Differential Modes of Nuclear Localization Signal (NLS) Recognition by Three Distinct Classes of NLS Receptors

The targeting of karyophilic proteins to nuclear pores is mediated via the formation of a nuclear pore-targeting complex, through the interaction of nuclear localization signal (NLS) with its NLS receptor. Recently, a novel human protein, Qip1, was identified from a yeast two-hybrid system with DNA helicase Q1. This study demonstrates that Qip1 is a novel third class of NLS receptor that efficiently recognizes the NLS of the helicase Q1. Moreover, the data obtained in this study show that the specific interaction between Qip1 and the NLS of the helicase Q1 requires its upstream sequence of the minimal essential NLS. By using purified recombinant proteins alone in the digitonin-permeabilized cell-free transport system, it was demonstrated that the two known human NLS receptors, Rch1 and NPI-1, are able to transport all the tested NLS substrates into the nucleus, while Qip1 most efficiently transports the helicase Q1-NLS substrates, which contain its upstream sequence in so far as we have examined the system. Furthermore, in HeLa cell crude cytosol, it was found that endogenous Rch1 binds to all the tested NLS substrates, while the binding of endogenous NPI-1 is restricted to only some NLSs, despite the fact that NPI-1 itself shows binding activity to a variety of NLSs. These results indicate that at least three structurally and functionally distinct NLS receptors exist in the human single cell population, and suggest that the nuclear import of karyophilic proteins may be controlled in a complex manner at the NLS recognition step by the existence of a variety of NLS receptors with various specificities to each NLS. The targeting of karyophilic proteins to nuclear pores is mediated via the formation of a nuclear pore-targeting complex, through the interaction of nuclear localization signal (NLS) with its NLS receptor. Recently, a novel human protein, Qip1, was identified from a yeast two-hybrid system with DNA helicase Q1. This study demonstrates that Qip1 is a novel third class of NLS receptor that efficiently recognizes the NLS of the helicase Q1. Moreover, the data obtained in this study show that the specific interaction between Qip1 and the NLS of the helicase Q1 requires its upstream sequence of the minimal essential NLS. By using purified recombinant proteins alone in the digitonin-permeabilized cell-free transport system, it was demonstrated that the two known human NLS receptors, Rch1 and NPI-1, are able to transport all the tested NLS substrates into the nucleus, while Qip1 most efficiently transports the helicase Q1-NLS substrates, which contain its upstream sequence in so far as we have examined the system. Furthermore, in HeLa cell crude cytosol, it was found that endogenous Rch1 binds to all the tested NLS substrates, while the binding of endogenous NPI-1 is restricted to only some NLSs, despite the fact that NPI-1 itself shows binding activity to a variety of NLSs. These results indicate that at least three structurally and functionally distinct NLS receptors exist in the human single cell population, and suggest that the nuclear import of karyophilic proteins may be controlled in a complex manner at the NLS recognition step by the existence of a variety of NLS receptors with various specificities to each NLS. In eukaryotic cells, the selective transport of karyophilic proteins to the nuclei is mediated by short amino acid sequences, which are commonly referred to as nuclear localization signals (NLSs) 1The abbreviations used are: NLS, nuclear localization signal; GST, glutathione S-transferase; PTAC, pore-targeting complex; MDBK, Madin-Darby bovine kidney; BSA, bovine serum albumin; bBSA, biotinylated BSA; APC, allophycocyanin; DTT, dithiothreitol; TB, transport buffer. and which are characteristically rich in basic amino acids (1Dingwall C. Laskey R.A. Trends Biochem. Sci. 1991; 16: 478-481Abstract Full Text PDF PubMed Scopus (1713) Google Scholar, 2Garcia-Bustos J. Heitman J. Hall M.N. Biochim. Biophys. Acta. 1991; 1071: 83-101Crossref PubMed Scopus (444) Google Scholar, 3Makkerh J.P.S. Dingwall C. Laskey R.A. Curr. Biol. 1996; 6: 1025-1027Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). NLSs can be classified into two major groups. The first is a single type containing 3–5 basic amino acids with the weak consensus Lys-Arg/Lys-X-Arg/Lys, which is similar to the simian virus 40 large T antigen (SV40 T) NLS. The other is a bipartite type NLS containing two clusters of basic regions of 3–4 residues, each separated by approximately 10 amino acids, similar to nucleoplasmin NLS. The NLS functions at various positions within the protein and is capable of directing a non-karyophilic protein into nuclei when conjugated genetically or chemically (4Yoneda Y. Arch. Histol. Cytol. 1996; 59: 97-107Crossref Scopus (15) Google Scholar). It is generally thought that the NLS-mediated nuclear transport of karyophilic proteins occurs in at least two steps (5Newmeyer D.D. Forbes D.J. Cell. 1988; 52: 641-653Abstract Full Text PDF PubMed Scopus (371) Google Scholar, 6Richardson W.D. Mills A.D. Dilworth S.M. Laskey R.A. Dingwall C. Cell. 1988; 52: 655-664Abstract Full Text PDF PubMed Scopus (374) Google Scholar, 7Akey C.W. Goldfarb D.S. J. Cell Biol. 1989; 109: 971-982Crossref PubMed Scopus (156) Google Scholar). The first step is the NLS-dependent, but energy- and temperature-independent, binding to the cytoplasmic face of the nuclear pore complex. The second step is an energy and temperature-dependent translocation through the nuclear pore complex. In earlier studies, we found that a karyophilic protein forms a stable complex, the nuclear pore-targeting complex (PTAC) in the cytoplasm to target nuclear pores (4Yoneda Y. Arch. Histol. Cytol. 1996; 59: 97-107Crossref Scopus (15) Google Scholar, 8Imamoto N. Tachibana T. Matsubae M. Yoneda Y. J. Biol. Chem. 1995; 270: 8559-8565Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). The complex consists of a karyophilic protein and two essential factors, PTAC58 and PTAC97, which were originally isolated from mouse Ehrlich ascites tumor cells. PTAC58, also named mouse pendulin, directly recognizes the NLS (9Imamoto N. Shimamoto T. Takao T. Tachibana T. Kose S. Matsubae M. Sekimoto T. Shimonishi Y. Yoneda Y. EMBO J. 1995; 14: 3617-3626Crossref PubMed Scopus (271) Google Scholar). A number of proteins related to PTAC58 have been identified from other species using various biological screening techniques. These include proteins such as SRP1p/Kap60 (10Yano R. Oakes M. Yamagishi M. Dodd J.A. Nomura M. Mol. Cell. Biol. 1992; 12: 5640-5651Crossref PubMed Scopus (156) Google Scholar, 11Enenkel C. Blobel G. Rexach M. J. Biol. Chem. 1995; 270: 16499-16502Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar) from yeast, importin-α (12Görlich D. Prehn S. Laskey R.A. Hartmann E. Cell. 1994; 79: 767-778Abstract Full Text PDF PubMed Scopus (601) Google Scholar) fromXenopus, Rch1/hSRP1α (13Cuomo C.A. Kirch S.A. Gyuris J. Brent R. Oettinger M.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6156-6160Crossref PubMed Scopus (164) Google Scholar, 14Weis K. Mattaj I.W. Lamond A.I. Science. 1995; 268: 1049-1053Crossref PubMed Scopus (309) Google Scholar) and NPI-1/karyopherin-α/hSRP1 (15Cortes P. Ye Z.-S. Baltimore D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7633-7637Crossref PubMed Scopus (170) Google Scholar, 16O'Neill R.E. Palese P. Virology. 1995; 206: 116-125Crossref PubMed Scopus (133) Google Scholar, 17Moroianu J. Blobel G. Radu A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2008-2011Crossref PubMed Scopus (251) Google Scholar) from human, OHO-31/pendulin (18Török I. Strand D. Schmitt R. Tick G. Török T. Kiss I. Mechler B.M. J. Cell Biol. 1995; 129: 1473-1489Crossref PubMed Scopus (100) Google Scholar,19Küssel P. Frasch M. J. Cell Biol. 1995; 129: 1491-1507Crossref PubMed Scopus (111) Google Scholar) from Drosophila, and alMPα (20Hicks G.R. Smith H.M. Lobreaux S. Raikhel N.V. Plant Cell. 1996; 8: 1337-1352Crossref PubMed Scopus (62) Google Scholar) fromArabidopsis. Rch1 and NPI-1 were found to have about 50% amino acid identity. These NLS receptors contain Armadillo repeating motifs in their primary structure (21Peifer M. Berg S. Reynolds A.B. Cell. 1994; 76: 789-791Abstract Full Text PDF PubMed Scopus (550) Google Scholar, 22Yano R. Oakes M.L. Tabb M.M. Nomura M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6880-6884Crossref PubMed Scopus (129) Google Scholar). The motifs consist of about 40 residues, which are rich in hydrophobic amino acids. On the other hand, PTAC97 (23Imamoto N. Shimamoto T. Kose S. Takao T. Tachibana T. Matsubae M. Sekimoto T. Shimonishi Y. Yoneda Y. FEBS Lett. 1995; 368: 415-419Crossref PubMed Scopus (156) Google Scholar), which is also called p97 (24Adam E.J. Adam S.A. J. Cell Biol. 1994; 125: 547-555Crossref PubMed Scopus (257) Google Scholar, 25Chi N.C. Adam E.J.H. Adam S.A. J. Cell Biol. 1995; 130: 265-274Crossref PubMed Scopus (247) Google Scholar), importin-β (26Görlich 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 (421) Google Scholar), and karyopherin-β (27Radu A. Blobel G. Moore M.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1769-1773Crossref PubMed Scopus (386) Google Scholar), does not recognize the NLS, but mediates the first step in the transport by binding directly to the PTAC58, which is bound to the karyophile, and nuclear pore complex. Moreover, it has been found that Ran/TC4 (28Melchior F. Paschal B. Evans J. Gerace L. J. Cell Biol. 1993; 123: 1649-1659Crossref PubMed Scopus (472) Google Scholar, 29Moore M.S. Blobel G. Nature. 1993; 365: 661-663Crossref PubMed Scopus (641) Google Scholar), which is a Ras superfamily of small GTPases, supports the translocation step of the transport in conjunction with its interacting protein, p10/NTF2 (30Moore M.S. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10212-10216Crossref PubMed Scopus (291) Google Scholar,31Paschal B.M. Gerace L. J. Cell Biol. 1995; 129: 925-937Crossref PubMed Scopus (342) Google Scholar). In the Ran-mediated translocation step, although importin-α enters the nucleus together with the cargo, importin-β remains on the nuclear envelope, suggesting that the two subunits of importin dissociate during the import reaction (32Moroianu J. Hijikata M. Blobel G. Radu A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6532-6536Crossref PubMed Scopus (249) Google Scholar, 33Görlich D. Vogel F. Mills A.D. Hartmann E. Laskey R.A. Nature. 1995; 377: 246-248Crossref PubMed Scopus (410) Google Scholar). It has been clearly demonstrated that guanosine triphosphate (GTP) hydrolysis by Ran is required for the translocation step, but the exact mechanism of translocation remains unclear (34Melchior F. Gerace L. Curr. Opin. Cell Biol. 1995; 7: 310-318Crossref PubMed Scopus (227) Google Scholar, 35Görlich D. Mattaj I.W. Science. 1996; 271: 1513-1518Crossref PubMed Scopus (1067) Google Scholar, 36Koepp D.M. Silver P. Cell. 1996; 87: 1-4Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Although, for example, two major groups, Rch1 and NPI-1, are found in the human single cell population, the number of groups that exist in the same species and the variety of NLS receptors that are actually required for the nuclear transport remains unknown. Further, if the variety is essential for cellular functions, then it is essential to know whether each isoform recognizes different classes of NLSs and how each one distinguishes the NLSs in the cytoplasm. Recently, it was shown by Northern blot analysis that mouse pendulin/PTAC58 is very highly expressed in thymus, spleen, and heart (37Prieve M.G. Guttridge K.L. Munguia J.E. Waterman M.L. J. Biol. Chem. 1996; 271: 7654-7658Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). This result suggests that the expression of the isoform is regulated in a tissue-specific manner, although the physiological significance for the tissue-specific expression is presently unknown. More recently, consistent with these results, it was found that Rch1/hSRP1α and NPI-1/hSRP1 are differentially expressed in various human leukocyte cell lines and can be induced in normal human peripheral lymphocytes (38Nadler S.G. Tritschler D. Haffar O.K. Blake J. Bruce A.G. Cleaveland J.S. J. Biol. Chem. 1997; 272: 4310-4315Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar), indicating that this expression is regulated in a cell type-specific manner. Very recently, a novel human protein, named DNA helicase Q1-interacting protein 1 (Qip1; GenBank accession number AB002533) was identified by using a yeast two-hybrid system with DNA helicase Q1 (39Seki T. Tada S. Katada T. Enomoto T. Biochem. Biophys. Res. Commun. 1997; 234: 48-53Crossref PubMed Scopus (82) Google Scholar). DNA helicase Q1 is a human homologue of Escherichia coli RecQ, which is known to be a member of the RecF recombination pathway and has an intrinsic DNA-dependent ATPase activity (40Seki M. Miyazawa H. Tada S. Yanagisawa J. Yamaoka T. Hoshino S. Ozawa K. Eki T. Nogami M. Okumura K. Taguchi H. Hanaoka F. Enomoto T. Nucleic Acids Res. 1994; 22: 4566-4573Crossref PubMed Scopus (144) Google Scholar, 41Seki M. Yanagisawa J. Kohda T. Sonoyama T. Ui M. Enomoto T. J. Biochem. (Tokyo). 1994; 115: 523-531Crossref PubMed Scopus (33) Google Scholar, 42Puranam K.L. Blackshear P.J. J. Biol. Chem. 1994; 269: 29838-29845Abstract Full Text PDF PubMed Google Scholar, 43Tada S. Yanagisawa J. Sonoyama T. Miyajima A. Seki M. Ui M. Enomoto T. Cell Struct. Funct. 1996; 21: 123-132Crossref PubMed Scopus (12) Google Scholar). Since Qip1 has Armadillo repeating motifs in its primary structure and shows about 50% amino acid identity to both Rch1 and NPI-1, it was suspected to be a third class of human NLS receptor. In this study, we demonstrate that Qip1 is, in fact, a novel third class of human NLS receptor, which efficiently recognizes the NLS of DNA helicase Q1. Further, we found that the recognition of helicase Q1-NLS by Qip1 requires the upstream amino acid sequences of helicase Q1-NLS as well as a single basic amino acid cluster of the NLS itself. Moreover, we found that PTAC58 (∼Rch1) interacts with all the NLSs tested, but NPI-1 can interact with only limited types of NLSs, as evidenced by solution binding assays of the crude cytosol. In contrast, NPI-1 showed binding ability for all the tested NLSs when the solution binding assays were performed using recombinant proteins alone. These findings provide evidence that at least three distinct classes of NLS receptors exist in human cells, each of which shows NLS recognition in a different manner. The results suggest that a regulatory mechanism may exist for nuclear transport of karyophilic proteins at the NLS recognition step, and that the binding of some NLS receptors to particular NLSs may be controlled by other cytosolic proteins. Madin-Darby bovine kidney (MDBK) cells were cultured in 5% CO2 at 37 °C in Dulbecco's modified Eagle's essential medium (Life Technologies, Inc.) supplemented with 5% fetal bovine serum (Dainippon Pharmaceutical Co., Ltd). Synthetic peptides used in this study are listed in Table I. ShortT has only the minimal essential sequence of SV40 T-antigen NLS, while LongT contains the NLS minimal sequence of SV40 T-antigen and its upstream 17 amino acids. ReverseT contains the amino acid sequence of ShortT, but arranged in the reverse order, and was used as a transport-negative control. ShortQ1 has the C-terminal 15 amino acids of DNA helicase Q1, which contain its C-terminal basic amino acid cluster, while LongQ1 contains the C-terminal 27 amino acids, including the ShortQ1 sequence and its upstream 12 amino acids. LongQ1(KK-AA) has amino acid substitutions (K625,626A) of LongQ1 as shown in Table I. UpstreamQ1 has the same sequence as LongQ1, but lacks the C-terminal 10 amino acids. CBP80 has the bipartite type NLSs of a nuclear CAP-binding protein 80 (14Weis K. Mattaj I.W. Lamond A.I. Science. 1995; 268: 1049-1053Crossref PubMed Scopus (309) Google Scholar, 45Izaurralde E. Lewis J. McGuigan C. Jankowska M. Darzynkiewicz E. Mattaj I.W. Cell. 1994; 78: 657-668Abstract Full Text PDF PubMed Scopus (435) Google Scholar). Q1/T has UpstreamQ1 sequence, combined with the minimal essential sequence of SV40 T-antigen NLS, and its most C-terminal 3 amino acids are the same as helicase Q1. N-terminal cysteine and tyrosine residues were added for the chemical coupling reaction with Sulfo-SMPB (Pierce). To protect cysteine from histidine attack to the N terminus, the cysteine residue of LongT or CBP80 had glycine added or was N-acetylated, respectively. ShortT and ShortQ1 peptides were purchased from Sawady Technology Co. (Japan). All other peptides were obtained from the Peptide Institute (Japan).Table ISynthetic peptide sequenceNameSequence645ShortQ1CYGSKNTGAKKRKIDDA626LongQ1 CYFQKKAANMLQQSGSKNTGAKKRKIDDALongQ1(KK-AA) CYFQAAAANMLQQSGSKNTGAKKRKIDDAUpstreamQ1 CYFQKKAANMLQQSGSKNTShortTCYGGPKKKRKVEDPLongTGCYMPSSDDEATADSQHSTPPKKKRKVEDPReverseTCYGGPDEVKRKKKPCBP80AcCYMSRRRHSDENDGGQPHKRRKTSDANETEDQ1/T CYFQKKAANMLQQSGSKNTPKKKRKVDDA Open table in a new tab Bovine serum albumin (BSA) (Sigma), biotinylated BSA (bBSA), or allophycocyanin (APC) (Calbiochem) was chemically conjugated to synthetic peptides as described previously (8Imamoto N. Tachibana T. Matsubae M. Yoneda Y. J. Biol. Chem. 1995; 270: 8559-8565Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 46Yoneda Y. Arioka T. Imamoto-Sonobe N. Sugawa H. Shimonishi Y. Uchida T. Exp. Cell Res. 1987; 170: 439-452Crossref PubMed Scopus (43) Google Scholar). All the conjugates contained 5–9 peptides/carrier molecule, as judged from SDS-polyacrylamide gel electrophoresis. Qip1 constructed into pGEX2T (Pharmacia Biotech Inc.), was expressed as glutathione S-transferase (GST)-fused Qip1 in E. coli BL21 (DE3). The E. coli cells were grown in LB medium containing 50 μg/ml ampicillin at 37 °C to a density ofA 550 = 1.0. Expression was induced by the addition of 0.5 mmisopropyl-1-thio-β-d-galactopyranoside, followed by incubation for 12 h at 20 °C. Lysis of bacteria and purification of the fusion protein with glutathione-Sepharose were performed as described previously (8Imamoto N. Tachibana T. Matsubae M. Yoneda Y. J. Biol. Chem. 1995; 270: 8559-8565Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). The GST portion of chimeras was cleaved by incubation for 2 h at 25 °C with 1 NIH unit of thrombin/100 μg of chimeras. GST and thrombin were separated from recombinant proteins by ion-exchange chromatography (MonoQ) column at a flow rate of 0.5 ml/min, with a linear gradient from 0.05 to 1.0m NaCl in 20 mm Hepes-NaOH, pH 7.3, 2 mm dithiothreitol (DTT), 1 μg/ml each of aprotinin, leupeptin, and pepstatin A, and checked on 10% SDS-polyacrylamide gel electrophoresis. The purified proteins were dialyzed against 20 mm Hepes, pH 7.3, 100 mm CH3COOK, 2 mm DTT, and 1 μg/ml each of aprotinin, leupeptin, and pepstatin A. Human NPI-1 gene was amplified from a HeLa cell cDNA library using the polymerase chain reaction with appropriate oligonucleotides. Polymerase chain reaction products were confirmed by DNA sequencing. Recombinant PTAC58 and NPI-1 were purified as described previously (9Imamoto N. Shimamoto T. Takao T. Tachibana T. Kose S. Matsubae M. Sekimoto T. Shimonishi Y. Yoneda Y. EMBO J. 1995; 14: 3617-3626Crossref PubMed Scopus (271) Google Scholar). Recombinant Ran (47Melchior F. Sweet D.J. Gerace L. Methods Enzymol. 1995; 257: 279-291Crossref PubMed Scopus (53) Google Scholar, 48Sekimoto T. Nakajima K. Tachibana T. Hirano T. Yoneda Y. J. Biol. Chem. 1996; 271: 31017-31021Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) and p10 (49Tachibana T. Hieda M. Sekimoto T. Yoneda Y. FEBS Lett. 1996; 397: 177-182Crossref PubMed Scopus (46) Google Scholar) were prepared as described previously. HeLa S3 cells were grown in culture medium (RPMI 1640 (Life Technologies, Inc.), 5% fetal bovine serum, 10 mm Hepes-NaOH, pH 7.3) using a spinner flask (Bellco). When the cell concentration of HeLa cells reached 5 × 105 cells/ml, they were harvested, washed twice in phosphate-buffered saline (137 mm NaCl, 2.7 mm KCl, 8.1 mm Na2HPO4, 1.5 mm KH2PO4, pH 7.2), and washed once in washing buffer (10 mm Hepes-NaOH, pH 7.3, 110 mm CH3COOK, 2 mm DTT). They were then homogenized with lysis buffer (5 mm Hepes-NaOH, pH 7.3, 10 mm CH3COOK, 2 mm DTT, 2 μg/ml cytochalasin B, 1 mm(p-amidinophenyl)methanesulfonyl fluoride hydrochloride, 1 μg/ml each of aprotinin, leupeptin, and pepstatin A). The extract was dialyzed against transport buffer (TB) (20 mm Hepes, pH 7.3, 110 mm CH3COOK, 2 mm(CH3COO)2Mg, 5 mmCH3COONa, 0.5 mm EGTA, 2 mm DTT, 1 μg/ml each of aprotinin, leupeptin, and pepstatin A). The purified recombinant Qip1 was used to immunize two rabbits. Immunizations were performed as described previously (9Imamoto N. Shimamoto T. Takao T. Tachibana T. Kose S. Matsubae M. Sekimoto T. Shimonishi Y. Yoneda Y. EMBO J. 1995; 14: 3617-3626Crossref PubMed Scopus (271) Google Scholar) except that 0.75 mg of recombinant protein was injected into each rabbit on each occasion. Anti-Qip1 antibodies were purified independently from antiserum of each rabbit, by passage over GST-Qip1 immobilized to glutathione-Sepharose with glutaraldehyde. Antibodies bound to Qip1-Sepharose were eluted with 100 mmglycine-HCl, pH 2.5, and neutralized. After dialysis against phosphate buffer, pH 7.2, containing 300 mm NaCl, the purified antibodies were concentrated in a Micro Centricon 30. Anti-PTAC58 and anti-NPI-1 antibodies were purified as described previously (9Imamoto N. Shimamoto T. Takao T. Tachibana T. Kose S. Matsubae M. Sekimoto T. Shimonishi Y. Yoneda Y. EMBO J. 1995; 14: 3617-3626Crossref PubMed Scopus (271) Google Scholar,50Imamoto N. Matsuoka Y. Kurihara T. Kohno K. Miyagi M. Sakiyama F. Okada Y. Tsunasawa S. Yoneda Y. J. Cell Biol. 1992; 119: 1047-1061Crossref PubMed Scopus (149) Google Scholar). MDBK cells were plated on marked coverslips, and microinjection experiments of DNA helicase Q1-related peptide-bBSA (1 mg/ml) were performed as described previously (46Yoneda Y. Arioka T. Imamoto-Sonobe N. Sugawa H. Shimonishi Y. Uchida T. Exp. Cell Res. 1987; 170: 439-452Crossref PubMed Scopus (43) Google Scholar). After injection into the cytoplasm and incubation for 30 min at 37 °C, cells were fixed with 3.7% HCHO in phosphate-buffered saline. The injected biotinylated peptide conjugates were detected by fluorescein isothiocyanate-avidin (Pierce). Ten μl of each peptide-conjugated bBSA (1 mg/ml) was immobilized on 15 μl of avidin-agarose gel, and mixed with affinity-purified recombinant GST-PTAC97 (100 pmol) in addition to GST-Qip1, GST-PTAC58, or GST-NPI-1 (100 pmol), respectively, and the total reaction volume was adjusted to 100 μl with TB. After incubation for 1 h at 4 °C, materials bound to the immobilized peptide conjugates were washed with TB, and eluted with elution buffer (10 mm Tris-HCl, pH 8.0, 1 mm EDTA, 0.2% SDS) for 30 min at 37 °C. Each NLS receptor immobilized peptide conjugate analyzed by Western blotting with anti-PTAC58, anti-NPI-1, or anti-Qip1 antibodies. Immunoblotting was performed as described previously (9Imamoto N. Shimamoto T. Takao T. Tachibana T. Kose S. Matsubae M. Sekimoto T. Shimonishi Y. Yoneda Y. EMBO J. 1995; 14: 3617-3626Crossref PubMed Scopus (271) Google Scholar). One ml of HeLa cell cytosol (8 mg/ml) containing a final concentration of 2 μg/ml cytochalasin B was added to 10 μl of peptide-bBSA conjugates (1 mg/ml), and the mixture was incubated with 15 μl of avidin-agarose gel for 1 h at 4 °C. After the agarose was washed with TB, the bound proteins were eluted with incubation with elution buffer for 30 min at 37 °C. NLS receptors bound to immobilized NLS substrates were detected by anti-NLS receptor antibodies. Digitonin-permeabilized MDBK cells were prepared as described previously (8Imamoto N. Tachibana T. Matsubae M. Yoneda Y. J. Biol. Chem. 1995; 270: 8559-8565Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 44Adam S.A. Sterne-Marr R. Gerace L. J. Cell Biol. 1990; 111: 807-816Crossref PubMed Scopus (771) Google Scholar). The 10-μl sample solution contained 1 μl of peptide-APC conjugate (1 mg/ml) and appropriate transport factors were diluted with TB. For the nuclear-binding (first step) assay, the incubation was performed on ice for 20 min in the presence of each NLS receptor (7 pmol) and PTAC97 (6 pmol), the concentration of which was adjusted with TB containing 2% BSA. For the nuclear import (second step) assay, the incubation was performed at 30 °C for 20 min in the presence of each NLS receptor (7 pmol), PTAC97 (6 pmol), GDP-Ran (42 pmol), p10/NTF2 (14 pmol), 1 mm ATP, ATP regeneration system (20 units/ml creatine phosphokinase, 5 mm creatine phosphate), and 1 mm GTP, the concentration of which was adjusted with TB containing 2% BSA. After incubation, cells were fixed with 3.7% HCHO in TB. Peptide-APC conjugates were detected by Axiophoto microscopy (Carl Zeiss, Inc.). To determine whether Qip1 actually functions as an NLS receptor, we first attempted to determine the NLS of DNA helicase Q1. Based on the observation that Qip1 failed to bind DNA helicase Q1, which lacks the C-terminal basic amino acid cluster in a yeast two hybrid system (39Seki T. Tada S. Katada T. Enomoto T. Biochem. Biophys. Res. Commun. 1997; 234: 48-53Crossref PubMed Scopus (82) Google Scholar), we postulated that DNA helicase Q1-NLS involves the 4 C-terminal basic amino acids (KKRK645). Furthermore, since another basic amino acid cluster (KK626) was located 16 amino acids upstream from this C-terminal basic cluster, we also considered the possibility that the helicase Q1-NLS is a bipartite type. Therefore, considering these two possibilities, we prepared four types of synthetic peptides containing only the C-terminal basic amino acid cluster (ShortQ1, termed in Table I), the C-terminal basic cluster and its 19-amino acid upstream sequence (LongQ1), a peptide identical to LongQ1 except for the amino acid substitution K625,626A (LongQ1(KK-AA)), and the 17 amino acids upstream from the C-terminal basic amino acid cluster containing KK626 (UpstreamQ1), and conjugated these to bBSA. We then examined whether these conjugates migrate into the nucleus, when injected into the cytoplasm of cultured mammalian cells. As shown in Fig. 1, we found that ShortQ1, LongQ1, and LongQ1(KK-AA) efficiently directed bBSA into the nucleus to the same extent, but UpstreamQ1 did not. From these findings, we conclude that the C-terminal basic amino acid cluster of DNA helicase Q1 is necessary and sufficient for NLS activity. As a result, the NLS of helicase Q1 can be classified into a conventional single basic type. We next examined whether Qip1 directly binds to the NLS of DNA helicase Q1 in a solution binding assay. Purified recombinant Qip1 was added to the peptide-bBSA conjugates immobilized to avidin-agarose, and the interaction was examined by Western blotting of bound materials with affinity-purified anti-Qip1 antibodies. Unexpectedly, as shown in Fig.2 A, Qip1 bound to LongQ1- and LongQ1(KK-AA)-bBSA efficiently, but only weakly to ShortQ1- and not at all to UpstreamQ1-bBSA. Differential binding activity of Qip1 with ShortQ1- and LongQ1-bBSA indicates that the upstream sequences contained in LongQ1 peptide play an important role in the recognition of helicase Q1-NLS by Qip1. To confirm whether these upstream sequences actually contribute to the NLS recognition by Qip1, we prepared three additional peptides containing minimal essential sequences of SV40 T-antigen NLS (ShortT), ShortT sequences plus its own upstream sequences of the same length as those of LongQ1 (LongT), and SV40 T-antigen NLS combined to the 17-amino acid upstream sequence of DNA helicase Q1-NLS (Q1/T). Furthermore, we prepared a synthetic peptide, CBP80, which contains a typical bipartite type NLS for a nuclear CAP-binding protein 80. As shown in Fig. 2 B, Qip1 was found to bind efficiently to Q1/T-bBSA, but only slightly to ShortT-, LongT-, and CBP80-bBSA, indicating that the NLS binding of Qip1 requires not only the C-terminal basic amino acid cluster of DNA helicase Q1, but also its upstream sequences, and that the basic amino acid sequence in the NLS is essential but not sufficient for the binding of Qip1. On the other hand, recombinant PTAC58 and NPI-1 efficiently bound to all the tested functional NLS substrates (ShortQ1-, LongQ1-, LongQ1(KK-AA)-, ShortT-, LongT-, CBP80-, and Q1/T-bBSA), suggesting that the upstream sequences are not required for the NLS recognition by PTAC58 and NPI-1. To further investigate whether Qip1 actually functions as an NLS receptor, we examined the transport activity of recombinant Qip1 using an in vitro digitonin-permeabilized cell-free transport assay. As expected, as shown in Fig. 3, Qip1 transported LongQ1- and Q1/T-APC conjugates into the nucleus efficiently, but ShortQ1-APC was transported only weakly, and UpstreamQ1-APC conjugate not at all (data not shown), which is consistent with the solution binding assay described above. These results indicate that Qip1 acts as a novel class of NLS receptor, requiring the upstream sequences of the helicase Q1-NLS for its NLS recognition. From this and previous studies, it is evident that at least three different classes of NLS receptors, Rch1, NPI-1, and Qip1, are present in HeLa cells. However, the issue of whether distinct classes of NLS receptors show different NLS recognition specificity to a variety of NLSs remains poorly understood. To address this question, we compared the ability of three classes of recombinant NLS receptors to transport various NLS substrates into the n