crossref PubMed Scopus (58) Google Scholar). Although relatively little is known regarding either protein, a Tof2p paralog, Net1p, is required for the proper nucleolar localization of Sir2 and Cdc14 (33Straight A.F. Shou W. Dowd G.J. Turck C.W. Deshaies R.J. Johnson A.D. Moazed D. Cell. 1999; 97: 245-256Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar, 34Shou W. Seol J.H. Shevchenko A. Baskerville C. Moazed D. Chen Z.W. Jang J. Charbonneau H. Deshaies R.J. Cell. 1999; 97: 233-244Abstract Full Text Full Text PDF PubMed Scopus (597) Google Scholar). Since helicases may generate supercoiling problems that are resolved by topoisomerases, a need for physical interactions between these two enzyme classes is easily understood. Indeed, binding of Top1 by T antigen is one of several examples of a physical interaction between a helicase and a topoisomerase (35Duguet M. J. Cell Sci. 1997; 110: 1345-1350Crossref PubMed Google Scholar). The biochemical relevance of interactions between Top1 and other proteins is less clear. With regard to the nucleolin-Top1 interaction, there are three major domains in nucleolin: an acidic N terminus that contains multiple putative phosphorylation sites, a middle region containing four RNA-binding domains (RBDs), and a C-terminal glycine-arginine-rich (GAR) domain. In addition to binding RNA and DNA (36Bugler B. Bourbon H. Lapeyre B. Wallace M.O. Chang J.H. Amalric F. Olson M.O. J. Biol. Chem. 1987; 262: 10922-10925Abstract Full Text PDF PubMed Google Scholar, 37Sapp M. Richter A. Weisshart K. Caizergues-Ferrer M. Amalric F. Wallace M.O. Kirstein M.N. Olson M.O.J. Eur. J. Biochem. 1989; 179: 541-548Crossref PubMed Scopus (29) Google Scholar), nucleolin was reported to be a helicase (38Tuteja N. Huang N.W. Skopac D. Tuteja R. Hrvatic S. Zhang J. Pongor S. Joseph G. Faucher C. Amalric F. Falashi A. Gene (Amst.). 1995; 160: 143-148Crossref PubMed Scopus (87) Google Scholar) and to function in nucleocytoplasmic transport by binding nuclear localization sequences (NLSs) (39Borer R.A. Lehner C.F. Eppenberger H.M. Nigg E.A. Cell. 1989; 56: 379-390Abstract Full Text PDF PubMed Scopus (921) Google Scholar, 40Xue Z. Shan X. Lapeyre B. Melese T. Eur. J. Cell Biol. 1993; 62: 13-21PubMed Google Scholar). Several lines of evidence indicate that both nucleolin and Top1 are involved in rRNA synthesis and processing (41Wu R.S. Kumar A. Warner J.R. Proc. Natl. Acad. Sci., U. S. A. 1971; 12: 3009-3014Crossref Scopus (89) Google Scholar, 42Abelson H.T. Penman S. Nat. New Biol. 1972; 237: 144-146Crossref PubMed Scopus (73) Google Scholar, 43Bonven B.J. Gocke E. Westergaard O. Cell. 1985; 41: 541-551Abstract Full Text PDF PubMed Scopus (216) Google Scholar, 44Brill S.J. DiNardo S. Voelkel-Meiman K. Sternglanz R. Nature. 1987; 326: 414-416Crossref PubMed Scopus (302) Google Scholar, 45Egyhazi E. Durban E. Mol. Cell. Biol. 1987; 7: 4308-4316Crossref PubMed Scopus (43) Google Scholar, 46Zhang H. Wang J.C. Liu L.F. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1060-1064Crossref PubMed Scopus (262) Google Scholar, 47Schultz M.C. Brill S.J. Ju Q. Sternglanz R. Reeder R.H. Genes Dev. 1992; 6: 1332-1341Crossref PubMed Scopus (107) Google Scholar, 48Gadal O. Mariotte-Labarre S. Chedin S. Quemeneur E. Carles C. Sentenac A. Thuriaux P. Mol. Cell. Biol. 1997; 17: 1787-1795Crossref PubMed Scopus (58) Google Scholar, 49Ginisty H. Sicard H. Roger B. Bouvet P. J. Cell Sci. 1999; 112: 761-772Crossref PubMed Google Scholar). To gain insight into the cellular role of the nucleolin-Top1 interaction, we studied both a recombinant form of nucleolin and the Saccharomyces cerevisiae nucleolin ortholog NSR1. We find that the N terminus of nucleolin is necessary and sufficient for Top1 binding. Moreover, our results indicate that NSR1 is involved in the cellular localization of yTop1p and that loss of NSR1 alters cellular sensitivity to topoisomerase-targeting drugs but not to DNA damaging agents in general. S. cerevisiae strains used in this work are listed in Table I. MRG13 is a MATα derivative of MRG5 (50Tsalik E.L. Gartenberg M.R. Yeast. 1998; 14: 847-852Crossref PubMed Scopus (40) Google Scholar). TE25 was generated by transformation of FW251 with plasmid pICS16, which is designed to replace a 0.35-kilobase region in the NSR1 coding sequence with the URA3 gene (51Kondo K. Inouye M. J. Biol. Chem. 1992; 267: 16252-16258Abstract Full Text PDF PubMed Google Scholar). Disruption ofNSR1 was confirmed by PCR using primers that flank the 5′ integration site (data not shown), as well as by immunoblotting using an Nsr1p antibody. An identical strategy was used to disruptNSR1 in strains MRG13 and JN284, yielding TE13 and TE284, respectively. Immunoblotting was used to confirm loss of Nsr1p in TE13 and TE284.Table IYeast strainsStrainGenotypeSource or Ref.FY251MATa ura3–52 leu2-Δ1 trp1-Δ63 his3-Δ20052Winston F. Dollard C. Ricupero-Hovasse S.L. Yeast. 1995; 11: 53-55Crossref PubMed Scopus (784) Google ScholarTE25FY251 Δnsr1∷URA3This studyJCW27FY251 Δtop1-h&r53Mirabella A. Gartenberg M.R. EMBO J. 1997; 16: 523-533Crossref PubMed Scopus (22) Google ScholarMRG13FY251 MATα Δade2 cir°50Tsalik E.L. Gartenberg M.R. Yeast. 1998; 14: 847-852Crossref PubMed Scopus (40) Google ScholarTE13MRG13 Δnsr1∷URA3This studyJN284MATa ise1 ura3–52 leu2 his73Nitiss J. Wang J.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7501-7505Crossref PubMed Scopus (446) Google ScholarTE284JN284 Δnsr1∷URA3This study Open table in a new tab Yeast expression plasmids encoding nucleolin deletion fragments linked to GST were constructed using PCR (primers for this, and subsequently described PCRs are available upon request) with the pKG-Nuc plasmid (25Haluska P. Saleem A. Edwards T.K. Rubin E.H. Nucleic Acids Res. 1998; 26: 1841-1847Crossref PubMed Scopus (36) Google Scholar) as a template. PCR products were ligated into the pKG vector (54Mitchell D.A. Marshall T.K. Deschenes R.J. Yeast. 1993; 9: 715-722Crossref PubMed Scopus (268) Google Scholar) using the SmaI and HindIII sites in this vector. The resulting plasmids were named according to the encoded nucleolin residues and include pKG-Nuc1–323, pKG-Nuc323–707, pKG-Nuc1–225, pKG-Nuc224–323, pKG-Nuc1–125, and pKG-Nuc125–225. Regions of these vectors corresponding to 5′-cloning sites were sequenced to confirm that the proper recombinants had been obtained. Plasmid pKG-Nsr1 expresses Nsr1p linked to the C terminus of GST and was constructed using SmaI/HindIII restriction sites and PCR with pYCB5 (51Kondo K. Inouye M. J. Biol. Chem. 1992; 267: 16252-16258Abstract Full Text PDF PubMed Google Scholar) as a source for the NSR1coding sequence. Plasmid pYX-GFP expresses GFP in yeast under the control of theGAL1–10 promoter and was constructed using PCR with the pEGFP-C1 vector (CLONTECH) as a source for GFP coding sequence. The GFP PCR product was ligated into theBamHI and HindIII sites of the pYX133 vector (Ingenius Co., Oxford, United Kingdom). Similar strategies were used to generate pYX-GFP-yTop1 and pYX-GFP-Nop1, which express GFP-yTop1p and GFP-Nop1p fusion proteins, respectively. For the former vector, pYCpScTOP1 (provided by Mary-Ann Bjornsti) was used as template to obtain a PCR product containing the S. cerevisiae TOP1coding sequence. The HindIII and XhoI sites in pYX-GFP were used to place TOP1 downstream from the GFP coding sequence in this vector. The same strategy and restriction sites were used to generate pYX-GFP-Nop1. The source of NOP1coding sequence was S. cerevisiae genomic DNA from strain W303-1A (55Cheng T.H. Li Y.C. Gartenberg M.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5521-5526Crossref PubMed Scopus (53) Google Scholar). Expression of an appropriately migrating GFP fusion protein in cells transformed with the pYX-GFP-yTop1 or pYX-GFP-Nop1 vectors was confirmed by SDS-PAGE and immunoblotting using a GFP antibody (data not shown). Monoclonal antibodies were generously provided by the listed individuals for the following proteins: nucleolin, Ning-Hsing Yeh (56Fang S. Yeh N. Exp. Cell Res. 1993; 208: 48-53Crossref PubMed Scopus (76) Google Scholar); Nsr1p, M. Snyder and C. Copeland 2M. Snyder and C. Copeland, unpublished data.; human Top1, Y. C. Cheng (57Chang J. Dethlefsen L.A. Barley L.R. Zhou B. Cheng Y. Biochem. Pharmacol. 1992; 43: 2443-2452Crossref PubMed Scopus (79) Google Scholar). A polyclonal yTop1p antibody was obtained from Mary-Ann Bjornsti. Monoclonal GFP and actin antibodies were obtained from Roche Molecular Biochemicals and Amersham Pharmacia Biotech, respectively, and a polyclonal GST antibody was obtained from Amersham Pharmacia Biotech. Camptothecin lactone (CPT), methyl methane sulfonate, m-AMSA, and 1,10-phenanthroline were obtained from Sigma. Stock solutions were prepared for CPT at 5 mg/ml in Me2SO, for m-AMSA at 10 mg/ml in Me2SO, and for phenanthroline at 100 mg/ml in ethanol. Bleomycin sulfate was obtained as a 3 units/ml stock from Bristol-Myers Squibb Co. Expression and purification of full-length nucleolin and nucleolin fragments using the pKG-Nuc plasmid and derivatives were performed as described previously (25Haluska P. Saleem A. Edwards T.K. Rubin E.H. Nucleic Acids Res. 1998; 26: 1841-1847Crossref PubMed Scopus (36) Google Scholar). HeLa cell nuclear extracts were prepared by lysing cells in 0.35% Triton X-100 followed by extraction with 0.5 m NaCl as described (58Rubin E. Pantazis P. Bharti A. Toppmeyer D. Giovanella B. Kufe D. J. Biol. Chem. 1994; 269: 2433-2439Abstract Full Text PDF PubMed Google Scholar). Pull-down assays using HeLa nuclear extracts and 10 μl of glutathione beads (Amersham Pharmacia Biotech) loaded with approximately 4 μg of purified GST, GST-nucleolin, or GST-nucleolin deletion fragments were performed in 1 ml of binding buffer (phosphate-buffered saline containing 0.2% Tween 20, 1 mm EDTA, 1 mm DTT, 1 mm PMSF, 0.5 μg/ml leupeptin, and 1 μg/ml pepstatin) with approximately 20 μg of nuclear extract. After a 1-h incubation at 4 °C, the beads were washed four times with binding buffer. Proteins remaining bound to the beads were examined by SDS-PAGE and immunoblotting using Top1 and GST antibodies. For pull-down assays using yeast extracts, cells were grown at 30 °C in appropriate media to an A 600 of 0.7–0.9, then pelleted and resuspended in 3 volumes of ice-cold RIPA buffer (50 mm Tris-HCl, pH 7.2, 150 mm NaCl, 0.1% SDS, 1% Triton X-100, 1 mm DTT, and 1% sodium deoxycholate) with freshly 1 mm PMSF, 0.5 μg/ml leupeptin, and 1 μg/ml The was with beads for 10 at 4 °C and the by at for 5 The was and of remaining by at for 5 followed by incubation with 20 μl of glutathione beads for at 4 After the beads with 1 ml of RIPA bound proteins were by the beads in followed by SDS-PAGE and Yeast transformed with or pYX-GFP-Nop1 were grown to 600 of in media containing but Expression of GFP fusion proteins was by the addition of to a followed by an was performed by and cells in followed by addition of to a μg/ml After a incubation at the cells were washed with and with then resuspended in were by and fluorescent using a fluorescent with a GFP and was using GFP and of and and and of and were using and using Yeast cell were prepared as described for pull-down For of nuclear and proteins, yeast cell were resuspended in buffer (50 mm Tris-HCl, pH 3 mm DTT, 10 mm and 1 m and at for The cells were then with buffer with mg/ml for at The resulting were in the following 10 mm Tris-HCl, pH 20 mm 5 mm 3 mm DTT, 1 mm EDTA, 1 mm PMSF, 0.5 mg/ml leupeptin, and 1 mg/ml The was at to cells and were then pelleted by at for 20 at 4 The containing was then using a 30 were by in SDS-PAGE For yeast were grown in in a 30 °C to After in media to an A 600 of approximately at 30 °C was by of and of A A were the were in media to that the A 600 with to cell with the by was used to the of the of for by A 600 as a function of in the strains were examined using a A of was For drug sensitivity cells were grown and to an A 600 of using of 4 μl obtained from were containing drug with the then at 30 °C for containing CPT were prepared with mm pH 7.2, and as described previously E.A. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). In other of cells from to were containing either drug or of After incubation at 30 °C for 1 mm in were To of nucleolin involved in Top1 we generated a recombinant protein of nucleolin to the C terminus of Since we were to express a fusion protein containing the full-length nucleolin protein in we used a yeast expression (54Mitchell D.A. Marshall T.K. Deschenes R.J. Yeast. 1993; 9: 715-722Crossref PubMed Scopus (268) Google Scholar). The GST-nucleolin fusion protein was purified from yeast extracts using glutathione and by SDS-PAGE and of GST-nucleolin a which is the of a GST-nucleolin fusion protein protein was by both nucleolin and GST antibodies 1 and not shown). To whether the recombinant nucleolin binds Top1, beads loaded with either purified GST-nucleolin or GST were with HeLa cell nuclear extracts in a buffer. containing GST-nucleolin, but not GST bound Top1 in HeLa nuclear extracts 1 with our previous results using purified nucleolin (23Bharti A.K. Olson M.O.J. Kufe D.W. Rubin E.H. J. Biol. Chem. 1996; 271: 1993-1997Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar), these indicate that both purified and recombinant nucleolin Top1 in we Top1 binding by a of nucleolin deletion We and purified nucleolin fragments containing the acidic N-terminal region or or the of the protein, of the four and the C-terminal domain In the purified a of the that was with a GST antibody In molecular that were with the GST antibody were also and products and In pull-down assays using HeLa nuclear extracts and GST-nucleolin an N-terminal nucleolin containing residues was identified as being necessary and sufficient for binding of Top1 Immunoblotting with a GST antibody that these results could not be by the of of GST-nucleolin fragments that two fragments the nucleolin N-terminal and were both of binding Top1, Top1 was bound by the by the Immunoblotting with a GST antibody that this was not to the a of the to the the nucleolin a of the a of In addition, the a of and acidic residues acidic domain in the and not the acidic to both the and nucleolin fragments may be involved in acidic is not sufficient to explain binding of nucleolin fragments to the relatively Top1 The N terminus of nucleolin is in binding and nucleocytoplasmic transport (39Borer R.A. Lehner C.F. Eppenberger H.M. Nigg E.A. Cell. 1989; 56: 379-390Abstract Full Text PDF PubMed Scopus (921) Google Scholar, 40Xue Z. Shan X. Lapeyre B. Melese T. Eur. J. Cell Biol. 1993; 62: 13-21PubMed Google Scholar). In addition, nucleolin binds an region in Top1 that is required for the nuclear localization of Top1 (23Bharti A.K. Olson M.O.J. Kufe D.W. Rubin E.H. J. Biol. Chem. 1996; 271: 1993-1997Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, J. E. B.S. Westergaard O. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar). with our that the N terminus of nucleolin is sufficient for Top1 these that nucleolin function in the nucleocytoplasmic transport of a of strategies be to this in the of yeast in previous studies of Top1 to this cerevisiae R.J. Benedetti P. 1998; PubMed Scopus Google Scholar). cerevisiae protein Nsr1p is similar to nucleolin in sequence and function (51Kondo K. Inouye M. J. Biol. Chem. 1992; 267: 16252-16258Abstract Full Text PDF PubMed Google Scholar, W. Z. Melese T. J. Cell Biol. PubMed Scopus Google Scholar, K. Inouye M. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar, D. Melese T. Mol. Cell. Biol. 1992; 12: PubMed Scopus Google Nsr1p an acidic N-terminal region with several putative phosphorylation sites, a middle region with two and a C-terminal domain. To whether yTop1p physically interacts with Nsr1p, we using yTop1p and Nsr1p but were not to either protein using antibody. We constructed a vector a fusion protein under the control of a In SDS-PAGE of from yeast that were to express an appropriately migrating protein was by an Nsr1p antibody (data not shown). from cells GST or the protein were with glutathione with bound proteins to immunoblotting using GST and yTop1p antibodies. In cells GST GST was by glutathione beads 3 By in cells both this protein and were by the beads 3 with the nucleolin-Top1 (23Bharti A.K. Olson M.O.J. Kufe D.W. Rubin E.H. J. Biol. Chem. 1996; 271: 1993-1997Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar), these results indicate that a physical interaction between nucleolin and Top1 is in both yeast and human To whether the interaction is important in the cellular localization of we studied yTop1p