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• W2018071491 abstract "The negative cofactor 2 (NC2) is a protein complex composed of two subunits, NC2α and NC2β, and plays a key role in transcription regulation. Here we investigate whether each subunit contains a nuclear localization signal (NLS) that permits individual crossing of the nuclear membrane or whether nuclear import of NC2α and NC2β depends on heterodimerization. Our results from in vitro binding studies and transfection experiments in cultured cells show that each subunit contains a classical NLS (cNLS) that is recognized by the importin α/β heterodimer. Regardless of the individual cNLSs the two NC2 subunits are translocated as a preassembled complex as co-transfection experiments with wild-type and cNLS-deficient NC2 subunits demonstrate. Ran-dependent binding of the nuclear export receptor Crm1/exportin 1 confirmed the presence of a leucine-rich nuclear export signal (NES) in NC2β. In contrast, NC2α does not exhibit a NES. Our results from interspecies heterokaryon assays suggest that heterodimerization with NC2α masks the NES in NC2β, which prevents nuclear export of the NC2 complex. A mutation in either one of the two cNLSs decreases the extent of importin α/β-mediated nuclear import of the NC2 complex. In addition, the NC2 complex can enter the nucleus via a second pathway, facilitated by importin 13. Because importin 13 binds exclusively to the NC2 complex but not to the individual subunits this alternative import pathway depends on sequence elements distributed among the two subunits. The negative cofactor 2 (NC2) is a protein complex composed of two subunits, NC2α and NC2β, and plays a key role in transcription regulation. Here we investigate whether each subunit contains a nuclear localization signal (NLS) that permits individual crossing of the nuclear membrane or whether nuclear import of NC2α and NC2β depends on heterodimerization. Our results from in vitro binding studies and transfection experiments in cultured cells show that each subunit contains a classical NLS (cNLS) that is recognized by the importin α/β heterodimer. Regardless of the individual cNLSs the two NC2 subunits are translocated as a preassembled complex as co-transfection experiments with wild-type and cNLS-deficient NC2 subunits demonstrate. Ran-dependent binding of the nuclear export receptor Crm1/exportin 1 confirmed the presence of a leucine-rich nuclear export signal (NES) in NC2β. In contrast, NC2α does not exhibit a NES. Our results from interspecies heterokaryon assays suggest that heterodimerization with NC2α masks the NES in NC2β, which prevents nuclear export of the NC2 complex. A mutation in either one of the two cNLSs decreases the extent of importin α/β-mediated nuclear import of the NC2 complex. In addition, the NC2 complex can enter the nucleus via a second pathway, facilitated by importin 13. Because importin 13 binds exclusively to the NC2 complex but not to the individual subunits this alternative import pathway depends on sequence elements distributed among the two subunits. The negative cofactor 2 (NC2) 2The abbreviations used are: NC2, negative cofactor 2; NLS, nuclear localization signal; NES, nuclear export signal; Crm1, chromosome region maintenance 1; HEAT, huntingtin, elongation factor 3, protein phosphatase 2A, TOR1; EGFP, enhanced green fluorescent protein; RFP, red fluorescent protein; GST, glutathione S-transferase; LMB, leptomycin B; cNLS, classical nuclear localiztion signal; NPC, nuclear pore complex. is a protein complex composed of two subunits, NC2α (DRAP1) and NC2β (Dr1). Both subunits are conserved in eukaryotes and essential for Saccharomyces cerevisae viability (1Kim S. Na J.G. Hampsey M. Reinberg D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 820-825Crossref PubMed Scopus (84) Google Scholar, 2Gadbois E.L. Chao D.M. Reese J.C. Green M.R. Young R.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3145-3150Crossref PubMed Scopus (54) Google Scholar). NC2α and NC2β heterodimerize via histone-fold domains and associate with the promotor-bound TATA-binding protein (3Goppelt A. Stelzer G. Lottspeich F. Meisterernst M. EMBO J. 1996; 15: 3105-3116Crossref PubMed Scopus (129) Google Scholar, 4Mermelstein F. Yeung K. Cao J. Inostroza J.A. Erdjument-Bromage H. Eagelson K. Landsman D. Levitt P. Tempst P. Reinberg D. Genes Dev. 1996; 10: 1033-1048Crossref PubMed Scopus (113) Google Scholar). The resulting NC2-TATA-binding protein-DNA complex sterically hinders the recruitment of transcription factor IIB and in part of transcription factor IIA (5Kamada K. Shu F. Chen H. Malik S. Stelzer G. Roeder R.G. Meisterernst M. Burley S.K. Cell. 2001; 106: 71-81Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar), and thus inhibits transcription initiation (6Inostroza J.A. Mermelstein F.H. Ha I. Lane W.S. Reinberg D. Cell. 1992; 70: 477-489Abstract Full Text PDF PubMed Scopus (292) Google Scholar, 7Meisterernst M. Roeder R.G. Cell. 1991; 67: 557-567Abstract Full Text PDF PubMed Scopus (227) Google Scholar). The NC2 complex is present on a substantial fraction of human genes (8Albert T.K. Grote K. Boeing S. Stelzer G. Schepers A. Meisterernst M. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 10000-10005Crossref PubMed Scopus (20) Google Scholar). Besides mediating TATA-binding protein binding to TATA-containing and TATA-less promoters (9Gilfillan S. Stelzer G. Piaia E. Hofmann M.G. Meisterernst M. J. Biol. Chem. 2005; 280: 6222-6230Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) the NC2 complex can also mobilize TATA-binding protein on the DNA (10Schluesche P. Stelzer G. Piaia E. Lamb D.C. Meisterernst M. Nat. Struct. Mol. Biol. 2007; 14: 1196-1201Crossref PubMed Scopus (58) Google Scholar). In addition to the well established function as transcriptional repressor (11White R.J. Khoo B.C. Inostroza J.A. Reinberg D. Jackson S.P. Science. 1994; 266: 448-450Crossref PubMed Scopus (57) Google Scholar) several studies have shown that NC2 activates transcription, in vitro and in vivo (12Cang Y. Prelich G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12727-12732Crossref PubMed Scopus (42) Google Scholar, 13Castano E. Gross P. Wang Z. Roeder R.G. Oelgeschlager T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7184-7189Crossref PubMed Scopus (32) Google Scholar, 14Willy P.J. Kobayashi R. Kadonaga J.T. Science. 2000; 290: 982-985Crossref PubMed Scopus (133) Google Scholar, 15Geisberg J.V. Holstege F.C. Young R.A. Struhl K. Mol. Cell. Biol. 2001; 21: 2736-2742Crossref PubMed Scopus (60) Google Scholar, 16Lemaire M. Xie J. Meisterernst M. Collart M.A. Mol. Microbiol. 2000; 36: 163-173Crossref PubMed Scopus (37) Google Scholar). The mechanism underlying the positive effects of NC2 on gene expression is not understood. Although the two NC2 subunits mostly function together, recent studies in S. cerevisae (17Creton S. Svejstrup J.Q. Collart M.A. Genes Dev. 2002; 16: 3265-3276Crossref PubMed Scopus (22) Google Scholar), Drosophila (18Giot L. Bader J.S. Brouwer C. Chaudhuri A. Kuang B. Li Y. Hao Y.L. Ooi C.E. Godwin B. Vitols E. Vijayadamodar G. Pochart P. Machineni H. Welsh M. Kong Y. Zerhusen B. Malcolm R. Varrone Z. Collis A. Minto M. Burgess S. McDaniel L. Stimpson E. Spriggs F. Williams J. Neurath K. Ioime N. Agee M. Voss E. Furtak K. Renzulli R. Aanensen N. Carrolla S. Bickelhaupt E. Lazovatsky Y. DaSilva A. Zhong J. Stanyon C.A. Finley Jr., R.L. White K.P. Braverman M. Jarvie T. Gold S. Leach M. Knight J. Shimkets R.A. McKenna M.P. Chant J. Rothberg J.M. Science. 2003; 302: 1727-1736Crossref PubMed Scopus (1915) Google Scholar), and human provided evidence that NC2α and NC2β can associate with different proteins (19Klejman M.P. Pereira L.A. van Zeeburg H.J. Gilfillan S. Meisterernst M. Timmers H.T. Mol. Cell. Biol. 2004; 24: 10072-10082Crossref PubMed Scopus (21) Google Scholar, 20Assmann E.M. Alborghetti M.R. Camargo M.E. Kobarg J. J. Biol. Chem. 2006; 281: 9869-9881Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 21Iratni R. Yan Y.T. Chen C. Ding J. Zhang Y. Price S.M. Reinberg D. Shen M.M. Science. 2002; 298: 1996-1999Crossref PubMed Scopus (64) Google Scholar). In this study, we have analyzed whether human NC2 subunits contain localization signals that permit individual crossing of the nuclear membrane or whether nuclear import of NC2α and NC2β depends on heterodimerization. During interphase the exclusive site of nucleocytoplasmic exchange is the nuclear pore complex (NPC) (22Fahrenkrog B. Aebi U. Nat. Rev. Mol. Cell Biol. 2003; 4: 757-766Crossref PubMed Scopus (335) Google Scholar). Although small molecules can traverse the NPC via diffusion the passage of molecules larger than 40 kDa is restricted by the permeability barrier of the NPC (23Gorlich D. Kutay U. Annu. Rev. Cell Dev. Biol. 1999; 15: 607-660Crossref PubMed Scopus (1662) Google Scholar). Recent work by Frey and Görlich (24Frey S. Richter R.P. Gorlich D. Science. 2006; 314: 815-817Crossref PubMed Scopus (428) Google Scholar, 25Frey S. Gorlich D. Cell. 2007; 130: 512-523Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar) provides evidence that the permeability barrier of the NPC consists of a hydrophobic meshwork with hydrogel-like properties. The passage of this physical barrier requires soluble nuclear transport receptors (also referred to as karyopherins) that recognize intrinsic signal elements displayed on transport cargoes (26Pemberton L.F. Paschal B.M. Traffic. 2005; 6: 187-198Crossref PubMed Scopus (567) Google Scholar). Import signals named nuclear localization signals (NLSs) can be categorized in classical (cNLS) and non-classical (ncNLS) types. Non-classical NLSs are directly recognized by import receptors, whereas the binding of cNLSs requires an additional importin α adapter protein (27Gorlich D. Kostka S. Kraft R. Dingwall C. Laskey R.A. Hartmann E. Prehn S. Curr. Biol. 1995; 5: 383-392Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar). The most abundant nuclear export signals (NESs) are leucine-rich and interact with the export receptor chromosome region maintenance 1 (Crm1), also known as exportin 1 (28Fornerod M. Ohno M. Yoshida M. Mattaj I.W. Cell. 1997; 90: 1051-1060Abstract Full Text Full Text PDF PubMed Scopus (1734) Google Scholar, 29Stade K. Ford C.S. Guthrie C. Weis K. Cell. 1997; 90: 1041-1050Abstract Full Text Full Text PDF PubMed Scopus (929) Google Scholar). A steep RanGTP gradient across the nuclear membrane controls the binding and release of transport cargoes (30Mattaj I.W. Englmeier L. Annu. Rev. Biochem. 1998; 67: 265-306Crossref PubMed Scopus (1004) Google Scholar). Here we demonstrate that the NC2 subunits are imported into the nucleus as a preassembled complex. Nuclear accumulation of the NC2 complex occurs via two alternative pathways, facilitated by the importin α/β heterodimer and importin 13. The cNLSs present in each subunit have a cumulative effect on the nuclear targeting efficiency of the NC2 complex. In addition to its cNLS the NC2β subunit exhibits also a leucine-rich NES, which is masked upon heterodimerization with NC2α. Cell Culture-NIH-3T3 (mouse embryonic fibroblast) cells obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ number ACC59) and HeLa P4 cells (31Charneau P. Mirambeau G. Roux P. Paulous S. Buc H. Clavel F. J. Mol. Biol. 1994; 241: 651-662Crossref PubMed Scopus (325) Google Scholar) were cultured in Dulbeccos modified Eagle’s medium (Invitrogen). Medium was supplemented with 10% (v/v) fetal bovine serum (Biochrom), antibiotics, and 2 mm glutamine. Cells were maintained in a humidified incubator with 5% CO2 atmosphere at 37 °C. Expression Constructs-The coding regions of the respective genes were amplified from plasmid DNA using specific primer pairs with appropriate restriction sites. The bacterial expression constructs were cloned as follows: the coding regions of human NC2α and NC2β as SpeI/HindIII fragments into the respective sites of pET-41a(+) (Novagen); the coding regions of human NC2α and NC2β as NcoI/HindIII fragments into the respective sites of pETM-30 (EMBL, Heidelberg); the coding regions of human NC2α and NC2β as NdeI/BamHI fragments together with six histidine residues as NcoI/NdeI fragments into the NcoI/BamHI sites of pET-11d (Novagen); and the coding region of murine Ubc9 as an EcoRI/XhoI fragment into the respective sites of pGEX-4T-1 (GE Healthcare). The eukaryotic expression constructs were cloned as follows: the coding regions or gene fragments of human NC2α and NC2β as SalI/BamHI fragments into the respective sites of pEGFP-C1 (Clontech) and pPW1 (modified pEGFP-C1 in which EGFP was replaced by RFP using the restriction sites NheI/BglII); the coding regions of human NC2α and NC2β as BglII/PstI fragments into the respective sites of pEGFP-EGFP-N1 (modified pEGFP-N1 (Clontech) in which a second EGFP was inserted C-terminal of the MCS as SalI/BamHI fragment); the coding regions of human NC2α and NC2β as BglII/SalI fragments from the pEGFP-EGFP-N1 expression constructs into the BglII/SalI sites of pmRFP-N1 (modified pEGFP-N1 in which EGFP was replaced by mono-RFP using restriction sites AgeI/NotI); the gene fragments of NC2α (1–10 amino acids) and NC2β (97–106 amino acids) as SalI/BamHI fragments into the respective sites of pEGFP-EGFP-GST-C1 (modified pEGFP-C1 in which GST was inserted N-terminal of the MCS as the BglII/XhoI fragment and in which a second EGFP was inserted at the N terminus as NheI fragment); the coding region and gene fragments of human importin 13 as EcoRI/XhoI fragments were inserted into the respective sites of pCS2flag (modified pCS2plus (32Rupp R.A. Snider L. Weintraub H. Genes Dev. 1994; 8: 1311-1323Crossref PubMed Scopus (565) Google Scholar, 33Turner D.L. Weintraub H. Genes Dev. 1994; 8: 1434-1447Crossref PubMed Scopus (951) Google Scholar) in which a FLAG tag was inserted N-terminal of the MCS as a NcoI/EcoRI fragment); the coding regions of human importin β, Xenopus importin 7, and murine importin 9 as NruI fragments were inserted into the StuI sites of pCS2flag; and the coding region of human importin 5 as the StuI/XhoI fragment was inserted into the respective sites of pCS2flag. All constructs were verified by DNA sequencing (Andreas Nolte, Abteilung Entwicklungsbiochemie, Universität Göttingen, Germany). Site-directed Mutagenesis-Nucleotide exchanges in RFP-NC2α-(K4A) and RFP-NC2α-(K5A) were inserted using sense amplification primers: 5′-TGCAGTCGACATGCCCTCCGCAAAGAAAAAGTACAATGCC-3′ for K4A, 5′-TGCAGTCGACATGCCCTCCAAGGCAAAAAAGTACAATGCC-3′ for K5A. To generate EGFP-NC2β-(K100A), EGFP-NC2β-(R101A), EGFP-NC2β-(L78A/F80A), NC2β-(L78A/F80A)-RFP, and GST-NC2β-(L78A/F80A) site-directed mutagenesis was performed according to the QuikChange site-directed mutagenesis kit protocol (Stratagene). The following oligonucleotides were used: 5′-TGTAAAACAGTAGCATTAGCAAGAAGAAAGGCCAGTTCT-3′ (sense) and 5′-AGAACTGGCCTTTCTTCTTGCTAATGCTACTGTTTTACA-3′ (antisense) for K100A, 5′-AAAACAGTAGCATTAAAAGCAAGAAAGGCCAGTTCTCGT-3′ (sense) and 5′-ACGAGAACTGGCCTTTCTTGCTTTTAATGCTACTGTTTT-3′ (antisense) for R101A, and 5′-GTCATACAAGCACTAGAAAGTGCTGGAGCAGGCTCTTACATCAGTGAAGTA-3′ (sense) and 5′-TACTTCACTGATGTAAGAGCCTGCTCCAGCACTTTCTAGTGCTTGTATGAC-3′ (antisense) for L78A/F80A. Transfection Experiments-Transfection into HeLa P4 cells was performed with the Effectene™ Transfection Reagent (Qiagen) according to the manufacturer’s instructions. The cells were fixed 24 h after transfection with 3% paraformaldehyde in phosphate-buffered saline for 15 min and either analyzed directly by fluorescence microscopy or subjected to indirect immunostaining first. For that purpose, fixed cells were permeabilized with 0.5% Triton X-100 in phosphate-buffered saline for 10 min, blocked with 3% bovine serum albumin in phosphate-buffered saline, and an anti-FLAG polyclonal (rabbit) antibody (Sigma) and anti-rabbit Alexa 488 antibody or anti-rabbit Alexa 555 antibody (Molecular Probes) used to detect FLAG-importin 13. After washing, cells were stained with 10 μg/ml Hoechst 33258 (Molecular Probes) and mounted in Histogel (Linaris Histogel). Heterokaryon Assays-Interspecies heterokaryons of human HeLa P4 cells and mouse NIH-3T3 cells were formed as described previously (34Michael W.M. Choi M. Dreyfuss G. Cell. 1995; 83: 415-422Abstract Full Text PDF PubMed Scopus (469) Google Scholar). Briefly, HeLa P4 cells were transiently (co-)transfected with plasmid DNA encoding either fluorescently labeled NC2α and NC2β or GFP-QKI-5. Thirty hours post-transfection, non-transfected mouse NIH-3T3 cells were co-plated with the HeLa P4 cells and co-cultured for 18 h. The co-culture was then incubated for 2 h in the presence of 50 μg/ml cycloheximide and another 30 min in 100 μg/ml cycloheximide. The co-cultured cells were washed with phosphate-buffered saline and fused with Sigma HybriMax® warmed to 23 °C for 2 min. Fused cells were washed with phosphate-buffered saline and incubated at 37 °C in medium containing 100 μg/ml cycloheximide for an additional 6 h. The cells were fixed with 3% paraformaldehyde in phosphate-buffered saline for 15 min and counterstained with Hoechst 33258 to distinguish between human and mouse cell nuclei. In Vitro Transcription and Translation-Transcription and translation of human importin 13 fragments (see also Fig. 6A) were performed from the corresponding SP6 promoter constructs (pCS2flag) in vitro. Using the TnT coupled reticulocyte lysate system (Promega) according to the manufacturer’s instructions the proteins were labeled with [35S]methionine (Amersham Biosciences). Reactions were performed at 30 °C for 2 h in a 12.5-μl volume and the samples were then directly used for GST pulldown assays. Recombinant Protein Expression and Purification-Epitope-tagged NC2 complexes were generated as follows: NC2α and NC2β were co-expressed in Escherichia coli BL21(DE3). The cultures were grown at 37 °C to an optical density of 1.0 at 600 nm. After shifting the temperature to 25 °C bacterial protein expression was induced with 0.4 mm isopropyl β-d-thiogalactopyranoside and the cultures were grown for 4 h. The collected bacteria were resuspended in buffer A (50 mm Tris-HCl, pH 7.5, 400 mm NaCl, 5 mm β-mercaptoethanol), lysed by sonication, and the recombinant NC2 complexes were purified on nickel nitrilotriacetic acid-agarose (Qiagen) followed by either gel filtration on Superdex 200 (GE Healthcare) or a second purification step on glutathione-Sepharose 4B (GE Healthcare). The following proteins were expressed in E. coli BL21(DE3) as indicated and subsequently purified on glutathione-Sepharose 4B or nickel nitrilotriacetic acid-agarose according to the manufacturer’s instructions: His6-GST-NC2α, His6-GST-NC2β, and His6-GST-NC2β-(L78A/F80A) at 25 °C for 4 h with 0.2 mm isopropylβ-d-thiogalactopyranoside; and GST-UBC9 at 30 °C for 3 h with 0.2 mm isopropyl β-d-thiogalactopyranoside. The following transport receptors were expressed in E. coli JM109 or TG1 as described in the literature indicated and were purified on nickel nitrilotriacetic acid-agarose (Qiagen): Xenopus importin α1 (35Gorlich D. Prehn S. Laskey R.A. Hartmann E. Cell. 1994; 79: 767-778Abstract Full Text PDF PubMed Scopus (599) Google Scholar), human importin β (36Kutay U. Izaurralde E. Bischoff F.R. Mattaj I.W. Gorlich D. EMBO J. 1997; 16: 1153-1163Crossref PubMed Scopus (310) Google Scholar), transportin 1 (37Izaurralde E. Kutay U. von Kobbe C. Mattaj I.W. Gorlich D. EMBO J. 1997; 16: 6535-6547Crossref PubMed Scopus (493) Google Scholar), Xenopus importin 7, human importin 5 (38Jakel S. Gorlich D. EMBO J. 1998; 17: 4491-4502Crossref PubMed Scopus (421) Google Scholar), murine importin 9 (39Muhlhausser P. Muller E.C. Otto A. Kutay U. EMBO Rep. 2001; 2: 690-696Crossref PubMed Scopus (117) Google Scholar), human importin 13 (40Mingot J.M. Kostka S. Kraft R. Hartmann E. Gorlich D. EMBO J. 2001; 20: 3685-3694Crossref PubMed Scopus (172) Google Scholar), and human exportin 1/Crm1 (41Guan T. Kehlenbach R.H. Schirmer E.C. Kehlenbach A. Fan F. Clurman B.E. Arnheim N. Gerace L. Mol. Cell. Biol. 2000; 20: 5619-5630Crossref PubMed Scopus (99) Google Scholar). Expression and purification of RanQ69L were performed as described (42Ribbeck K. Lipowsky G. Kent H.M. Stewart M. Gorlich D. EMBO J. 1998; 17: 6587-6598Crossref PubMed Scopus (355) Google Scholar). GST Pulldown Assays-GST fusion proteins (or complexes containing GST fusions) immobilized on glutathione-Sepharose 4B were used as affinity matrix for binding experiments. Appropriate amounts of affinity matrix were incubated for 3 h at 4 °C with bacterial lysates containing expressed import receptors in buffer B (50 mm Tris-HCl, pH 7.5, 200 mm NaCl, 5 mm MgCl2, and 5 mm β-mercaptoethanol). The binding experiments were performed in the absence or presence of 2 μm RanQ69L(GTP). After washing three times with ice-cold buffer B, the affinity matrix was boiled in SDS-PAGE sample buffer and the matrix-bound proteins were analyzed by SDS-PAGE followed by Coomassie staining. For binding to importin 13 fragments, purified GST-NC2α/His6-NC2β and GST-UBC9 were immobilized on glutathione-Sepharose 4B that had been preincubated with 10% bovine serum albumin in buffer C (50 mm Tris-HCl, pH 7.5, 300 mm NaCl, 5 mm MgCl2, and 5 mm β-mercaptoethanol). The resulting affinity matrix was washed and incubated with 5 μl of the TnT coupled reticulocyte lysate containing in vitro transcribed and translated 35S-labeled importin 13 fragments in 300 μl of buffer C supplemented with 3% bovine serum albumin. After 3 h at 4 °C, the matrix was washed three times with buffer C, boiled in SDS-PAGE sample buffer, and matrix-bound proteins were analyzed by SDS-PAGE followed by phosphorimaging (Amersham Biosciences). For interaction studies with exportin 1, purified His6-GST-NC2β (0.4 μm), His6-GST-NC2β-(L78A/F80A) (0.4 μm), and epitope-tagged NC2 complexes (supplemental Fig. S3) were incubated for 3 h at 4 °C with purified recombinant exportin 1 (0.2 μm) in buffer D (50 mm Tris-HCl, pH 7.5, 130 mm NaCl, 2% glycerol, 5 mm MgCl2, and 5 mm β-mercaptoethanol). Binding of exportin 1 was performed in the absence or presence of 2 μm RanQ69L(GTP). Subsequent binding to glutathione-Sepharose 4B (20 μl of matrix) was carried out at 4 °C for 12 h. After washing three times with ice-cold buffer D, the matrix was boiled in SDS-PAGE sample buffer and matrix-bound proteins were analyzed by SDS-PAGE followed by Coomassie staining. Both NC2 Subunits Exhibit a Monopartite cNLS-Classical NLSs are characterized as short stretches enriched in basic amino acids (43Dingwall C. Laskey R.A. Trends Biochem. Sci. 1991; 16: 478-481Abstract Full Text PDF PubMed Scopus (1708) Google Scholar). Based on the consensus motifs for cNLSs (44Conti E. Kuriyan J. Structure Fold Des. 2000; 8: 329-338Abstract Full Text Full Text PDF Scopus (253) Google Scholar) each NC2 subunit contains one putative monopartite cNLS. This type of signal follows the four-residue consensus K-(K/R)-X-(K/R) (45Chelsky D. Ralph R. Jonak G. Mol. Cell. Biol. 1989; 9: 2487-2492Crossref PubMed Scopus (312) Google Scholar) with additional sequence requirements up- and downstream (46Conti E. Results Probl. Cell Differ. 2002; 35: 93-113Crossref PubMed Scopus (18) Google Scholar). The predicted basic stretch in NC2α (4KKKK7) is located in the unstructured N-terminal region, whereas the relevant sequence in NC2β (100KRRK103) is found in the predicted random coiled C-terminal half of the fourth α helix (5Kamada K. Shu F. Chen H. Malik S. Stelzer G. Roeder R.G. Meisterernst M. Burley S.K. Cell. 2001; 106: 71-81Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). To functionally characterize the putative signals we first compared the subcellular localization of wild-type and mutated NC2 subunits heterologously expressed in HeLa P4 cells (Fig. 1). To visualize the subcellular distribution, both NC2 subunits were fused to green (EGFP) and red (RFP) fluorescent proteins. Surprisingly, the subcellular distribution of the NC2 subunits showed a strong dependence on the position of the fluorescent fusion protein. Tandem EGFP or RFP fused to the C terminus caused a dominant cytoplasmic distribution of the individual NC2 subunits (supplemental Fig. S1A). In contrast, fusion of EGFP or RFP to the N terminus caused a nuclear localization pattern of NC2α and a rather homogeneous distribution of NC2β (Fig. 1 and supplemental Fig. S1B). Because the subcellular distribution of N-terminal fusion proteins was similar to the localization of endogenous NC2 subunits (data not shown) they were used for further analysis. We observed that wild-type RFP-NC2α no longer accumulated in the nucleus when either lysine residue 4 (K4A) or lysine residue 5 (K5A) was mutated (Fig. 1A). Similar results were found for NC2β, the homogeneous distribution of wild-type EGFP-NC2β was blocked when either lysine residue 100 (K100A) or arginine residue 101 (R101A) of the putative cNLS was substituted for alanine (Fig. 1B). Because the absence of neither critical lysine residues at the second position nor basic amino acid residues at the third position of the four-residue consensus sequence were tolerated, both sequences appeared to represent cNLSs. To analyze whether the predicted sequences are sufficient to mediate nuclear transport of a reporter protein, amino acids 1–10 of NC2α (1MPSKKKKYNA10) and 97–106 of NC2β (97VALKRRKASS106) containing the putative cNLSs (underlined) were fused to EGFP-EGFP-GST (EEG). EEG alone resides exclusively in the cytoplasm when expressed in mammalian cells, because it is too large to enter the nucleus by passive diffusion and does not contain a NLS (Fig. 1C). Fusion of amino acids 1–10 of NC2α or amino acids 97–106 of NC2β to EEG led to efficient nuclear import of the resulting proteins (Fig. 1C). To identify nuclear transport receptors that interact with the individual NC2 subunits, in vitro binding studies were performed. GST-tagged NC2α and NC2β were expressed in E. coli and immobilized on glutathione-Sepharose. The immobilized fusion proteins were incubated with the importin α/β heterodimer, importin β, transportin 1, importin 5, importin 7, and importin 13, all from bacterial lysates (Fig. 1D). After washing, the bound proteins were analyzed by SDS-PAGE followed by Coomassie staining. The importin α/β heterodimer was bound to both NC2 subunits. In the nucleus direct binding of RanGTP to β-family import receptors disintegrates the receptor-substrate interaction. The binding of importin α/β to the NC2 subunits was abolished in the presence of RanGTP, demonstrating its specificity (Fig. 1D). Additionally, weak binding of transportin 1 was observed. However, this interaction was not RanGTP-dependent (data not shown). None of the other import receptors, including importin β alone, bound significantly to the individual NC2 subunits. Together, these results demonstrate that each subunit exhibits a monopartite cNLS, 4KKKK7 in NC2α and 100KRRK103 in NC2β, which are necessary and sufficient for nuclear uptake of the respective NC2 subunit. NC2β Contains Also a Leptomycin B-sensitive NES That Is Recognized by Exportin 1-Next we examined whether the NC2 subunits exhibit NESs in addition to the characterized cNLSs. The first hint toward the existence of an NES was provided by the subcellular localization of N-terminal-tagged NC2β. Despite the presence of a cNLS, wild-type EGFP-NC2β showed a homogeneous localization in transfected cells (Fig. 2A). Furthermore, the first 110 amino acids of NC2β fused to EGFP (EGFP-NC2β-(1–110)) distributed homogeneously in transfected cells similar to the pattern of wild-type NC2β. In contrast, the first 100 amino acids of NC2β fused to EGFP (EGFP-NC2β-(1–100)) showed a cytoplasmic localization at steady state. This loss of nuclear uptake can be explained by the missing cNLS (100KRRK103) of NC2β. This fusion protein, however, is not homogeneously distributed like EGFP alone but strictly cytoplasmically, which can only be explained by the presence of an NES within the first 100 amino acids of NC2β. The most abundant and also the best-characterized NESs are leucine-rich (enriched in hydrophobic amino acids) and interact with the export receptor Crm 1/exportin 1 (28Fornerod M. Ohno M. Yoshida M. Mattaj I.W. Cell. 1997; 90: 1051-1060Abstract Full Text Full Text PDF PubMed Scopus (1734) Google Scholar, 29Stade K. Ford C.S. Guthrie C. Weis K. Cell. 1997; 90: 1041-1050Abstract Full Text Full Text PDF PubMed Scopus (929) Google Scholar). Thus, we examined whether the cytoplasmic localization of fluorescently labeled NC2β was lost on the application of leptomycin B (LMB), a specific inhibitor of exportin 1 (28Fornerod M. Ohno M. Yoshida M. Mattaj I.W. Cell. 1997; 90: 1051-1060Abstract Full Text Full Text PDF PubMed Scopus (1734) Google Scholar). We observed that wild-type EGFP-NC2β and NC2β-RFP largely accumulated in the nucleus on LMB treatment for 2 h (Fig. 2, A and B). In contrast to NC2β, the strong cytoplasmic distribution of C-terminal-labeled NC2α was not affected by LMB treatment (data not shown) and N-terminal-labeled NC2α showed a largely nuclear localization in transfected HeLa P4 cells (see Fig. 1A and supplemental Fig. S1B). Hence, these results do not provide evidence for the existence of an NES in NC2α. As LMB impairs various cellular pathways the effects of LMB were additionally verified. The sequence analysis of the first 100 amino acids of NC2β revealed one sequence element (71VIQALESLGF80) that fulfills the loosely defined consensus motif for leucine-rich NES: ΦX2–3ΦX2–3ΦXΦ (Φ= Leu, Ile, Val, Phe, Met) (47Bogerd H.P. Fridell R.A. Benson R.E. Hua J. Cullen B.R. Mol. Cell. Biol. 1996; 16: 4207-4214Crossref PubMed Scopus (321) Google Scholar, 48Kim F.J. Beeche A.A. Hunter J.J. Chin D.J. Hope T.J. Mol. Cell. Biol. 1996; 16: 5147-5155Crossref PubMed Scopus (84) Google Scholar). This putative NES was initiall" @default.
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• W2018071491 date "2009-04-01" @default.
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• W2018071491 title "Regulation of Nuclear Import and Export of Negative Cofactor 2" @default.
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