Discovering Novel Interactions at the Nuclear Pore Complex Using Bead Halo
2007; Elsevier BV; Volume: 7; Issue: 1 Linguagem: Inglês
10.1074/mcp.m700407-mcp200
ISSN1535-9484
AutoresSamir S. Patel, Michael Rexach,
Tópico(s)Genomics and Chromatin Dynamics
ResumoA highly sensitive, equilibrium-based binding assay termed "Bead Halo" was used here to identify and characterize interactions involving components of the nucleocytoplasmic transport machinery in eukaryotes. Bead Halo uncovered novel interactions between the importin Kap95 and the nucleoporins (nups) Nic96, Pom34, Gle1, Ndc1, Nup84, and Seh1, which likely occur during nuclear pore complex biogenesis. Bead Halo was also used to characterize the molecular determinants for binding between Kap95 and the family of nups that feature multiple phenylalanine-glycine motifs (FG nups). Binding was sensitive to the number of FG motifs present and to amino acid (AA) residues immediately flanking the FG motifs. Also, binding was reduced but not abolished when phenylalanine residues in all FG motifs were replaced by tyrosine or tryptophan. These results suggest flexibility in the binding pockets of Kap95 and synergism in binding FG motifs. The hypothesis that Nup53 and Nup59 bind directly to membranes through a C-terminal amphipathic alpha helix and to DNA via an RNA recognition motif domain was also tested and validated using Bead Halo. The results support a role for these nups in nuclear pore membrane biogenesis and in gene expression. Finally, Bead Halo detected binding of the nups Gle1, Nup60, and Nsp1 to phospholipid bilayers. This may reflect the known interaction between Gle1 and phosphoinositides and suggests similar interactions for Nup60 and Nsp1. As the Bead Halo assay detected molecular interactions in cell lysates, as well as between purified components, it can be adapted for large-scale proteomic studies using automated robotics and microscopy. A highly sensitive, equilibrium-based binding assay termed "Bead Halo" was used here to identify and characterize interactions involving components of the nucleocytoplasmic transport machinery in eukaryotes. Bead Halo uncovered novel interactions between the importin Kap95 and the nucleoporins (nups) Nic96, Pom34, Gle1, Ndc1, Nup84, and Seh1, which likely occur during nuclear pore complex biogenesis. Bead Halo was also used to characterize the molecular determinants for binding between Kap95 and the family of nups that feature multiple phenylalanine-glycine motifs (FG nups). Binding was sensitive to the number of FG motifs present and to amino acid (AA) residues immediately flanking the FG motifs. Also, binding was reduced but not abolished when phenylalanine residues in all FG motifs were replaced by tyrosine or tryptophan. These results suggest flexibility in the binding pockets of Kap95 and synergism in binding FG motifs. The hypothesis that Nup53 and Nup59 bind directly to membranes through a C-terminal amphipathic alpha helix and to DNA via an RNA recognition motif domain was also tested and validated using Bead Halo. The results support a role for these nups in nuclear pore membrane biogenesis and in gene expression. Finally, Bead Halo detected binding of the nups Gle1, Nup60, and Nsp1 to phospholipid bilayers. This may reflect the known interaction between Gle1 and phosphoinositides and suggests similar interactions for Nup60 and Nsp1. As the Bead Halo assay detected molecular interactions in cell lysates, as well as between purified components, it can be adapted for large-scale proteomic studies using automated robotics and microscopy. In the context of the cellular milieu, protein interactions of high and low affinity are key for the survival of organisms. Current proteomic studies aim to uncover all such interactions with the ultimate goal of reconstructing, understanding and predicting all cellular behavior at a system-wide level. Despite the great advances in cellular proteomics, our ability to detect protein interactions of low affinity, in particular, is limited. Current proteomic analyses using biochemical tools, such as Protein A-tag (also termed ZZ-tag; TAP tag) pull-downs or co-immunoprecipitations, are limited to only detecting high affinity interactions. This is because the necessary wash steps that remove nonspecific interactors (i.e. from the agarose or magnetic beads used as solid support during the isolations) also remove specific low affinity interactions. It is estimated that pull-down methods can identify protein interactions stronger than the Kd ∼5 μm range (1Pyhtila B. Rexach M. A gradient of affinity for the karyopherin Kap95p along the yeast nuclear pore complex.J. Biol. Chem. 2003; 278: 42699-42709Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 2Patel S.S. Belmont B.J. Sante J.M. Rexach M.F. Natively unfolded nucleoporins gate protein diffusion across the nuclear pore complex.Cell. 2007; 129: 83-96Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). The sensitivity can be improved through extended incubation times followed by rapid collection and washing of the beads, but nonspecific interactions also increase (3Cristea I.M. Williams R. Chait B.T. Rout M.P. Fluorescent proteins as proteomic probes.Mol. Cell. Proteomics. 2005; 4: 1933-1941Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). To detect protein interactions of low affinity (beyond the Kd ∼5 μm range), we recently reported the development of an assay termed Bead Halo, which can detect molecular interactions of high and low affinity in real time at equilibrium (2Patel S.S. Belmont B.J. Sante J.M. Rexach M.F. Natively unfolded nucleoporins gate protein diffusion across the nuclear pore complex.Cell. 2007; 129: 83-96Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). Here, we expand the repertoire of interactions tested using this novel technique to include protein–DNA and protein–phospholipid interactions. The nucleocytoplasmic transport machinery of yeast was used as the experimental landscape. The yeast nucleocytoplasmic transport machinery relies on nucleoporins to form the nuclear pore complex, karyopherins (kaps) 1The abbreviations used are: kaps, karyopherins; AA, amino acid; CFP, cyan fluorescent protein; GTP, guanosine triphosphate; MBP, maltose-binding protein; NLS, nuclear localization signal; NPC, nuclear pore complex; nups, nucleoporins; POMs, pore membrane nucleoporins; RRM, RNA-recognition motif; WT, wild-type; YFP, yellow fluorescent protein. , to ferry cargo across the nuclear pore complex (NPC) and the RanGTPase system to load or unload cargos from kaps (supplemental Fig. 1) (reviewed in Ref. 4Stewart M. Molecular mechanism of the nuclear protein import cycle.Nat. Rev. Mol. Cell. Biol. 2007; 8: 195-208Crossref PubMed Scopus (671) Google Scholar). The NPC forms and maintains the sole aqueous conduit between the cytoplasm and nucleoplasm of cells and gates all macromolecular transport between these two compartments. It is composed of ∼33 nups, and some (the FG nups) contain large, natively unfolded domains with multiple FG repeats (42Denning D.P. Patel S.S. Uversky V. Fink A.L. Rexach M. Disorder in the Nuclear Pore Complex: The FG repeat regions of nucleoporins are natively unfolded.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 2450-2455Crossref PubMed Scopus (378) Google Scholar). The FG nups function as binding sites for kaps during their stochastic translocation across the NPC (5Rexach M. Blobel G. Protein import into nuclei: association and dissociation reactions involving transport substrate, transport factors, and nucleoporins.Cell. 1995; 83: 683-692Abstract Full Text PDF PubMed Scopus (665) Google Scholar), and as the structural elements of a permeability barrier that prevents entry of large (>30 kDa) non-karyophilic particles into the nucleus (2Patel S.S. Belmont B.J. Sante J.M. Rexach M.F. Natively unfolded nucleoporins gate protein diffusion across the nuclear pore complex.Cell. 2007; 129: 83-96Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar, 6Shulga N. Mosammaparast N. Wozniak R. Goldfarb D.S. Yeast nucleoporins involved in passive nuclear envelope permeability.J. Cell Biol. 2000; 149: 1027-1038Crossref PubMed Scopus (89) Google Scholar, 7Ribbeck K. Gorlich D. The permeability barrier of nuclear pore complexes appears to operate via hydrophobic exclusion.EMBO J. 2002; 21: 2664-2671Crossref PubMed Scopus (426) Google Scholar, 8Shulga N. Goldfarb D.S. Binding dynamics of structural nucleoporins govern nuclear pore complex permeability and may mediate channel gating.Mol. Cell. Biol. 2003; 23: 534-542Crossref PubMed Scopus (90) Google Scholar, 9Frey S. Gorlich D. A saturated FG-repeat hydrogel can reproduce the permeability properties of nuclear pore complexes.Cell. 2007; 130: 512-523Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar). Recombinant nups were expressed as GST fusions in the Escherichia coli expression vector pGEX-2TK (GE Healthcare) and purified as described (10Allen N.P. Huang L. Burlingame A. Rexach M.F. Proteomic analysis of nucleoporin interacting proteins.J. Biol. Chem. 2001; 276: 29268-29274Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 11Denning D. Mykytka B. Allen N.P. Huang L. Al B. Rexach M. The nucleoporin Nup60p functions as a Gsp1p-GTP-sensitive tether for Nup2p at the nuclear pore complex.J. Cell Biol. 2001; 154: 937-950Crossref PubMed Scopus (73) Google Scholar). GST-Nup116 (AA 348–458)-6xHIS was modified by site-directed mutagenesis to generate F>A mutations of some or all phenylalanine residues in FG motifs. The DNA encoding other mutants of Nup116 or Nup100 were synthesized de novo (GenScript) and cloned into pGEX-2TK. The GST-Nup53ΔC mutant lacks the C-terminal 15 AAs and was created by deletion mutagenesis of pGEX-2TK NUP53. All yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP)-fusions were initially purified as GST fusions, and the GST was later removed by thrombin cleavage when indicated (GST Handbook, GE Healthcare). The fluorescent fusion proteins were purified by gel filtration in a FPLC Superdex-200 column pre-equilibrated in binding buffer (20 mm HEPES pH 6.8, 150 mm KOAc, 2 mm Mg(OAc)2, 1 mm DTT, 0.1% Tween-20). Protein in the eluates was concentrated to 0.4–1 mg/ml using a Centricon-10 (Millipore). Purified GST fusions were loaded onto glutathione–Sepharose beads (GE Healthcare) at a concentration of 1–10 μg per μl of packed beads, as indicated. GST fusions in crude E. coli extracts could also be used if the extracts were titrated in advance to ascertain the amount of GST fusion present per unit volume of crude extract (10Allen N.P. Huang L. Burlingame A. Rexach M.F. Proteomic analysis of nucleoporin interacting proteins.J. Biol. Chem. 2001; 276: 29268-29274Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Beads loaded with GST fusion from crude extracts were washed 8 times with binding buffer, with washes 2–4 containing 1 m NaCl, and washes 5–7 containing 0.1 mm ATP. After the final wash, an equal volume of binding buffer was added to create a 50% slurry of "loaded" beads. Typically, 10–20 μl of 50% bead slurry was prepared for each experiment. The loaded beads were stored at −70 °C after freezing in liquid N2. Yeast expressing fluorescent fusion proteins were grown at 30 °C to log phase in 50 ml of YPD or minimal growth media, as indicated. Cells were harvested by centrifugation, transferred to a 1.5 ml microcentrifuge tube, and washed once with binding buffer. Cell pellets were resuspended with 50 μl of binding buffer, which contained 5 mm DTT and protease inhibitors (1 μg/ml pepstatin, 2 μg/ml leupeptin, 2 μg/ml aprotinin, 0.1 mg/ml PMSF), but no Tween-20. The suspension was frozen at −70 °C for 3 min and then thawed quickly to aid in fracturing the cells. An equal volume of acid-washed glass beads was added and the mixture was vortexed vigorously for 2 min, then placed on ice for 2 min for a total of 3 cycles. Tween-20 was added to 0.1%, and the samples were subjected to centrifugation at 20,000 × g for 10 min to remove insoluble cell debris and glass beads. The cleared cell lysates (the supernatants) were centrifuged immediately before each use to remove any insoluble material. Liposomes were prepared using a mixture of pure phospholipids: 49 mol/dl 1,2-oleoyl-sn-glycero-3-phosphocholine, 21 mol/dl 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 8 mol/dl 1,2-dioleoyl-sn-glycero-3-phosphoserine, 5 mol/dl 1,2-dioleoyl-sn-glycero-3-phosphate, 8 mol/dl phosphatidylinositol, 2.2 mol/dl phosphatidylinositol 4-phosphate, 0.8 mol/dl phosphatidylinositol bisphosphate, 2 mol/dl cytidine diphosphate-diacylglycerol, 2 mol/dl nitrobenzoxadiazole phosphatidylcholine, 2 mol/dl Texas Red-phosphatidylethanolamine that generally mimic what is found in yeast microsomal membranes (12Matsuoka K. Orci L. Amherdt M. Bednarek S.Y. Hamamoto S. Schekman R. Yeung T. COPII-coated vesicle formation reconstituted with purified coat proteins and chemically defined liposomes.Cell. 1998; 93: 263-275Abstract Full Text Full Text PDF PubMed Scopus (502) Google Scholar, 13Lee M.C. Orci L. Hamamoto S. Futai E. Ravazzola M. Schekman R. Sar1p N-terminal helix initiates membrane curvature and completes the fission of a COPII vesicle.Cell. 2005; 122: 605-617Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar). This "major-minor" mix of phospholipids was hydrated in 20 mm HEPES-KOH pH 7.0, 150 mm KOAc, 250 mm sorbitol at room temperature, and the resulting suspension of liposomes was extruded through a 400 nm pore size polycarbonate filter (Poretics). A 41 base oligonucleotide was synthesized with a 5′-fluorescent Cy5 modification (IDT) and dissolved in TE buffer to 100 μm. A small aliquot of the DNA was diluted 10-fold into binding buffer, and centrifuged at 20,000 × g for 10 min before use in the Bead Halo assay. The free Cy5 dye used as a control was prepared by quenching a Cy5 mono NHS ester (GE Healthcare) with hydroxylamine. A 10 μm stock solution of Cy5 was prepared in binding buffer. For experiments involving all purified proteins, an aliquot of GST fusion coated beads (0.75 μl portion of a 50% slurry) was mixed with 0.5 μl of EHBN 4 × stock buffer (40 mm EDTA, 2% 1,6-hexanediol, 40 mg/ml bovine serum albumin, 500 mm NaCl) and 0.75 μl of purified soluble fluorescent protein, to obtain a 2 μl sample suitable for imaging on a microscope slide. The EHBN buffer supplement controls the stringency of the assay, with the final working solution containing 10 mg/ml bovine serum albumin (as a blocking and crowding agent), 10 mm EDTA (to prevent divalent cation-dependent interactions), 240 mm salt in total (to disrupt weak ionic interactions), and 0.5% 1,6-hexanediol (to disrupt weak hydrophobic interactions). For experiments involving cleared yeast lysate, 0.5 μl of a 50% bead slurry was mixed with 0.5 μl EHBN 4× stock buffer and 1 μl soluble yeast lysate. For experiments involving purified fluorescent liposomes, a 0.75 μl portion of a 50% bead slurry was mixed directly with 0.75 μl of a liposome solution. For experiments involving fluorescent DNA, 0.75 μl of the 50% slurry was mixed with 0.5 μl EHBN 4× stock buffer and 0.75 μl 10 μm DNA or free Cy5 dye. In all cases, beads were imaged under a Nikon fluorescence microscope with a 20× air objective using fluorophore-specific filters and a 2× binned CCD for image acquisition. Image exposure settings were adjusted for each sample to obtain maximum signal intensity without saturation. A portion of the Saccharomyces cerevisiae NSP1 gene encoding AA 377–471 was cloned into vector pGEX-2TK, expressed as GST fusion protein in BL21 Codon Plus E. coli (Novagen), and purified as described (10Allen N.P. Huang L. Burlingame A. Rexach M.F. Proteomic analysis of nucleoporin interacting proteins.J. Biol. Chem. 2001; 276: 29268-29274Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Mutants of Nsp1 (AA 377–471), where every instance of phenylalanine was replaced with tyrosine (F>Y) or with serine (F>S), were synthesized de novo (GenScript) and prepared the same way as the wild-type (WT) protein. For the affinity capture experiments, 5 μg of each GST-Nsp1 (WT or mutants) was immobilized onto 10 μl of glutathione–Sepharose beads. The beads were incubated with ∼4 mg of yeast cytosol in a total volume of 1 ml for 2 h at 4 °C in the absence or presence of 12 μg RanGTP (HIS-Gsp1Q71L loaded with GTP). Beads were recovered and washed twice with binding buffer. Bound proteins were extracted with 2% SDS, resolved by SDS-PAGE, transferred to PVDF membrane, and analyzed by Western blotting using specific rabbit polyclonal antibodies. Agarose microbeads are widely used for biochemical chromatography. One popular use is as a solid phase support for the capture (immobilization) of affinity-tagged fusion proteins (such as GST, 6xHIS, Protein A) from cell extracts. Such pull-down experiments typically involve a binding phase, a washing phase to remove unbound proteins and nonspecific interactions, and an analysis phase, which follows protein elution or extraction from the beads. In all cases, the technique precludes detection of specific low affinity interactions because they are lost during the wash steps, due to their generally fast off rates. To overcome this limitation, we recently developed a nonquantitative method termed Bead Halo, which can detect both high and low affinity interactions. Bead Halo detects low affinity interactions because it monitors the samples in real time under equilibrium binding conditions without the need for wash steps. The assay is based on the simple principle of observing by microscopy a soluble, fluorescently labeled macromolecule binding to the surface of microbeads, onto which a second macromolecule had been pre-immobilized via an affinity tag (Fig. 1). The visual appearance of the surface-bound, fluorescently labeled macromolecules is as a bright circle or halo around microscopic cross-sections of beads, which reflects the location on the bead surface where the affinity-tagged protein is immobilized. The Bead Halo assay detects interactions over a wide range of affinities (Fig. 2). The sensitivity is determined by the concentration of the bead-immobilized protein. Depending on the molar ratio between the bead-immobilized "receptor" and the soluble fluorescent "ligand," high affinity interactions are typically manifested as the complete titration of the ligand out of solution onto the bead surface. Low affinity interactions are observed as a fluorescent halo around beads with surrounding fluorescence in the solution. To illustrate, we reconstituted a known high affinity interaction (Kd = 0.22 μm) between the Nup100 FG domain and Kap95 (1Pyhtila B. Rexach M. A gradient of affinity for the karyopherin Kap95p along the yeast nuclear pore complex.J. Biol. Chem. 2003; 278: 42699-42709Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar), and compared it to a known low affinity interaction (Kd > 5 μm) between the Nup100 and Nup116 FG domains (2Patel S.S. Belmont B.J. Sante J.M. Rexach M.F. Natively unfolded nucleoporins gate protein diffusion across the nuclear pore complex.Cell. 2007; 129: 83-96Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). The FG domain of Nup100 (AA 1–640) was immobilized as a GST-tagged fusion protein on glutathione–Sepharose beads at a concentration of 1, 3, 5, or 10 μg/μl beads, and was subsequently mixed with Kap95-YFP (a fusion between the importin Kap95 and the Yellow Fluorescent Protein) or with CFP-Nup116 AA 165–716 (a fusion between the CFP and the FG domain of Nup116) or with CFP-Nup116 AA 348–458 (a similar fusion, but with a smaller portion of the Nup116 FG domain containing fewer FG motifs, as indicated in Fig. 2A). As negative controls, we used GST and CFP-maltose binding protein (MBP). GST-coated beads did not capture the soluble fluorescent proteins tested (Fig. 2B, top row), and CFP-MBP did not bind to beads coated with the GST fusions used (Fig. 2B, left column). For the high affinity interaction between the GST-Nup100 FG domain and Kap95-YFP (Kd = 0.22 μm) (Fig. 2B, second column), bright fluorescent halos were observed around the beads and no fluorescence was observed free in solution, indicating that all of the Kap95-YFP was captured by the nup-coated beads. For the low affinity interaction between the Nup100 and Nup116 FG domains (Kd > 5 μm) (2Patel S.S. Belmont B.J. Sante J.M. Rexach M.F. Natively unfolded nucleoporins gate protein diffusion across the nuclear pore complex.Cell. 2007; 129: 83-96Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar), fluorescence was observed in the solution and as a halo around the beads, indicating that some but not all of the CFP-Nup116 was captured by the nup-coated beads (Fig. 2B, right columns). We had characterized this low affinity interaction previously, and several other of the same type, and concluded that some (but not all) FG nups contain "cohesive" FG domains that interact via hydrophobic FG motifs to form a meshwork of filaments at the NPC center, which regulates the diffusion of macromolecules across the nuclear envelope (2Patel S.S. Belmont B.J. Sante J.M. Rexach M.F. Natively unfolded nucleoporins gate protein diffusion across the nuclear pore complex.Cell. 2007; 129: 83-96Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). The in vitro data shown here demonstrate that the low affinity interaction between the cohesive FG domains is sensitive to the length of the FG domain and/or to the number of FG motifs. This is similar to what was observed in vivo (2Patel S.S. Belmont B.J. Sante J.M. Rexach M.F. Natively unfolded nucleoporins gate protein diffusion across the nuclear pore complex.Cell. 2007; 129: 83-96Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). Specifically, the Nup100 FG domain bound better to a 552 AA FG domain of Nup116 (AA 165–716; containing 34 FG motifs) than to a smaller 111 AA fragment (Nup116 AA 348–458; containing 10 FG motifs). Likely, the combined avidity of interactions between multiple FG motifs strengthens the FG domain interactions. Although kaps are best known for their ability to bind nuclear import or export signals on cargos, and FG motifs on FG nups, their interaction with other nucleoporins such as the pore membrane nups (POMs) or the non-FG nups (supplemental Fig. 1) may also be physiologically relevant and could explain some outstanding issues in nucleocytoplasmic transport and/or NPC biogenesis. For example, a direct binding interaction between Kap95 and non-FG nups would explain 1) the Kap95-dependent recruitment of non-FG nups (e.g. Nic96) into new or preexisting NPCs (14Ryan K.J. Zhou Y. Wente S.R. The karyopherin Kap95 regulates nuclear pore complex assembly into intact nuclear envelopes in vivo.Mol. Biol. Cell. 2007; 18: 886-898Crossref PubMed Scopus (42) Google Scholar) and 2) the importin β-regulated assembly of the Nup107 complex (a non-FG nup oligomer) unto NPCs after mitosis (15Harel A. Chan R.C. Lachish-Zalait A. Zimmerman E. Elbaum M. Forbes D.J. Importin beta negatively regulates nuclear membrane fusion and nuclear pore complex assembly.Mol. Biol. Cell. 2003; 14: 4387-4396Crossref PubMed Scopus (124) Google Scholar, 16Walther T.C. Askjaer P. Gentzel M. Habermann A. Griffiths G. Wilm M. Mattaj I.W. Hetzer M. RanGTP mediates nuclear pore complex assembly.Nature. 2003; 424: 689-694Crossref PubMed Scopus (178) Google Scholar). The Kap95 protein is the yeast homolog of the vertebrate importin β and the Nup84 complex is the yeast homolog of the vertebrate Nup107 complex (supplemental Fig. 1). Despite these functional connections, a direct binding interaction between Kap95 (importin β) and these nups has not been documented. To test if Kap95 binds directly to non-FG nups, we coated beads with various GST-non-FG nups and monitored their ability to capture soluble Kap95-YFP using Bead Halo (Fig. 3). Of the 13 non-FG nups (or large soluble fragments thereof) tested, six bound to Kap95-YFP directly (namely Nic96, Nup84, Seh1, Pom34, Gle1, and Ndc1) and seven did not (namely Nup82, Nup85, Gle2, Nup157, Nup120, Nup192, and Nup170). In control experiments, GST and CFP-MBP were completely inert (as before), and GST-Kap60 bound to all of the available Kap95-YFP, as expected for this high affinity interaction (Kd = 0.15 nm) (17Gilchrist D. Mykytka B. Rexach M. Accelerating the rate of disassembly of karyopherin-cargo complexes.J. Biol. Chem. 2002; 277: 18161-18172Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). These results demonstrate a direct interaction between Kap95 and non-FG nups, and between Kap95 and POMs, and suggest a direct role for Kap95 in NPC biogenesis. The binding between kaps and FG domains of nups is well documented (examples in (10Allen N.P. Huang L. Burlingame A. Rexach M.F. Proteomic analysis of nucleoporin interacting proteins.J. Biol. Chem. 2001; 276: 29268-29274Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar)), but important details regarding their interaction remain unanswered. For example, it is known that kaps bind directly to phenylalanine (F) side chains in GLFG and FXFG motifs (18Bayliss R. Littlewood T. Stewart M. Structural basis for the interaction between FxFG nucleoporin repeats and importin-beta in nuclear trafficking.Cell. 2000; 102: 99-108Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar, 19Bayliss R. Littlewood T. Strawn L.A. Wente S.R. Stewart M. GLFG and FxFG nucleoporins bind to overlapping sites on importin-beta.J. Biol. Chem. 2002; 277: 50597-50606Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar), but it is unknown whether the adjacent leucine residues in GLFG motifs play a role in binding, or more generally, whether any aromatic AA such as tyrosine or tryptophan can substitute for the phenylalanine residue. It is also unclear how many FG motifs are needed for effective kap–nup binding interactions. Lastly, it is known that Kap95/importin β binds FG motifs at various locations on its surface (18Bayliss R. Littlewood T. Stewart M. Structural basis for the interaction between FxFG nucleoporin repeats and importin-beta in nuclear trafficking.Cell. 2000; 102: 99-108Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar, 19Bayliss R. Littlewood T. Strawn L.A. Wente S.R. Stewart M. GLFG and FxFG nucleoporins bind to overlapping sites on importin-beta.J. Biol. Chem. 2002; 277: 50597-50606Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 20Bednenko J. Cingolani G. Gerace L. Importin beta contains a COOH-terminal nucleoporin binding region important for nuclear transport.J. Cell Biol. 2003; 162: 391-401Crossref PubMed Scopus (115) Google Scholar, 21Isgro T.A. Schulten K. Binding dynamics of isolated nucleoporin repeat regions to importin-beta.Structure. 2005; 13: 1869-1879Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar), but it remains unclear whether the binding is simultaneous and/or cooperative, since only short peptides with one FG motif were generally used in those studies. Native FG domains are typically 150–700 AA long and contain 15–40 FG motifs each (22Denning D.P. Rexach M.F. Rapid evolution exposes the boundaries of domain structure and function in natively unfolded FG nucleoporins.Mol. Cell. Proteomics. 2006; 6: 272-282Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Here, we provide answers to some of these questions using Bead Halo. The contribution of specific AA residues in GLFG motifs to Kap95 binding was analyzed using point mutants in the Nup100 (AA 300–400) and/or Nup116 (AA 348–458) FG domains (Figs. 2A and 4A), where every instance of a target AA was replaced by another residue. For example, in the Nup116 F>A and F>W mutants every phenylalanine in FG motifs was substituted by alanine or tryptophan, respectively. As a positive control, beads coated with the WT FG domains captured Kap95-YFP efficiently, as evidenced by the fluorescent halo around beads and by the absence of fluorescence in the surrounding solution (Fig. 4). Also as expected for the negative control, none of the immobilized GST fusions captured the inert CFP-MBP fusion (see (2Patel S.S. Belmont B.J. Sante J.M. Rexach M.F. Natively unfolded nucleoporins gate protein diffusion across the nuclear pore complex.Cell. 2007; 129: 83-96Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar) and data not shown). Mutation of every phenylalanine to alanine abolished binding of the FG domains to Kap95 (Fig. 4A). This was expected, given the prominent role of the phenylalanine residue in mediating GLFG motif–Kap95 interactions (19Bayliss R. Littlewood T. Strawn L.A. Wente S.R. Stewart M. GLFG and FxFG nucleoporins bind to overlapping sites on importin-beta.J. Biol. Chem. 2002; 277: 50597-50606Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). More interestingly, mutation of every phenylalanine to tryptophan or tyrosine resulted in a reduction of Kap95 binding to the FG domains, but in general, these mutants were still able to bind Kap95 effectively, though at a lower affinity (Fig. 4A). Thus, it appears that any of the three AAs with aromatic side chains (Phe, Tyr, or Trp) can be accommodated in the Kap95 binding pockets. Mutation of all leucine residues to alanine in the GLFG motifs of Nup100 and Nup116 weakened the interaction with Kap95 but did not abolish the binding (Fig. 4A). In essence, the L>A mutation converted the GLFG motifs into degenerate XXFG motifs, which are less hydrophobic than GLFG motifs but appear to be sufficient for kap binding. In contrast, the same L>A mutation completely abolished the interaction between GLFG-rich domains of nups (2Patel S.S. Belmont B.J. Sante J.M. Rexach M.F. Natively unfolded nucleoporins gate protein diffusion across the nuclear pore complex.Cell. 2007; 129: 83-96Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). Yeast FG nups contain a variety of evolut
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