Molecular Basis for the Rapid Dissociation of Nuclear Localization Signals from Karyopherin α in the Nucleoplasm
2003; Elsevier BV; Volume: 278; Issue: 51 Linguagem: Inglês
10.1074/jbc.m307371200
ISSN1083-351X
AutoresDaniel A. Gilchrist, Michael Rexach,
Tópico(s)Genomics and Chromatin Dynamics
ResumoThe yeast karyopherin heterodimer Kap60p·Kap95p facilitates nuclear import of proteins bearing a classic nuclear localization signal (NLS). The α subunit Kap60p binds to the NLS of cargo molecules in the cytoplasm, forming stable complexes that must ultimately dissociate in the nucleoplasm. Although Kap60p can release NLSs on its own using an autoinhibitory sequence (AIS) motif that can occupy the NLS binding site, that mechanism is too slow to support rapid nuclear import. We previously showed that the nuclear basket nucleoporin Nup2p and the exportin complex Cse1p·Gsp1p·GTP function as karyopherin release factors (KaRFs) because they can accelerate the rate of dissociation of NLSs from Kap60p. Here we dissect the molecular mechanics of their KaRF activity. We show that Cse1p accelerates dissociation of Kap60p·NLS-cargo complexes and Kap60p·Nup2p complexes by increasing the affinity of Kap60p for its AIS motif. In contrast, Nup2p uses a conserved sequence motif (VMXXRKIA) coupled to an AIS-like motif to accelerate dissociation of Kap60p·NLS complexes in a vectorial reaction mechanism. Mutation of either motif in Nup2p leads to a loss of KaRF activity and to the accumulation of Kap60p·NLS-cargo complexes in the nucleoplasm of yeast. We discuss a model whereby Nup2p, Cse1p, and Gsp1p cooperate to establish directionality in the movement of Kap60p and NLS-cargos across the nuclear pore complex. The yeast karyopherin heterodimer Kap60p·Kap95p facilitates nuclear import of proteins bearing a classic nuclear localization signal (NLS). The α subunit Kap60p binds to the NLS of cargo molecules in the cytoplasm, forming stable complexes that must ultimately dissociate in the nucleoplasm. Although Kap60p can release NLSs on its own using an autoinhibitory sequence (AIS) motif that can occupy the NLS binding site, that mechanism is too slow to support rapid nuclear import. We previously showed that the nuclear basket nucleoporin Nup2p and the exportin complex Cse1p·Gsp1p·GTP function as karyopherin release factors (KaRFs) because they can accelerate the rate of dissociation of NLSs from Kap60p. Here we dissect the molecular mechanics of their KaRF activity. We show that Cse1p accelerates dissociation of Kap60p·NLS-cargo complexes and Kap60p·Nup2p complexes by increasing the affinity of Kap60p for its AIS motif. In contrast, Nup2p uses a conserved sequence motif (VMXXRKIA) coupled to an AIS-like motif to accelerate dissociation of Kap60p·NLS complexes in a vectorial reaction mechanism. Mutation of either motif in Nup2p leads to a loss of KaRF activity and to the accumulation of Kap60p·NLS-cargo complexes in the nucleoplasm of yeast. We discuss a model whereby Nup2p, Cse1p, and Gsp1p cooperate to establish directionality in the movement of Kap60p and NLS-cargos across the nuclear pore complex. Transport between the cytoplasm and nucleoplasm of eukaryotic cells occurs at the nuclear membrane and proceeds through nuclear pore complexes (NPCs) 1The abbreviations used are: NPCnuclear pore complexNLSnuclear localization signalcNLSclassical NLSNupnucleoporinAISautoinhibitory sequenceIBBimportin β bindingKaRFkaryopherin release factorGSTglutathione S-transferaseDTTdithiothreitolONPGo-nitrophenyl p-d-galactopyranoside. (1Mattaj I.W. Englmeier L. Annu. Rev. Biochem. 1998; 67: 265-306Crossref PubMed Scopus (1011) Google Scholar, 2Gorlich D. Kutay U. Annu. Rev. Cell Dev. Biol. 1999; 15: 607-660Crossref PubMed Scopus (1676) Google Scholar, 3Barry D.M. Wente S.R. Essays Biochem. 2000; 36: 89-103Crossref PubMed Scopus (11) Google Scholar, 4Quimby B.B. Corbett A.H. Cell Mol. Life Sci. 2001; 58: 1766-1773Crossref PubMed Scopus (47) Google Scholar, 5Chook Y.M. Blobel G. Curr. Opin. Struct. Biol. 2001; 11: 703-715Crossref PubMed Scopus (427) Google Scholar). Proteins that need to be imported into nuclei (cargos) contain nuclear localization signals (NLSs) that are recognized by mobile receptors termed karyopherins (importins/transportin). In Saccharomyces cerevisiae, the karyopherin αβ heterodimer Kap60p·Kap95p is responsible for import of proteins bearing a cNLS (6Enenkel C. Blobel G. Rexach M. J. Biol. Chem. 1995; 270: 16499-16502Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). The Kap60p subunit (karyopherin α; Srp1p) binds simultaneously to the NLS-cargo and to the Kap95p subunit (karyopherin β), whereas Kap95p facilitates transport of Kap60p·NLS-cargo complexes across the NPC by interacting with nuclear pore complex proteins (nucleoporins; Nups) (7Rexach M. Blobel G. Cell. 1995; 83: 683-692Abstract Full Text PDF PubMed Scopus (665) Google Scholar). nuclear pore complex nuclear localization signal classical NLS nucleoporin autoinhibitory sequence importin β binding karyopherin release factor glutathione S-transferase dithiothreitol o-nitrophenyl p-d-galactopyranoside. A subset of Nups and the Gsp1p GTPase contribute to the efficiency and directionality of Kap95p and Kap60p translocation across the NPC. Nup1p and Nup2p reside in the nuclear basket of the yeast NPC and promote efficient import of Kap95p·Kap60p·NLS-cargo complexes (8Rout M.P. Aitchison J.D. Suprapto A. Hjertaas K. Zhao Y. Chait B.T. J. Cell Biol. 2000; 148: 635-651Crossref PubMed Scopus (1156) Google Scholar, 9Dilworth D.J. Suprapto A. Padovan J.C. Chait B.T. Wozniak R.W. Rout M.P. Aitchison J.D. J. Cell Biol. 2001; 153: 1465-1478Crossref PubMed Scopus (131) Google Scholar, 10Solsbacher J. Maurer P. Vogel F. Schlenstedt G. Mol. Cell. Biol. 2000; 20: 8468-8479Crossref PubMed Scopus (79) Google Scholar, 11Hood J.K. Casolari J.M. Silver P.A. J. Cell Sci. 2000; 113: 1471-1480PubMed Google Scholar, 12Bogerd A.M. Hoffman J.A. Amberg D.C. Fink G.R. Davis L.I. J. Cell Biol. 1994; 127: 319-332Crossref PubMed Scopus (79) Google Scholar). Gsp1p (Ran in vertebrates) (13Weis K. Cell. 2003; 112: 441-451Abstract Full Text Full Text PDF PubMed Scopus (580) Google Scholar, 14Dasso M. Curr. Biol. 2002; 12: 502-508Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar) imparts directionality to their translocation by executing a terminal step in Kap95p·Kap60p-mediated nuclear import and by serving as a cofactor in the initial step of Kap60p nuclear export. In nuclear import, Gsp1p·GTP binds to Kap95p (7Rexach M. Blobel G. Cell. 1995; 83: 683-692Abstract Full Text PDF PubMed Scopus (665) Google Scholar) and accelerates release of the Kap60p·NLS-cargo complex from Kap95p (15Gilchrist D. Mykytka B. Rexach M. J. Biol. Chem. 2002; 277: 18161-18172Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). In nuclear export, Gsp1p·GTP binds to the exportin Cse1p and enhances its affinity for Kap60p (its cargo) as the first step in Kap60p export out of the nucleus (16Solsbacher J. Maurer P. Bischoff F.R. Schlenstedt G. Mol. Cell. Biol. 1998; 18: 6805-6815Crossref PubMed Google Scholar, 17Hood J.K. Silver P.A. J. Biol. Chem. 1998; 273: 35142-35146Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Karyopherins bind cargos with high affinity to ensure the integrity of Kap·cargo complexes during transit through the NPC (15Gilchrist D. Mykytka B. Rexach M. J. Biol. Chem. 2002; 277: 18161-18172Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 18Catimel B. Teh T. Fontes M. Jennings I. Jans D.A. Howlett G. Nice E. Kobe B. J. Biol. Chem. 2001; 276: 34189-34198Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 19Hodel M.R. Corbett A.H. Hodel A.E. J. Biol. Chem. 2001; 276: 1317-1325Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, 20Jans D.A. Xiao C.Y. Lam M.H. BioEssays. 2000; 22: 532-544Crossref PubMed Scopus (478) Google Scholar). Consequently, these interactions may have half-lives of several minutes, as in the case of Kap60p·NLS-cargo complexes (15Gilchrist D. Mykytka B. Rexach M. J. Biol. Chem. 2002; 277: 18161-18172Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). However, karyopherins need to release cargos rapidly upon completion of translocation to ensure efficient nuclear import. In the case of most importins, binding of Gsp1p·GTP disrupts their association with cargos as the terminal step of transport (13Weis K. Cell. 2003; 112: 441-451Abstract Full Text Full Text PDF PubMed Scopus (580) Google Scholar, 14Dasso M. Curr. Biol. 2002; 12: 502-508Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 21Gorlich D. Pante N. Kutay U. Aebi U. Bischoff F.R. EMBO J. 1996; 15: 5584-5594Crossref PubMed Scopus (535) Google Scholar). However, in the case of Kap95p·Kap60p·NLS complexes, Gsp1p·GTP only accelerates the rate of dissociation of Kap60p·NLS from Kap95p, leaving the Kap60p·NLS complex intact (15Gilchrist D. Mykytka B. Rexach M. J. Biol. Chem. 2002; 277: 18161-18172Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). An essential autoinhibitory sequence (AIS) motif in the importin β binding (IBB) domain of Kap60p (aa 1-61) can occupy the NLS binding pocket of Kap60p and has therefore been proposed to play a key role in the dissociation of Kap60p·NLS-cargo complexes (22Harreman M.T. Hodel M.R. Fanara P. Hodel A.E. Corbett A.H. J. Biol. Chem. 2003; 278: 5854-5863Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 23Harreman M.T. Cohen P.E. Hodel M.R. Truscott G.J. Corbett A.H. Hodel A.E. J. Biol. Chem. 2003; 278: 21361-21369Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 24Kobe B. Nat. Struct. Biol. 1999; 6: 388-397Crossref PubMed Scopus (323) Google Scholar). For some cargos, however, that mechanism is too slow and can take up to several minutes (15Gilchrist D. Mykytka B. Rexach M. J. Biol. Chem. 2002; 277: 18161-18172Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). We showed previously that Nup2p and Cse1p function as karyopherin release factors (KaRFs) that accelerate the dissociation of Kap60p·NLS-cargo complexes (15Gilchrist D. Mykytka B. Rexach M. J. Biol. Chem. 2002; 277: 18161-18172Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). In the case of Cse1p, its KaRF activity was strictly dependent on forming a complex with Gsp1p·GTP. Here, we define the molecular basis of Nup2p and Cse1p KaRF activities and explore their relationship to the Kap60p AIS motif. Preparation and Purification of Recombinant Proteins—Genes encoding the proteins or protein fragments used were amplified from S. cerevisiae genomic DNA using Taq or Pfu-driven polymerase chain reactions with designed oligonucleotides that incorporate restriction enzyme sites compatible for ligation into pGEX-2TK in frame with the 3′ end of the gene encoding glutathione S-transferase (GST). Construction of Kap60p, Kap95p, Cse1p, Gsp1p, Nup2p, and NLS-cargo as GST fusions has been described previously (15Gilchrist D. Mykytka B. Rexach M. J. Biol. Chem. 2002; 277: 18161-18172Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). The Kap60ΔIBB construct lacks aa 1-61. Recombinant proteins were expressed in Escherichia coli strains BLR or BL21 Codon Plus (Novagen) and were purified on glutathione-coated Sepharose beads (Amersham Biosciences). In each case, a bacterial cell extract prepared from 1000 A600 units of cells was incubated with 1 ml of glutathione-coated Sepharose beads in batch for 1 h at 4 °C. After extensive washing of the column, GST proteins were eluted with elution buffer (50 mm Tris, pH 8, 110 mm KOAc, 2 mm Mg(OAc)2, 2 mm DTT, 0.1% Tween 20 plus 15 mm reduced glutathione), and proteins were concentrated in a Centricon 30 unit (Amicon). Concentrated proteins were aliquoted in 1-mg portions, frozen in liquid nitrogen, and stored at -70 °C. To remove GST, thawed GST fusions were treated with thrombin (Calbiochem) at room temperature for specific times. After adding hirudin (Calbiochem) to neutralize thrombin, the samples were applied separately to fast protein liquid chromatography Superdex 200 or Superose 6 sizing columns (Amersham Biosciences), which were equilibrated in 20 mm Hepes, pH 6.8, 150 mm KOAc, 2 mm Mg(OAc)2, and 2 mm DTT. Peak fractions containing the purified karyopherin, nucleoporin, or NLS-cargo were pooled, Tween 20 was added to 0.1%, and aliquots were frozen in liquid nitrogen and stored at -70 °C. His-tagged Gsp1p was purified and charged with GTP as described previously (25Allen N. Huang L. Burlingame A. Rexach M. J. Biol. Chem. 2001; 276: 29268-29274Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Point mutations in Nup2p and Kap60p were incorporated using the QuikChange site-directed mutagenesis kit (Stratagene), and all mutant constructs were verified by DNA sequencing. Peptides containing the sequences GGGVMANRKIARMRHSKR (Nup1p aa 1059-1076) and CTPPKKKRKVEDP (SV40 T-antigen NLS) were obtained by chemical synthesis. Solution Binding Assay—All assays were performed using recombinant proteins in binding buffer (20 mm Hepes, pH 6.8, 150 mm KOAc, 2 mm Mg(OAc)2, 2 mm DTT, and 0.1% Tween 20). For each experiment, the GST-Nup, GST-Kap, or GST-NLS-cargo was incubated in batch with glutathione-Sepharose beads (1-2 μg of GST fusion per 10 μl of packed beads) (Amersham Biosciences) in 1 ml of binding buffer for 15 min at 4 °C. The beads were collected by centrifugation at 2,000 × g for 30 s and were washed six times by resuspension in 0.5 ml of binding buffer and sedimentation as before. Two washes were done at room temperature and contained 100 μm ATP to remove any E. coli heat shock proteins that bound to the GST chimera. Washed collected beads were resuspended in a 50% slurry, and the bead slurry was aliquoted in 20-μl portions into siliconized 0.5-ml microtubes (Sigma) that contained protein additions, for a total volume of 40 μl. Tubes were then tumbled for 1 h at 4 °C. At the end of incubations, beads were sedimented at 2,000 × g for 30 s, and unbound proteins in the supernatant fractions were collected by removing 30 μl from the meniscus; this constitutes the unbound fraction. Beads were washed twice and were resuspended with 30 μl of buffer. All samples were finally processed by adding 10 or 12 μl of 6× sample buffer with β-mercaptoethanol to unbound and bound fractions, respectively. Samples were heated at 95 °C for 10 min, and proteins in one-half of each sample were resolved by SDS-PAGE and stained with Coomassie Blue. Molecular Dissociation Assay and KaRF Assay—Dissociation rates were assayed using purified radiolabeled proteins in a GST-based solution binding assay as described previously (15Gilchrist D. Mykytka B. Rexach M. J. Biol. Chem. 2002; 277: 18161-18172Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Briefly, GST-Nup2p and GST-NLS-cargo were immobilized separately on beads and incubated for 2 h with radiolabeled Kap60p at room temperature or 4 °C in 20 mm Hepes, pH 6.8, 150 mm KOAc, 2 mm Mg(OAc)2, 1 mm DTT, 0.1% Tween 20, and 10 mg/ml bovine serum albumin. 12 μl of the bead mix was then diluted 100 times into 1.2 ml of 20 mm Hepes, pH 6.8, 150 mm KOAc, 2 mm Mg(OAc)2,1mm DTT, 0.1% Tween 20, and 10 mg/ml bovine serum albumin containing excess unlabeled Kap60p as competitor. In a KaRF assay, an unlabeled effector protein was added instead of the competitor. Beginning 10 s after dilution, beads were flash-collected on filters using a vacuum manifold. Bound radiolabeled Kap60p was eluted from filters with 1% SDS and quantified by scintillation counting. Single-exponential dissociation curves were fit to the data using GraphPad Prism™ software (Biosoft). Half-lives of complexes (t) were calculated using the equation t = ln2/koff. Quantitative Yeast Two-hybrid Assay—Yeasts expressing Kap60p fused to the C terminus of the Gal4p DNA binding domain (Kap60pDBD) and the SV40 T-antigen NLS fused to the Gal4p activation domain (NLSAD) were grown to midlog phase in selective medium. Cells were pelleted, washed once with 1 ml of H2O, and frozen at -70 °C. Cells were thawed on ice, resuspended in 60 μl of breaking buffer (0.1 m Tris, pH 8, 20% glycerol, 1 mm DTT, 5 mm phenylmethylsulfonyl fluoride, 1 μg/ml pepstatin) and lysed by vortexing with glass beads for five 1-min intervals with 1-min rest periods between intervals. Extracts were spun at 4 °C for 15 min at 15,000 rpm in a microcentrifuge, the supernatants were collected, and the protein concentrations were determined using a Bradford assay. Liquid β-galactosidase assays contained 90 μl of Z-buffer (Na2HPO4·7H2O (16.1 mg/ml), NaH2PO4·H2O (5.5 mg/ml), KCl (0.75 mg/ml), MgSO4·7H2O (0.246 mg/ml)) and 20 μl of supernatant and were initiated by the addition of 20 μl of 4 mg/ml ONPG (Sigma) in Z-buffer. Hydrolysis of ONPG by β-galactosidase was monitored at room temperature for 90 min by measuring ΔA415 in a Bio-Rad 96-well spectrophotometer. The rate of ΔA415 for each supernatant was normalized to the protein concentration, and data sets were compared with the largest rate obtained, which was assigned a value of 100% for convenience. Assays were performed in triplicate, and graphs are representative of at least two independent experiments. Fluorescence Microscopy—Yeast expressing Kap60p-GFP were grown to midlog phase at 30 °C in selective media with 2% glucose. 2 μl of culture was spotted on a slide, and yeasts were visualized immediately with a Nikon Eclipse E600 epifluorescence microscope equipped with a CCD camera. Images were captured and adjusted using QED Imaging software and Photoshop 4.0. Nuclear import assays were performed as described in Ref. 26Denning D. Mykytka B. Allen N. Huang L. Burlingame A. Rexach M. J. Cell Biol. 2001; 154: 937-950Crossref PubMed Scopus (73) Google Scholar. Briefly, yeasts carrying the plasmid pCu413 cNLS-YFP (encoding YFP with the cNLS of large T-antigen under a copper-inducible promoter) were grown at 30 °Cto A600 = 1.0 in synthetic growth medium. Cultures were diluted to A600 = 0.4 into medium with or without 100 μm CuSO4 and grown for 2 h. Yeasts were then sedimented, washed once with 1 ml of water, and resuspended in 1 ml of medium containing 20 mm sodium azide and 20 mm 2-deoxyglucose. Following incubation for 1 h at 4 °C yeast were sedimented and resuspended in 100 μl of ice-cold medium containing 2% dextrose. Import assays were initiated by placing 2 μl of cells onto room temperature microscope slides, and in five 2-min intervals cells were categorized as either displaying nuclear cNLS-YFP accumulation or diffuse cytoplasmic and nucleoplasmic fluorescence. Nup2p immunofluorescence was performed as described in Ref. 26Denning D. Mykytka B. Allen N. Huang L. Burlingame A. Rexach M. J. Cell Biol. 2001; 154: 937-950Crossref PubMed Scopus (73) Google Scholar. After growth in synthetic medium cells were fixed for 10 min with 3.7% formaldehyde. Cells were sedimented and resuspended in 100 mm KH2PO4, followed by digestion with 0.5 mg/ml zymolyase for 15 min at room temperature. Spheroplasts were adhered to polylysine-coated slides and postfixed for 6 min in ice-cold methanol and 30 s in ice-cold acetone. Nup2p was detected using affinity-purified anti-Nup2p antibodies and fluorescein isothiocyanate-conjugated anti-rabbit IgG. We previously showed that Nup2p and Cse1p function as KaRFs (karyopherin release factors) to accelerate the dissociation of Kap60p·NLS-cargo complexes (15Gilchrist D. Mykytka B. Rexach M. J. Biol. Chem. 2002; 277: 18161-18172Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). We proposed that their reaction mechanisms proceed through a transient KaRF intermediate where Nup2p or Cse1p·Gsp1p·GTP bind to Kap60p and trigger an allosteric change in its structure that accelerates dissociation of the NLS-cargo. A diagram of these KaRF reaction mechanisms is shown in Fig. 1A (models i and iii). As a starting point in defining the molecular basis of Nup2p and Cse1p KaRF activity, we used a KaRF assay to test if the Kap60p AIS motif in the IBB domain is required by Nup2p or Cse1p·Gsp1p·GTP to accelerate release of Kap60p from NLS-cargo. In a sample KaRF assay (diagramed in Fig. 1B), the dissociation of radiolabeled Kap60p from NLS-cargo is monitored in the absence (line 1) or presence (line 2) of excess unlabeled Kap60p (control) or in the presence of a possible effector/KaRF (such as Nup2p or the Cse1p·Gsp1p·GTP complex) (line 3). The unlabeled Kap60p serves to prevent rebinding of radiolabeled Kap60p to the NLS-cargo-coated beads during their dissociation reaction, allowing accurate determination of their dissociation rate (15Gilchrist D. Mykytka B. Rexach M. J. Biol. Chem. 2002; 277: 18161-18172Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). If the effector is active as a KaRF (line 3), then the dissociation rate of the Kap60p·NLS-cargo complex is accelerated beyond its intrinsic rate. In all of the KaRF assays shown below, the 5-min data point of control dissociation curves (circled in Fig. 1B, line 2) was normalized to a 0% value, and the dissociation of all radiolabeled Kap60p (circled in Fig. 1B, line 3) was assigned a 100% value. These values are plotted in a histogram format as a qualitative measure of the KaRF activity of possible effectors. In all cases where Gsp1p was used, it was preloaded with GTP; for convenience, however, Gsp1p·GTP is simply referred to as Gsp1p throughout. To test whether Nup2p and Cse1p·Gsp1p can accelerate release of an NLS-cargo from a Kap60p mutant lacking the IBB domain (Kap60pΔIBB; Δaa 1-61) or from a Kap60p point mutant lacking autoinhibitory function (Kap60p K54A) (23Harreman M.T. Cohen P.E. Hodel M.R. Truscott G.J. Corbett A.H. Hodel A.E. J. Biol. Chem. 2003; 278: 21361-21369Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), we first had to measure the intrinsic rate of dissociation of these mutant Kap60p·NLS-cargo complexes (Fig. 2, A and B). Radiolabeled Kap60pΔIBB and Kap60p K54A were incubated separately with NLS-cargo-coated beads and allowed to form complexes for 2 h at room temperature. Incubations were then flash-diluted 100-fold into buffer containing excess Kap60pΔIBB (Fig. 2A) or Kap60p K54A (Fig. 2B) as unlabeled competitors. At time points after dilution, beads in aliquots were flash-collected in a manifold filter, and the amount of radiolabeled Kap60p remaining in beads was determined by liquid scintillation. For reference, wild type Kap60p binds the NLS-cargo (the NLS of Cbp80p fused to the maltose-binding protein) with an affinity of KD = 2.8 nm, and a dissociation rate constant of koff = 1.8 × 10-3 s-1 (15Gilchrist D. Mykytka B. Rexach M. J. Biol. Chem. 2002; 277: 18161-18172Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). We found that Kap60pΔIBB and Kap60p K54A dissociate more slowly from the NLS-cargo, with dissociation rate constants of koff = 1.3 × 10-4 s-1 and koff = 2.4 × 10-4 s-1, respectively (Fig. 2, A and B). This result suggests that the AIS motif in the Kap60p IBB domain enhances the dissociation of NLS-cargo from Kap60p by up to 13-fold. This enhancement is significant but is lower than the enhanced rate of dissociation when Nup2p or Cse1p·Gsp1p is present (koff ≥ 3.9 × 10-2 s-1 and koff ≥ 6.9 × 10-2 s-1, respectively) (15Gilchrist D. Mykytka B. Rexach M. J. Biol. Chem. 2002; 277: 18161-18172Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). As expected from previous measurements, Cse1p·Gsp1p and Nup2p were capable of releasing all bound radiolabeled Kap60p from NLS-cargo within 5 min in a KaRF assay (Fig. 2C, black bars). In contrast, Cse1p·Gsp1p was ineffective in accelerating the dissociation of Kap60pΔIBB or Kap60p K54A from NLS-cargo (Fig. 2C, gray bars), despite being able to bind Kap60pΔIBB and Kap60p K54A as well as wild type Kap60p (data not shown) (22Harreman M.T. Hodel M.R. Fanara P. Hodel A.E. Corbett A.H. J. Biol. Chem. 2003; 278: 5854-5863Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Combined, these results suggest that the AIS motif within the IBB domain of Kap60p is utilized by the Cse1p·Gsp1p KaRF reaction mechanism. In stark contrast, Nup2p was able to accelerate the dissociation of Kap60pΔIBB and Kap60p K54A from NLS-cargo (Fig. 2C), demonstrating that Nup2p does not need the Kap60p AIS motif or the IBB domain as part of its KaRF reaction mechanism. Stabilization of a Transient KaRF Intermediate (the NLS-cargo·Kap60p·Cse1p·Gsp1p Complex)—The accelerated release of NLS-cargo from Kap60p by Cse1p·Gsp1p was predicted to proceed through a transient tetrameric intermediate, where Kap60p binds simultaneously to NLS-cargo and Cse1p·Gsp1p (Fig. 1A, model iii) (15Gilchrist D. Mykytka B. Rexach M. J. Biol. Chem. 2002; 277: 18161-18172Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). This intermediate was not detected previously, presumably because Kap60p releases the NLS-cargo very rapidly upon binding Cse1p·Gsp1p. However, if the release of the NLS-cargo was impeded by a defect in Cse1p KaRF mechanism, a tetrameric intermediate complex might be stabilized. Indeed, removal of the Kap60p IBB domain allowed detection of this intermediate in a solution binding assay (Fig. 2D). Beads coated with NLS-cargo were incubated with Kap60p or Kap60pΔIBB for 30 min, washed once, and incubated for 1 h in the presence or absence of Cse1p·Gsp1p. In the absence of Cse1p·Gsp1p, most of the Kap60p was recovered bound to the immobilized NLS-cargo (lanes 2 and 3), and in the presence of Cse1p·Gsp1p, nearly all Kap60p was recovered in the unbound fraction (lane 4). In contrast, Kap60pΔIBB remained bound to the NLS-cargo in the presence of Cse1p·Gsp1p (lanes 5-7), and a portion of the Cse1p·Gsp1p added was recovered in the bound fraction in a stable complex with NLS-cargo and Kap60pΔIBB (lane 7). As expected, Cse1p·Gsp1p did not bind directly to immobilized NLS-cargo (lane 8). These data suggest that Cse1p·Gsp1p binds to the Kap60pΔIBB·NLS-cargo complex in a stalled KaRF intermediate, as depicted in Fig. 1A (model iii). Cse1p·Gsp1p Enhances the Affinity of Kap60p for Its IBB Domain—How does Cse1p·Gsp1p employ the Kap60p IBB domain to accelerate the release of NLS-cargo from Kap60p? In one scenario, Cse1p·Gsp1p might trigger an allosteric change in Kap60p structure that reduces its affinity toward the NLS-cargo, allowing the IBB domain to compete more effectively for the NLS-binding site. That scenario is unlikely, since Cse1p·Gsp1p cannot accelerate dissociation of NLS-cargo from Kap60pΔIBB (Fig. 2C). In a second scenario, Cse1p·Gsp1p might accelerate release of an NLS from Kap60p by increasing the affinity of Kap60p toward the AIS motif in its IBB domain. To test this, we examined the effect of Cse1p·Gsp1p on Kap60p binding to an isolated IBB domain in a solution binding assay (Fig. 2E). The IBB domain of Kap60p was immobilized on beads as a GST fusion (GST-IBB) and was incubated with Kap60p or Kap60pΔIBB in the presence or absence of Cse1p·Gsp1p for 1 h at 4 °C. Neither Kap60p nor Kap60pΔIBB bound to the isolated IBB domain (Fig. 2E, lanes 2 and 4), suggesting that Kap60p is normally in a conformation that exhibits low affinity toward the IBB domain. However, in the presence of Cse1p·Gsp1p, Kap60pΔIBB bound stably to the immobilized IBB domain and formed a complex with Cse1p·Gsp1p (lane 5). In contrast, Cse1p·Gsp1p did not promote binding of full-length Kap60p to the immobilized IBB domain (lane 3), suggesting that wild type Kap60p binds its IBB domain in cis more effectively than in trans. In a similar experiment, Nup2p did not stimulate binding of Kap60p or Kap60pΔIBB to the immobilized IBB domain (data not shown). Altogether, the data suggest that Kap60p is normally in a conformation that binds weakly to its IBB domain, but Cse1p·Gsp1p binding can trigger an allosteric change in Kap60p structure that significantly enhances its affinity for the IBB domain. This allosteric conformation switch may be the molecular basis for Cse1p·Gsp1p KaRF activity (i.e. changing the Kap60p NLS-cargo binding pocket from a pro-NLS to a pro-IBB conformation). Cse1p Also Accelerates Dissociation of Kap60p from Nup2p via a KaRF Mechanism—It has been previously shown that Cse1p·Gsp1p antagonizes the interaction of Kap60p with Nup2p, and it was proposed that Nup2p serves as a platform for Cse1p-mediated export of Kap60p from the nucleus (10Solsbacher J. Maurer P. Vogel F. Schlenstedt G. Mol. Cell. Biol. 2000; 20: 8468-8479Crossref PubMed Scopus (79) Google Scholar, 11Hood J.K. Casolari J.M. Silver P.A. J. Cell Sci. 2000; 113: 1471-1480PubMed Google Scholar, 28Booth J.W. Belanger K.D. Sannella M.I. Davis L.I. J. Biol. Chem. 1999; 274: 32360-32367Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). We therefore examined whether Cse1p·Gsp1p can also accelerate the rate of dissociation of Kap60p from Nup2p. To test this notion, we first had to measure the dissociation rate of radiolabeled Kap60p from immobilized Nup2p (Fig. 3A). At room temperature, this complex had a very short half-life (t ∼25 s; koff = 3 × 10-2 s-1) (data not shown) despite its high affinity (KD = 0.3 nm), making the analysis difficult. To solve that problem, we monitored the same reaction at low temperature, which slows down the dissociation rate. At 4 °C, the Kap60p·Nup2p interaction had a half-life of 4.5 min in the presence of excess unlabeled competitor Kap60p (koff = 2.5 × 10-3 s-1) (Fig. 3A). In comparison, the Kap60pΔIBB·Nup2p complex dissociated more slowly (data not shown). The dissociation of Kap60p and Kap60pΔIBB from Nup2p was monitored in a KaRF assay in the presence of Cse1p·Gsp1p or Nup2p as possible effectors (Fig. 3B). Cse1p·Gsp1p accelerated the dissociation of Kap60p but not Kap60pΔIBB from Nup2p (Fig. 3B), consistent with a KaRF mechanism whereby Cse1p·Gsp1p binds to Kap60p (in a Kap60p·Nup2p complex) and triggers an allosteric change in Kap60p structure that accelerates the release of Nup2p in an IBB-dependent mechanism (Fig. 1A, model ii). A stable KaRF intermedi
Referência(s)