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. 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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 which is also S.A. J. Cell Biol. 1994; PubMed Scopus Google Scholar, S.A. J. Cell Biol. 1995; PubMed Scopus Google D. S. R. Dingwall C. Laskey R.A. Hartmann E. Prehn S. Curr. Biol. 1995; Full Text Full Text PDF PubMed Scopus Google and A. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: PubMed Scopus Google the NLS, but the first step in the transport by binding directly to the PTAC58, which is to the and nuclear pore complex. Moreover, it been found that J. J. Cell Biol. PubMed Scopus Google Scholar, Blobel G. PubMed Scopus Google which is a of the translocation step of the transport in with its protein, Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: PubMed Scopus Google B.M. J. Cell Biol. 1995; 129: PubMed Scopus Google Scholar). In the translocation importin-α the with the the nuclear that the two of the import J. M. Blobel G. Radu A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: PubMed Scopus Google Scholar, D. Mills A.D. Hartmann E. Laskey R.A. 1995; PubMed Scopus Google Scholar). It been demonstrated that by is the translocation but the of translocation Curr. Cell Biol. 1995; PubMed Scopus Google Scholar, D. Mattaj I.W. Science. 1996; PubMed Scopus Google Scholar, P. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). two major Rch1 and NPI-1, are found in the human single cell population, the number of that exist in the species and the variety of NLS receptors that are the nuclear transport the variety is essential it is essential to each recognizes of NLSs and each the NLSs in the Recently, it was by that mouse is in and M.L. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). This that the of the is in a the the is with it was found that Rch1/hSRP1α and are in various human cell and can be in human D. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google that this is in a cell a novel human protein, named DNA helicase protein number was identified by using a yeast two-hybrid system with DNA helicase Q1 T. S. T. T. Biochem. Biophys. PubMed Scopus Google Scholar). DNA helicase Q1 is a human of which is known to be a of the and an activity M. S. J. T. S. K. T. M. K. T. 1994; PubMed Scopus Google Scholar, M. J. T. T. M. T. J. Biochem. 1994; PubMed Scopus Google Scholar, J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, S. J. T. A. M. M. T. Cell 1996; PubMed Scopus Google Scholar). Qip1 Armadillo repeating motifs in its primary structure and shows about 50% amino acid to Rch1 and NPI-1, it was to be a third class of human NLS receptor. In this we that Qip1 in a novel third class of human NLS which efficiently recognizes the NLS of DNA helicase Q1. we found that the recognition of helicase Q1-NLS by Qip1 requires the upstream amino acid of helicase Q1-NLS as as a single basic amino acid of the NLS Moreover, we found that PTAC58 with all the NLSs but NPI-1 can with only of NLSs, as by binding of the crude In NPI-1 binding all the tested NLSs when the binding were using recombinant proteins These that at least three distinct of NLS receptors exist in human cells, each of which shows NLS recognition in a The results suggest that a may exist nuclear transport of karyophilic proteins at the NLS recognition and that the binding of some NLS receptors to NLSs may be controlled by other Madin-Darby bovine were in at in essential with bovine serum used in this study are in I. only the minimal essential sequence of NLS, while the NLS minimal sequence of and its upstream amino acids. the amino acid sequence of but in the and was used as a the amino acids of DNA helicase which contain its basic amino acid while the amino acids, the sequence and its upstream amino acids. amino acid of as in I. the sequence as but the 10 amino acids. the bipartite type NLSs of a nuclear protein K. Mattaj I.W. Lamond A.I. Science. 1995; 268: 1049-1053Crossref PubMed Scopus (309) Google Scholar, E. J. C. M. E. Mattaj I.W. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar). with the minimal essential sequence of NLS, and its most amino acids are the as helicase Q1. and were the with from to the the of or or was and were from other were obtained from the in a serum biotinylated or was chemically conjugated to as N. Tachibana T. Matsubae M. Yoneda Y. J. Biol. Chem. 1995; 270: 8559-8565Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, Y. T. N. Shimonishi Y. T. Cell PubMed Scopus Google Scholar). the as from Qip1 into was as glutathione Qip1 in E. The E. were in containing at to a was by the of by at of and of the protein with were as 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 of was by at with of of and were separated from recombinant proteins by at a of with a from to in each of and and The purified proteins were DTT, and each of and A. NPI-1 was from a HeLa cell using the with were by DNA PTAC58 and NPI-1 were purified as (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). D.J. 1995; PubMed Scopus Google Scholar, T. K. Tachibana T. T. Yoneda Y. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar) and T. M. Sekimoto T. Yoneda Y. FEBS Lett. 1996; PubMed Scopus Google Scholar) were as HeLa were in bovine 10 using a the cell of HeLa were in and in were with 10 DTT, each of and The was transport DTT, each of and The purified recombinant Qip1 was used to two were as (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) that of recombinant protein was into each each were purified from of each by to with to were with and containing the purified were in a and were purified as (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 N. Y. T. K. M. Y. S. Yoneda Y. J. Cell Biol. 1992; PubMed Scopus Google Scholar). were and of DNA helicase were as Y. T. N. Shimonishi Y. T. Cell PubMed Scopus Google Scholar). into the cytoplasm and at were with in The biotinylated were by of each was of and with recombinant in to or and the was to with at to the were with TB, and with at NLS receptor by with or was as (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). of HeLa cell containing a of was to 10 of and the was with of at the was with TB, the proteins were with with at NLS receptors to NLS substrates were by receptor were as N. Tachibana T. Matsubae M. Yoneda Y. J. Biol. Chem. 1995; 270: 8559-8565Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, S.A. R. J. Cell Biol. PubMed Scopus Google Scholar). The of and transport were with the the was in the of each NLS receptor and PTAC97 the of which was with containing the nuclear import the was at in the of each NLS receptor PTAC97 system and the of which was with containing were with in were by Qip1 functions as an NLS we first to the NLS of DNA helicase Q1. the that Qip1 to DNA helicase which the basic amino acid in a yeast two system T. S. T. T. Biochem. Biophys. PubMed Scopus Google we that DNA helicase Q1-NLS the basic amino acids Furthermore, basic amino acid was amino acids upstream from this basic we also the that the helicase Q1-NLS is a bipartite two we of containing only the basic amino acid in the basic and its acid upstream sequence a to the amino acid and the amino acids upstream from the basic amino acid containing and conjugated to examined into the nucleus, when into the cytoplasm of cells. in we found that and efficiently into the to the but we that the basic amino acid of DNA helicase Q1 is and NLS a the NLS of helicase Q1 can be classified into a single basic examined Qip1 directly binds to the NLS of DNA helicase Q1 in a binding recombinant Qip1 was to the to and the interaction was examined by of with as in Qip1 to and but only to and at all to binding activity of Qip1 with and that the upstream in an in the recognition of helicase Q1-NLS by upstream to the NLS recognition by Qip1, we three containing minimal essential of NLS its upstream of the as of and NLS to the acid upstream sequence of DNA helicase Q1-NLS Furthermore, we a which a bipartite type NLS a nuclear protein in Qip1 was found to efficiently to but only to and that the NLS binding of Qip1 requires only the basic amino acid of DNA helicase but also its upstream sequences, and that the basic amino acid sequence in the NLS is essential but the binding of On the other hand, recombinant PTAC58 and NPI-1 efficiently to all the tested NLS substrates and that the upstream are the NLS recognition by PTAC58 and Qip1 functions as an NLS we examined the transport activity of recombinant Qip1 using an in digitonin-permeabilized cell-free transport as in Qip1 and into the but was only and at all which is with the binding These results indicate that Qip1 as a novel class of NLS the upstream of the helicase Q1-NLS its NLS this and studies, it is that at least three of NLS receptors, NPI-1, and Qip1, are in HeLa cells. the of distinct of NLS receptors show NLS recognition to a variety of NLSs this we the of three of recombinant NLS receptors to transport various NLS substrates into the using the digitonin-permeabilized cell-free transport system. mouse PTAC58 is related to human an amino acid of this recombinant mouse PTAC58 was used of human The data and transport are their were the as of and the recombinant Qip1 and to the nuclear pores in the of recombinant PTAC97 the first step but and at all Moreover, the second step the recombinant Qip1 efficiently and into the On the other hand, the recombinant PTAC58 efficiently all the tested NLS substrates into the nucleus, in the first and second step of NPI-1, PTAC58, all the NLS substrates examined to nuclear pores in the first step of the transport the second step all the transport substrates were efficiently by the recombinant NPI-1 into the nucleus, some of the transport substrates and an of within the The was when the was in the of a that the in the in transport may be to some in the translocation These indicate that the NLS receptors can be into two major from the of their specificities NLS Rch1 and NPI-1 have a binding a variety of NLSs, while Qip1 binding to only NLS. These results also to the of Rch1 and NPI-1 may as the NLS receptor in cells. examined endogenous NLS receptors to the NLS substrates in the crude as efficiently as the purified recombinant proteins NLS were with crude from HeLa cells, and the were with and These have specificities each and with human Rch1 but NPI-1 and Qip1 in HeLa cells. in it was found that endogenous Rch1 by efficiently to all the tested substrates and These results show that Rch1 is capable of binding to of NLSs, in the of other endogenous which is with the the in transport On the other hand, it was found that endogenous NPI-1 only to and purified recombinant NPI-1 recombinant Rch1 efficiently into the to the as in the of the other tested NLS substrates in transport These results indicate that three of NLS receptors in in a variety of NLSs in cells. This that NLS receptors can be structurally and functionally into three distinct Moreover, the the NLS recognition as was NPI-1 in the or of other proteins it is to that the NLS recognition of the NLS receptors may be by other proteins The study the of the NLS of DNA helicase Q1. the primary of helicase Q1 suggest that the NLS may be a bipartite as in its amino acids containing only a single basic amino acid and its amino acids, which amino acid were found to as an NLS in cells. the upstream amino acids alone the which that the NLS of helicase Q1 can be classified into a single basic similar to the NLS. These in results were by using the in cell-free transport system. The was efficiently into the in the of the known transport or crude HeLa cell Qip1 an Armadillo repeating of the known NLS receptors, in its primary and shows about 50% amino acid to of the known human NLS receptors, Rch1 and NPI-1, it be that Qip1 with the NLS of helicase in the data show that Qip1 binds to and but only to in the binding we examined Qip1 transports substrates into the nucleus, in the of the known transport alone in the in transport in Qip1 efficiently but into the nucleus, which is with the binding in A. These results indicate that Qip1 as an NLS receptor helicase Q1 but that its NLS recognition requires that are upstream from the NLS. it was that Qip1 is a NLS receptor helicase we examined Qip1 functions as an NLS receptor other NLS we containing as a single basic type NLS and as that bipartite basic type NLS. in Qip1 only to and and of and into the in the in These results suggest the that Qip1 as a novel NLS which recognition helicase the that Qip1 is capable of other karyophilic proteins into the as efficiently as helicase Q1. the that the and specific NLS recognition by Qip1 requires the NLS to only from the we containing its upstream is as that of and a in which the basic amino acid of the was by the minimal essential sequence of in and it was found that efficiently to Qip1, and was by Qip1 in the in On the other hand, and were efficiently and into the by The number of conjugated to was the and as from it is most that the upstream sequence of helicase Q1 NLS recognition by Qip1 to the NLS from the but a in the recognition of NLS by which contain only the upstream amino acids, were by Qip1, the upstream is but NLS recognition by that Qip1 efficiently recognizes and that the upstream the basic of the NLS, a in the recognition of helicase Q1-NLS by Qip1, are Qip1 to as an NLS receptor the helicase Q1. The of which of the acid upstream sequence is the NLS recognition by Qip1 is this and earlier studies, it that are at least three of NLS receptors, NPI-1, and Qip1 in HeLa cells. the results their recombinant it is also that Rch1 and NPI-1 can and transport all the substrates into the as by to the NLS receptors can be into two major a class of NLS receptors, such as Rch1 and and a class of NLS receptors, such as The of NLS recognition by Qip1 was found to the upstream of the NLS, the NLS Qip1 recognizes this upstream the helicase Q1-NLS and it is capable of other NLSs or Furthermore, the of Qip1 the recognition of the upstream is from that the binding of the basic amino acid of the NLS to be Rch1 and NPI-1 were functionally when their recombinant proteins were used in the in which the that Rch1 and NPI-1 as NLS receptors in cells. this we examined endogenous Rch1 and NPI-1 in a manner similar to that of recombinant NLS in endogenous Rch1 efficiently to all the tested NLS substrates, while the binding of endogenous NPI-1 to but other tested NLSs, was in the HeLa cell crude are three to endogenous Rch1 may have a NPI-1 with endogenous it that receptors be by substrates, of the NLS substrates were to the crude endogenous NPI-1 may to in the crude cytosol, which have a NPI-1 the In this the binding of other substrates, such as and to endogenous NPI-1, also be the of NPI-1 is the other may exist that the binding of NPI-1 to a variety of NLSs. D. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) that the two NLS receptors, Rch1 and NPI-1, with specificities in a manner to NLSs and that the sequence is by other proteins in cell the NLSs used in this study are from of as a we results with results are with their Moreover, the substrates related to the helicase Q1 such as an within the only when recombinant NPI-1 was the in results using purified recombinant proteins when the substrates were into the cytoplasm of cells, this was to within the which may to other of NLS receptors with to such as the of the substrates from The study that are at least three distinct of NLS receptors, which have NLS The of such a of NLS receptors in is an and of show that the NLS of each receptor is in a cell is a that the NLS receptor transports into the when it in of cells. Furthermore, Qip1 is a that binds efficiently to number of NLSs, it is that other NLS receptors that and specific NLSs may be identified in the On the other hand, it been that the of various NLS receptors is in and cell M.L. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google D. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). S. and Y. in The suggest that nuclear protein transport is at the NLS recognition step through the the of NLS binding specificities of NLS receptors in various cells, in may to the of or cell it is to of NLS receptors are in the their and NLS specificities are controlled at the and which transport into the in each cell or