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• W2019944861 abstract "Karyopherin β2 (Kapβ2, transportin) binds the M9 sequence of human ribonucleoprotein A1 and mediates its nuclear import. Here we show a role for the nucleoporin Nup98 in the disassembly of Kapβ2 import complexes at the nuclear side of the nuclear pore complex (NPC). Kapβ2 bound to a region at the N terminus of Nup98 that contains an M9-like sequence. The human ribonucleoprotein A1 M9 sequence competed with Nup98 for binding to Kapβ2, indicating that Nup98 can dissociate Kapβ2 from its substrate. Binding of Kapβ2 to Nup98 was inhibited by Ran loaded with guanylyl imidophosphate, suggesting that RanGTP dissociates Kapβ2 from Nup98. RanGTP is produced from RanGDP through nucleotide exchange mediated by RanGEF (RCC1). Immunoelectron microscopy and nucleotide exchange assays revealed functional RanGEF on both sides of the NPC. On the nuclear side, the localization of RanGEF coincided with that of Nup98. RanGEF bound to Nup98 at a region adjacent to the Kapβ2-binding site. These findings suggest a model where 1) import substrate is released from Kapβ2 at the nucleoplasmic side of the NPC by competition with the Nup98 M9-like site, 2) Nup98-bound RanGEF catalyzes the formation of RanGTP, and 3) RanGTP dissociates Kapβ2 from Nup98 allowing repeated cycles of import. Karyopherin β2 (Kapβ2, transportin) binds the M9 sequence of human ribonucleoprotein A1 and mediates its nuclear import. Here we show a role for the nucleoporin Nup98 in the disassembly of Kapβ2 import complexes at the nuclear side of the nuclear pore complex (NPC). Kapβ2 bound to a region at the N terminus of Nup98 that contains an M9-like sequence. The human ribonucleoprotein A1 M9 sequence competed with Nup98 for binding to Kapβ2, indicating that Nup98 can dissociate Kapβ2 from its substrate. Binding of Kapβ2 to Nup98 was inhibited by Ran loaded with guanylyl imidophosphate, suggesting that RanGTP dissociates Kapβ2 from Nup98. RanGTP is produced from RanGDP through nucleotide exchange mediated by RanGEF (RCC1). Immunoelectron microscopy and nucleotide exchange assays revealed functional RanGEF on both sides of the NPC. On the nuclear side, the localization of RanGEF coincided with that of Nup98. RanGEF bound to Nup98 at a region adjacent to the Kapβ2-binding site. These findings suggest a model where 1) import substrate is released from Kapβ2 at the nucleoplasmic side of the NPC by competition with the Nup98 M9-like site, 2) Nup98-bound RanGEF catalyzes the formation of RanGTP, and 3) RanGTP dissociates Kapβ2 from Nup98 allowing repeated cycles of import. nuclear pore complex nuclear localization signal classical nuclear localization signal karyopherin Ran-binding protein GDP-bound Ran GTP-bound Ran guanylyl imidophosphate Ran guanine nucleotide exchange factor regulator of chromatin condensation 1 Ran GTPase activating protein 1 Phe-Gly repeats glutathioneS-transferase human ribonucleoprotein polymerase chain reaction maltose-binding protein dithiothreitol polyacrylamide gel electrophoresis bovine serum albumin Transport of proteins and nucleic acids between the nucleus and cytoplasm occurs through nuclear pore complexes (NPCs)1 (1Davis L.I. Annu. Rev. Biochem. 1995; 64: 865-896Crossref PubMed Scopus (287) Google Scholar). The vertebrate NPC is a supramolecular structure of 125 million daltons, which is approximately 30 times the size of a ribosome (2Reichelt R. Holzenburg A. Buhle Jr., E.L. Jarnik M. Engel A. Aebi U. J. Cell Biol. 1990; 110: 883-894Crossref PubMed Scopus (369) Google Scholar). Each NPC is composed of a central cylinder surrounded by a spoke-ring structure that anchors the cylinder to the nuclear envelope (3Akey C.W. Radermacher M. J. Cell Biol. 1993; 122: 1-19Crossref PubMed Scopus (337) Google Scholar, 4Hinshaw J.E. Carragher B.O. Milligan R.A. Cell. 1992; 69: 1133-1141Abstract Full Text PDF PubMed Scopus (350) Google Scholar). Filaments extending 50–100 nm emanate from this central core into the cytoplasm and nucleoplasm (5Goldberg M.W. Allen T.D. J. Mol. Biol. 1996; 257: 848-865Crossref PubMed Scopus (106) Google Scholar, 6Cordes V.C. Reidenbach S. Rackwitz H.R. Franke W.W. J. Cell Biol. 1997; 136: 515-529Crossref PubMed Scopus (190) Google Scholar). On the nucleoplasmic side, these filaments form a basket-like structure (7Ris H. EMSA Bull. 1991; 21: 54-56Google Scholar). In the mammalian NPC, all these structures together consist of approximately 50 different proteins termed nucleoporins (8Doye V. Hurt E. Curr. Opin. Cell Biol. 1997; 9: 401-411Crossref PubMed Scopus (213) Google Scholar). A subset of nucleoporins that contain Phe-Gly repeats (FG nucleoporins) constitute docking sites for transport factors at the NPC (9Buss F. Kent H. Stewart M. Bailer S. Hanover J. J. Cell Sci. 1994; 107: 631-638Crossref PubMed Google Scholar, 10Radu A. Moore M.S. Blobel G. Cell. 1995; 81: 215-222Abstract Full Text PDF PubMed Scopus (389) Google Scholar, 11Katahira J. Strasser K. Podtelejnikov A. Mann M. Jung J.U. Hurt E. EMBO J. 1999; 18: 2593-2609Crossref PubMed Scopus (341) Google Scholar, 12Kehlenbach R.H. Dickmanns A. Kehlenbach A. Guan T. Gerace L. J. Cell Biol. 1999; 145: 645-657Crossref PubMed Scopus (175) Google Scholar). Some of these nucleoporins are localized asymmetrically at the NPC. Nup358 and Nup214, for example, are associated with the cytoplasmic filaments, whereas Nup98 and Nup153 are localized at or near the basket on the nucleoplasmic side (10Radu A. Moore M.S. Blobel G. Cell. 1995; 81: 215-222Abstract Full Text PDF PubMed Scopus (389) Google Scholar, 13Kraemer D. Wozniak R.W. Blobel G. Radu A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1519-1523Crossref PubMed Scopus (198) Google Scholar, 14Yokoyama N. Hayashi N. Seki T. Pante N. Ohba T. Nishii K. Kuma K. Hayashida T. Miyata T. Aebi U. Fukui M. Nishimoto T. Nature. 1995; 376: 184-188Crossref PubMed Scopus (411) Google Scholar, 15Wu J. Matunis M.J. Kraemer D. Blobel G. Coutavas E. J. Biol. Chem. 1995; 270: 14209-14213Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar, 16Sukegawa J. Blobel G. Cell. 1993; 72: 29-38Abstract Full Text PDF PubMed Scopus (242) Google Scholar). On the other hand, p62 appears to be symmetrically localized on both sides close to the midplane of the NPC (17Guan T. Muller S. Klier G. Pante N. Blevitt J.M. Haner M. Paschal B. Aebi U. Gerace L. Mol. Biol. Cell. 1995; 6: 1591-1603Crossref PubMed Scopus (134) Google Scholar, 18Hu T. Guan T. Gerace L. J. Cell Biol. 1996; 134: 589-601Crossref PubMed Scopus (151) Google Scholar). The asymmetric distribution of nucleoporins and their different affinities for import and export complexes may be important in determining the directionality of transport. Nup98 contains a number of FG repeats in its N-terminal portion that act as docking sites during import (10Radu A. Moore M.S. Blobel G. Cell. 1995; 81: 215-222Abstract Full Text PDF PubMed Scopus (389) Google Scholar). In addition, Nup98 appears to be involved in multiple RNA export pathways (19Pritchard C.E. Fornerod M. Kasper L.H. van Deursen J.M. J. Cell Biol. 1999; 145: 237-254Crossref PubMed Scopus (194) Google Scholar, 20Powers M.A. Forbes D.J. Dahlberg J.E. Lund E. J. Cell Biol. 1997; 136: 241-250Crossref PubMed Scopus (180) Google Scholar). Interestingly, Nup98 has been described as a frequent target for chromosomal rearrangements in acute leukemia, and its N-terminal FG repeat region is present in all leukemia-associated Nup98 fusions that have been characterized to date (21–32). These findings suggest a link between the function of Nup98 in nuclear transport and its role in leukemogenesis. Nuclear import and export of molecules involve interactions of soluble transport factors with their cargoes and with the NPC. Transport receptors bind import or export signals present in their cargoes and are collectively known as karyopherins (also called importins or exportins). Nuclear import of proteins bearing a classical nuclear localization signal (cNLS) was the first to be characterized at the molecular level. Import of a cNLS-bearing protein is mediated by a heterodimer of karyopherin α (Kapα) and karyopherin β1 (Kapβ1). Kapα interacts directly with the cNLS, whereas Kapβ1 binds to nucleoporins resulting in the docking of the complex to the NPC. Other soluble factors are involved in the translocation process through the NPC, including the small GTPase Ran, p10 (also known as NTF2), and the Ran-binding protein RanBP1 (33Gorlich D. Curr. Opin. Cell Biol. 1997; 9: 412-419Crossref PubMed Scopus (257) Google Scholar, 34Pemberton L.F. Blobel G. Rosenblum J.S. Curr. Opin. Cell Biol. 1998; 10: 392-399Crossref PubMed Scopus (211) Google Scholar, 35Nakielny S. Dreyfuss G. Cell. 1999; 99: 677-690Abstract Full Text Full Text PDF PubMed Scopus (648) Google Scholar). Other members of the Kapβ family serve as import receptors for other classes of proteins that have non-classical NLSs. In many of these cases the Kapβ binds directly to the NLS of its import cargo rather than through a Kapα-like adapter. For example, hnRNP A1 has an NLS rich in aromatic residues and glycine, called M9, that is directly bound by Kapβ2 (transportin1) resulting in docking of the complex at the NPC and its import into the nucleus (36Bonifaci N. Moroianu J. Radu A. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5055-5060Crossref PubMed Scopus (148) Google Scholar, 37Pollard V.W. Michael W.M. Nakielny S. Siomi M.C. Wang F. Dreyfuss G. Cell. 1996; 86: 985-994Abstract Full Text Full Text PDF PubMed Scopus (577) Google Scholar). Additional import and export pathways have been identified and are reviewed elsewhere (33Gorlich D. Curr. Opin. Cell Biol. 1997; 9: 412-419Crossref PubMed Scopus (257) Google Scholar, 34Pemberton L.F. Blobel G. Rosenblum J.S. Curr. Opin. Cell Biol. 1998; 10: 392-399Crossref PubMed Scopus (211) Google Scholar, 35Nakielny S. Dreyfuss G. Cell. 1999; 99: 677-690Abstract Full Text Full Text PDF PubMed Scopus (648) Google Scholar). Ran is a small GTPase that cycles between a GDP-bound form (RanGDP) and a GTP-bound form (RanGTP) and plays an important role in both import and export (38Cole C.N. Hammell C.M. Curr. Biol. 1998; 8: R368-R372Abstract Full Text Full Text PDF PubMed Google Scholar, 39Moore M.S. J. Biol. Chem. 1998; 273: 22857-22860Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 40Rush M.G. Drivas G. D'Eustachio P. BioEssays. 1996; 18: 103-112Crossref PubMed Scopus (91) Google Scholar). Interconversion of RanGDP and RanGTP is regulated by the Ran GTPase-activating protein RanGAP1 and the Ran guanine nucleotide exchange factor RanGEF (also called RCC1). RanGAP1 is localized in the cytosol and at the cytoplasmic face of the NPC and catalyzes nucleotide hydrolysis by RanGTP to form RanGDP (41Matunis M.J. Coutavas E. Blobel G. J. Cell Biol. 1996; 135: 1457-1470Crossref PubMed Scopus (952) Google Scholar). RanGEF is localized predominantly in the nucleus and catalyzes nucleotide exchange favoring the generation of RanGTP, since the intracellular concentration of GTP is higher than that of GDP (39Moore M.S. J. Biol. Chem. 1998; 273: 22857-22860Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 40Rush M.G. Drivas G. D'Eustachio P. BioEssays. 1996; 18: 103-112Crossref PubMed Scopus (91) Google Scholar, 42Seki T. Hayashi N. Nishimoto T. J. Biochem. (Tokyo ). 1996; 120: 207-214Crossref PubMed Scopus (59) Google Scholar, 43Koepp D.M. Silver P.A. Cell. 1996; 87: 1-4Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 44Avis J.M. Clarke P.R. J. Cell Sci. 1996; 109: 2423-2427Crossref PubMed Google Scholar). These findings suggest that the nucleus has a higher concentration of RanGTP than the cytoplasm. Ran appears to be imported primarily in the GDP-bound form, but functional RanGEF is required in order for Ran to accumulate in the nucleus, presumably as RanGTP (45Ribbeck K. Lipowsky G. Kent H.M. Stewart M. Gorlich D. EMBO J. 1998; 17: 6587-6598Crossref PubMed Scopus (356) Google Scholar). The mechanisms by which import complexes travel through the NPC and are disassembled in the nucleus are not well understood. The process is thought to involve several steps. Kapβs interact strongly with RanGTP but not with RanGDP. Binding of RanGTP to import Kapβs results in the release of their cargo. Since a higher concentration of RanGTP is predicted in the nucleus, it is thought that this reaction results in the release of cargo from import Kapβs in the nucleus (46Rexach M. Blobel G. Cell. 1995; 83: 683-692Abstract Full Text PDF PubMed Scopus (663) Google Scholar, 47Siomi M.C. Eder P.S. Kataoka N. Wan L. Liu Q. Dreyfuss G. J. Cell Biol. 1997; 138: 1181-1192Crossref PubMed Scopus (202) Google Scholar, 48Izaurralde E. Kutay U. von Kobbe C. Mattaj I.W. Gorlich D. EMBO J. 1997; 16: 6535-6547Crossref PubMed Scopus (493) Google Scholar). However, there is evidence that Kapβ2 can deliver its cargo to the nucleus in the absence of Ran when Kapβ2 and the cargo are provided in equimolar concentrations (49Ribbeck K. Kutay U. Paraskeva E. Gorlich D. Curr. Biol. 1999; 9: 47-50Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Thus the release of cargo from the nuclear side of the nuclear pore complex may also occur by Ran-independent mechanisms. In this study, we have focused on the role of the nucleoporin Nup98 in the termination of Kapβ2-mediated nuclear import at the nuclear side of the NPC. We show that Kapβ2 binds to the N terminus of Nup98 at a region that contains an hnRNP A1 M9-like sequence. This binding is prevented by an M9-containing protein, indicating that Nup98 binds to the cargo-binding site of Kapβ2. Thus, Nup98 may disassemble the Kapβ2-M9 import complex at the nuclear side of the NPC providing a mechanism for the Ran-independent import by Kapβ2 mentioned above. The resulting Kapβ2-Nup98 complex would need to be disassembled before Kapβ2 can be recycled for further rounds of import. This function is probably performed by RanGTP since our data show that RanGMPPNP inhibits binding of Kapβ2 to Nup98. However, since Ran traverses the NPC on its way to the nucleus primarily in the GDP-bound form (45Ribbeck K. Lipowsky G. Kent H.M. Stewart M. Gorlich D. EMBO J. 1998; 17: 6587-6598Crossref PubMed Scopus (356) Google Scholar, 50Smith A. Brownawell A. Macara I.G. Curr. Biol. 1998; 8: 1403-1406Abstract Full Text Full Text PDF PubMed Google Scholar), a mechanism is needed for the local production of RanGTP in order to dissociate Kapβ2 from Nup98. RanGEF is the only known nucleotide exchange factor for Ran and is thought to be confined to the inside of the nucleus in association with chromatin (51Ohtsubo M. Okazaki H. Nishimoto T. J. Cell Biol. 1989; 109: 1389-1397Crossref PubMed Scopus (286) Google Scholar). Here we show that RanGEF, in addition to its intranuclear localization, is also associated with the NPC. By using immunoelectron microscopy, we show that RanGEF is present on both the cytoplasmic and nucleoplasmic sides of the NPC. On the nucleoplasmic side, the localization of RanGEF coincides with the previously determined localization of Nup98 (10Radu A. Moore M.S. Blobel G. Cell. 1995; 81: 215-222Abstract Full Text PDF PubMed Scopus (389) Google Scholar). By using in vitro nucleotide exchange assays we found that nuclear envelope-associated RanGEF is functional. Consistent with the ultrastructural data, RanGEF indeed bound to Nup98, at the FG repeat region immediately downstream of the Kapβ2-binding site. These data are integrated into a model for the termination of Kapβ2-mediated nuclear import at the nuclear side of the NPC. Wild-type Nup98 precursor was cloned into pAlter-MAX as described previously (52Fontoura B.M. Blobel G. Matunis M.J. J. Cell Biol. 1999; 144: 1097-1112Crossref PubMed Scopus (195) Google Scholar). All truncated mutants were generated by PCR using the wild-type Nup98 precursor as template. An oligonucleotide complementary to the 5′ end of the indicated region for each mutant (see Fig. 3) and containing aSalI site was used in conjunction with an antisense oligonucleotide complementary to sequences at the 3′ end of the indicated regions in Fig. 3 and containing a NotI site. The resulting PCR products were digested with SalI andNotI and ligated into the SalI/NotI sites of myc-pAlter-MAX. The human NUP98 open reading frame was amplified by PCR from a bone marrow cDNA library (CLONTECH, Palo Alto, CA) and subcloned into theBamHI and SalI sites of pGEX4T3 (Amersham Pharmacia Biotech) to produce a GST-Nup98 fusion. The GST-Nup98 fusion protein was purified from bacterial lysate by binding to glutathione-Sepharose beads (Amersham Pharmacia Biotech). The human KAPβ2 gene subcloned into pGEX4T3 containing a Tev protease cleavage site, and the maltose-binding protein fused with M3 (MBP-M3) was a kind gift from Yuh-Min Chook (53Chook Y.M. Blobel G. Nature. 1999; 399: 230-237Crossref PubMed Scopus (289) Google Scholar). Expression and purification of GST-Kapβ2 was performed as originally described (53Chook Y.M. Blobel G. Nature. 1999; 399: 230-237Crossref PubMed Scopus (289) Google Scholar). The maltose-binding protein (MBP) was obtained from New England Biolabs (Beverly, MA). The humanRANGEF open reading frame was amplified by PCR from a bone marrow cDNA library (CLONTECH, Palo Alto, CA) and subcloned into the BamHI and SalI sites of pGEX4T3 to produce a GST-RanGEF fusion. The GST-RanGEF fusion protein was purified from bacterial lysate by binding to glutathione-Sepharose beads and either kept immobilized on the beads or eluted by thrombin cleavage. Human recombinant Ran protein loaded with either GDP or GMPPNP was a kind gift from Yuh-Min Chook (53Chook Y.M. Blobel G. Nature. 1999; 399: 230-237Crossref PubMed Scopus (289) Google Scholar). All wild-type and Nup98 mutant proteins were in vitro transcribed and translated using a coupled reticulocyte lysate transcription/translation system (Promega Corp., Madison, WI), in the presence of [35S]methionine, according to manufacturer's instructions. Binding reactions were carried out as described (54Yaseen N.R. Blobel G. J. Biol. Chem. 1999; 274: 26493-26502Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) using 10 μl (unless otherwise indicated) of in vitro transcribed and translated proteins as indicated in the figure legends. Bound and unbound fractions were separated on SDS-PAGE, and the gels were analyzed by autoradiography. Binding reactions with bacterially expressed recombinant proteins were carried out as described previously (54Yaseen N.R. Blobel G. J. Biol. Chem. 1999; 274: 26493-26502Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Rat liver nuclei were isolated as described (55Blobel G. Potter V.R. Science. 1966; 154: 1662-1665Crossref PubMed Scopus (983) Google Scholar) and stored frozen at −80 °C in 100-unit aliquots (1 unit = 3 × 106 nuclei). Nuclear envelopes were prepared by a modification of the procedure described by Dwyer and Blobel (56Dwyer N. Blobel G. J. Cell Biol. 1976; 70: 581-591Crossref PubMed Scopus (270) Google Scholar). Nuclei were thawed and pelleted at 500 rpm in a tabletop microcentrifuge for 1 min. After removing the supernatant, the pellet was resuspended to a final concentration of 100 units/ml by dropwise addition of cold buffer A, 0.1 mm MgCl2, protease inhibitors (0.5 mm phenylmethanesulfonyl fluoride, 1 μg/ml leupeptin, 1 μg/ml pepstatin A, and 18 μg/ml aprotinin), 5 μg/ml DNase I (Sigma), and 5 μg/ml RNase A (Sigma) with constant vortexing. The nuclei were then immediately diluted to 20 units/ml by addition of ice-cold buffer B, 10% sucrose, 20 mmtriethanolamine (pH 8.5), 0.1 mm MgCl2, 1 mm dithiothreitol (DTT), and protease inhibitors, again with constant vortexing. Following a 15-min incubation on ice, the suspension was underlaid with 5 ml of ice-cold buffer C, 30% sucrose, 20 mm triethanolamine (pH 7.5), 0.1 mmMgCl2, 1 mm DTT, and protease inhibitors, and centrifuged at 4,100 × g in a swinging bucket rotor (Sorvall type HB-4) for 15 min at 4 °C. After removing the supernatant and sucrose cushion, the pellet was resuspended to a final concentration of 100 units/ml in ice cold buffer D, 10% sucrose, 20 mm triethanolamine (pH 7.5), 0.1 mmMgCl2, 1 mm DTT, and protease inhibitors. The suspension was immediately underlaid with 5 ml of buffer C and pelleted as above. The pellet resulting from this second extraction is operationally defined as the nuclear envelope fraction. Isolated nuclear envelopes were fixed for 15 min in 2.5% formaldehyde in STM (10% sucrose, 20 mm triethanolamine HCl (pH 7.5), 0.1 mmMgCl2) and centrifuged at 2,000 × g for 5 min onto 35-mm tissue culture dishes. The pelleted nuclear envelopes were washed three times with 1% BSA, 68 mm NaCl, 13 mm KCl, 15 mm KH2PO4, 40 mm Na2HPO4, 0.5 mmphenylmethanesulfonyl fluoride and incubated with two goat polyclonal anti-RanGEF antibodies, RCC1(N-19) and RCC1(C-20), diluted 1:10 (Santa Cruz Biotechnology). Bound antibodies were detected with rabbit anti-goat IgG conjugated with 5-nm gold (Ted Pella, Redding, CA) diluted 1:50. Recombinant human Ran was prepared and loaded with [α-32P]GTP as described (57Floer M. Blobel G. J. Biol. Chem. 1996; 271: 5313-5316Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Exchange reactions were carried out at room temperature for 30 min in 300 μl of STM in the presence of 1 mm GDP. The reactions were stopped by adding 300 μl of cold transport buffer with 1 mm DTT and 1 mg/ml BSA on ice and applied onto BA85 filters (Schleicher & Schuell) by vacuum aspiration. The filters were washed with 5 ml of cold transport buffer, 1 mm DTT, 1 mg/ml BSA and allowed to dry before scintillation counting. We have previously shown that Nup98 is synthesized as a precursor derived from at least two alternatively spliced mRNAs (52Fontoura B.M. Blobel G. Matunis M.J. J. Cell Biol. 1999; 144: 1097-1112Crossref PubMed Scopus (195) Google Scholar). The Nup98 precursor is cleaved at the C terminus, generating Nup98 (residues 1–863) and a 6-kDa C-terminal fragment (residues 864–920), and the Nup98-Nup96 precursor is cleaved at the same site generating Nup98 (residues 1–863) and Nup96-(residues 864–1712) (52Fontoura B.M. Blobel G. Matunis M.J. J. Cell Biol. 1999; 144: 1097-1112Crossref PubMed Scopus (195) Google Scholar). Nup98 can be divided into two domains (Fig.1 A). The N-terminal half contains the FG-containing repeat domain (residues 1–497), and the second half of the molecule contains the highly conserved cleavage site domain.Figure 1Nup98 and Nup153 contain sequences similar to hnRNPA1 M9. A, schematic representation of the Nup98 precursor protein and hnRNP A1. The hatched region represents the FG-containing repeat domain in Nup98. Thearrow indicates the Nup98 cleavage site. The locations of the hnRNP A1 M9 and M3 sequences and the Nup98 M9-like sequence are indicated by thick lines. B, amino acid sequence alignments of the M9-like sequences of Nup98 and Nup153 with the hnRNPA1 M9. Amino acids are numbered from the initiating methionine in each protein. Boxes and shadings indicate sequence identity and homology, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT) An alignment search showed that the N-terminal domain of Nup98 contains an hnRNP A1 M9-like sequence between residues 25–60 (Fig. 1). Using the Clustal method, the M9 sequence of hnRNP A1 and the M9-like sequence of Nup98 are approximately 30% identical and 56% similar (Fig. 1 B). No other sequence similarity was observed between Nup98 and hnRNP A1. An M9-like sequence has previously been reported on Nup153 (58Nakielny S. Shaikh S. Burke B. Dreyfuss G. EMBO J. 1999; 18: 1982-1995Crossref PubMed Scopus (181) Google Scholar). This sequence has approximately 20% identity and 47% similarity to that of hnRNP A1 M9 (Fig. 1 B) and is a site for Kapβ2 binding (58Nakielny S. Shaikh S. Burke B. Dreyfuss G. EMBO J. 1999; 18: 1982-1995Crossref PubMed Scopus (181) Google Scholar). Previous evidence from overlay assays suggests that Nup98 also binds to Kapβ2 (36Bonifaci N. Moroianu J. Radu A. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5055-5060Crossref PubMed Scopus (148) Google Scholar). To determine whether the M9-like sequence in Nup98 is indeed a Kapβ2-binding site,in vitro binding assays were performed using bacterially expressed recombinant Kapβ2. Full-length Nup98 could be expressed in soluble form either with a GST tag in bacteria (rGST-Nup98) or in a rabbit reticulocyte lysate in vitrotranscription/translation system (ivNup98). With purified rGST-Nup98 a direct interaction between Nup98 and Kapβ2 could be demonstrated (see below). However, as some of the truncated Nup98 mutants were insoluble when expressed in bacteria (data not shown), a rabbit reticulocytein vitro transcription and translation system was used to map the Kapβ2-binding site on Nup98. GST-tagged recombinant Kapβ2 was immobilized on glutathione-Sepharose beads, incubated with ivNup98, and the bound and unbound fractions were visualized by SDS-PAGE and autoradiography (Fig.2 A). In vitrotranscribed/translated Nup96 and luciferase were used as controls. Nup98 bound to GST-Kapβ2, whereas Nup96 and luciferase showed no significant binding (Fig. 2 A). The observed binding of Nup98 to Kapβ2 could be either direct or it could be indirect,i.e. mediated by one or more of the numerous proteins present in the reticulocyte lysate. To determine whether the binding is direct, rGST-Nup98 was immobilized on glutathione-Sepharose beads and incubated with recombinant Kapβ2. Kapβ2 bound to rGST-Nup98 (Fig.2 B) showing that the two proteins interact directly. As a control, Kapβ2 was incubated with GST immobilized on glutathione-Sepharose beads to show that Kapβ2 does not interact with GST moiety (Fig. 2 B). We conclude that the binding of Kapβ2 to Nup98 is both specific and direct. To map the Kapβ2-binding site on Nup98, a series of Nup98 truncation mutants were constructed and expressed in the reticulocyte lysate system. These truncation mutants were then incubated with immobilized GST-Kapβ2 (Fig. 3). Amino acids 1–316 were shown to constitute the necessary region for binding of Nup98 to Kapβ2. As mentioned above (see Fig. 1), this region contains the M9-like sequence (amino acids 25–60) as well as FG repeats. Thus, Kapβ2 binds to the FG-containing repeat region of Nup98, a region that includes and extends beyond the M9-like sequence. The presence of an M9-like sequence in Nup98 led us to investigate whether Nup98 would compete with M9 for binding to Kapβ2. The M9 sequence of hnRNP A1 is contained within a larger sequence, called M3 (Fig.1 A), that appears to bind to Kapβ2 more efficiently (37Pollard V.W. Michael W.M. Nakielny S. Siomi M.C. Wang F. Dreyfuss G. Cell. 1996; 86: 985-994Abstract Full Text Full Text PDF PubMed Scopus (577) Google Scholar). The M3 fragment was expressed in bacteria as a fusion protein with MBP and used in the competition studies. In vitro binding assays were performed with immobilized GST-Kapβ2 and in vitroexpressed Nup98 in the absence or presence of either MBP or MBP fused to M3 (Fig. 4). Most of the binding of Nup98 to Kapβ2 was abolished in the presence of MBP-M3 but not in the presence of MBP alone (Fig. 4). These results indicate that Nup98 is able to compete for the substrate-binding site on Kapβ2. Thus, when Kapβ2 with its bound cargo reaches the nuclear side of the NPC, Nup98 might release the cargo. In order to complete the import cycle, Kapβ2 would then need to be released from Nup98. Since Ran is known to regulate the assembly and disassembly of nuclear transport complexes, we sought to determine the effect of Ran on the binding of Kapβ2 to Nup98. In vitro binding assays were performed with immobilized GST-Kapβ2 and in vitro expressed Nup98 in the absence or presence of Ran loaded with either GDP or GMPPNP (a non-hydrolyzable GTP analogue). As shown in Fig. 5, RanGMPPNP inhibited the binding of Kapβ2 to Nup98, whereas RanGDP had no such effect. These results indicate that RanGTP could dissociate Kapβ2 from Nup98 at the nucleoplasmic side of the NPC. Thus, during import, the Kapβ2-substrate complex could be dissociated by binding of Kapβ2 to the M9-like sequence of Nup98, leading to the release of substrate. The next step would be to dissociate Kapβ2 from Nup98, which can be accomplished by RanGTP. However, Ran is imported through the NPC primarily in its GDP-bound form, and conversion of RanGDP to RanGTP can only be accomplished by RanGEF (45Ribbeck K. Lipowsky G. Kent H.M. Stewart M. Gorlich D. EMBO J. 1998; 17: 6587-6598Crossref PubMed Scopus (356) Google Scholar, 50Smith A. Brownawell A. Macara I.G. Curr. Biol. 1998; 8: 1403-1406Abstract Full Text Full Text PDF PubMed Google Scholar). RanGEF is known to be present in the nuclear interior in association with chromatin (51Ohtsubo M. Okazaki H. Nishimoto T. J. Cell Biol. 1989; 109: 1389-1397Crossref PubMed Scopus (286) Google Scholar) but has not been shown in association with the NPC. It was therefore of interest to determine whether RanGEF is also found at the NPC where it would catalyze the conversion of RanGDP to RanGTP in order to release Kapβ2 from Nup98. Isolated rat liver nuclear envelopes were probed with antibodies to RanGEF followed by secondary gold-conjugated antibody. RanGEF was found associated with both sides of the NPC (Fig. 6 A). On the cytoplasmic side, the distribution of gold particles gave a mean distance of 42.9 nm from the midplane of the nuclear envelope (n = 171). On the nucleoplasmic side, the mean distance was 39.6 nm (n = 164) (Fig. 6 B). Interestingly, the localization of RanGEF at the nucleoplasmic side of the NPC coincides with the previously determined localization of Nup98" @default.
• W2019944861 created "2016-06-24" @default.
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• W2019944861 date "2000-10-01" @default.
• W2019944861 modified "2023-10-17" @default.
• W2019944861 title "The Nucleoporin Nup98 Is a Site for GDP/GTP Exchange on Ran and Termination of Karyopherin β2-mediated Nuclear Import" @default.
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