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• W2091386365 abstract "Nuclear transport factor 2 (NTF2) is a small homodimeric protein that interacts simultaneously with both RanGDP and FxFG nucleoporins. The interaction between NTF2 and Ran is essential for the import of Ran into the nucleus. Here we use mutational analysis to dissect the in vivo role of the interaction between NTF2 and nucleoporins. We identify a series of surface residues that form a hydrophobic patch on NTF2, which when mutated disrupt the NTF2-nucleoporin interaction. Analysis of these mutants in vivo demonstrates that the strength of this interaction can be significantly reduced without affecting cell viability. However, cells cease to be viable if the interaction between NTF2 and nucleoporins is abolished completely, indicating that this interaction is essential for the function of NTF2 in vivo. In addition, we have isolated a dominant negative mutant of NTF2, N77Y, which has increased affinity for nucleoporins. Overexpression of the N77Y protein blocks nuclear protein import and concentrates Ran at the nuclear rim. These data support a mechanism in which NTF2 interacts transiently with FxFG nucleoporins to translocate through the pore and import RanGDP into the nucleus. Nuclear transport factor 2 (NTF2) is a small homodimeric protein that interacts simultaneously with both RanGDP and FxFG nucleoporins. The interaction between NTF2 and Ran is essential for the import of Ran into the nucleus. Here we use mutational analysis to dissect the in vivo role of the interaction between NTF2 and nucleoporins. We identify a series of surface residues that form a hydrophobic patch on NTF2, which when mutated disrupt the NTF2-nucleoporin interaction. Analysis of these mutants in vivo demonstrates that the strength of this interaction can be significantly reduced without affecting cell viability. However, cells cease to be viable if the interaction between NTF2 and nucleoporins is abolished completely, indicating that this interaction is essential for the function of NTF2 in vivo. In addition, we have isolated a dominant negative mutant of NTF2, N77Y, which has increased affinity for nucleoporins. Overexpression of the N77Y protein blocks nuclear protein import and concentrates Ran at the nuclear rim. These data support a mechanism in which NTF2 interacts transiently with FxFG nucleoporins to translocate through the pore and import RanGDP into the nucleus. nuclear pore complex nuclear transport factor 2 rat and yeast NTF2 nuclear localization signal green fluorescent protein polymerase chain reaction phosphate-buffered saline with 2.5 mm MgCl2 phosphate-buffered saline with 2.5 mm MgCl2 and 0.5% Triton X-100 monoclonal antibody 414 synthetic dextrose media 5-fluoroorotic acid Nucleocytoplasmic transport occurs through nuclear pore complexes (NPCs),1 large proteinaceous channels that perforate the nuclear membrane. Protein cargoes to be imported into the nucleus contain internal sequences, termed nuclear localization signals (NLS), that target them to the nucleus (1Görlich D. Kutay U. Annu. Rev. Cell Dev. Biol. 1999; 15: 607-660Crossref PubMed Scopus (1676) Google Scholar, 2Adam S.A. Curr. Opin. Cell Biol. 1999; 11: 402-406Crossref PubMed Scopus (77) Google Scholar, 3Bayliss R. Corbett A.H. Stewart M. Traffic. 2000; 1: 448-456Crossref PubMed Scopus (64) Google Scholar). However, cargo does not interact directly with the NPC but is transported bound to soluble transport receptors of the importin-β family of proteins (4Görlich D. EMBO J. 1998; 17: 2721-2727Crossref PubMed Scopus (289) Google Scholar, 5Melchior F. Gerace L. Trends Cell Biol. 1998; 8: 175-179Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). The recent purification and analysis of the yeast NPC indicates that the majority of the ∼40 proteins that make up the NPC, collectively termed nucleoporins, are localized symmetrically throughout the pore complex (6Rout M.P. Aitchison J.D. Suprapto A., K. Hjertaas Zhao Y. Chait B.T. J. Cell Biol. 2000; 148: 635-652Crossref PubMed Scopus (1156) Google Scholar). A subset of these proteins contains a phenylalanine-glycine (FG) repeat motif. These proteins appear to line the nuclear pore channel and can be subdivided further into either GLFG repeat-containing proteins or FxFG proteins (6Rout M.P. Aitchison J.D. Suprapto A., K. Hjertaas Zhao Y. Chait B.T. J. Cell Biol. 2000; 148: 635-652Crossref PubMed Scopus (1156) Google Scholar, 7Rout M.P. Wente S.R. Trends Cell Biol. 1994; 4: 357-365Abstract Full Text PDF PubMed Scopus (247) Google Scholar). Although the precise mechanism by which the transport receptor-cargo complex translocates through the NPC is largely unknown, importin-β family transport receptors have been shown to interact with both the GLFG repeat and the FxFG repeat nucleoporins (8Radu A. Blobel G. Moore M.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1769-1773Crossref PubMed Scopus (386) Google Scholar, 9Rexach M. Blobel G. Cell. 1995; 83: 683-692Abstract Full Text PDF PubMed Scopus (665) Google Scholar, 10Chi N.C. Adam J.H.E. Adam S.A. J. Cell Biol. 1995; 130: 265-274Crossref PubMed Scopus (247) Google Scholar, 11Hu T. Guan T. Gerace L. J. Cell Biol. 1996; 134: 589-601Crossref PubMed Scopus (152) Google Scholar, 12Shah S. Forbes D.J. Curr. Biol. 1998; 8: 1376-1386Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 13Kose S. Imamoto N. Yoneda Y. FEBS Lett. 1999; 463: 327-330Crossref PubMed Scopus (13) Google Scholar, 14Seedorf M. Damelin M. Kahana J. Taura T. Silver P.A. Mol. Cell. Biol. 1999; 19: 1547-1557Crossref PubMed Scopus (118) Google Scholar, 15Kehlenbach R.H. Dickmanns A. Kehlenbach A. Guan T. Gerace L. J. Cell Biol. 1999; 145: 645-657Crossref PubMed Scopus (177) Google Scholar, 16Damelin M. Silver P.A. Mol. Cell. 2000; 5: 133-140Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar) suggesting that these proteins are involved directly in the translocation process. The small GTPase Ran is central to nucleocytoplasmic transport. Although Ran is localized throughout the cell, ∼80% is concentrated within the nucleus (17Moore M.S. J. Biol. Chem. 1998; 273: 22857-22860Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). The Ran guanine nucleotide exchange factor is tethered to DNA in the nucleus (18Ohtsubo M. Okazaki H. Nishimoto T. J. Cell Biol. 1989; 109: 1389-1397Crossref PubMed Scopus (288) Google Scholar, 19Nemergut M.E. Mizzen C.A. Stukenberg T. Allis C.D. Macara I.G. Science. 2001; 292: 1540-1543Crossref PubMed Scopus (190) Google Scholar), suggesting that the majority of nuclear Ran is GTP-bound. On the other hand, RanGAP (Ran GTPase-activating protein) is predominantly cytoplasmic (20Hopper A.K. Traglia H.M. Dunst R.W. J. Cell Biol. 1990; 111: 309-321Crossref PubMed Scopus (167) Google Scholar, 21Melchior F. Weber K. Gerke V. Mol. Biol. Cell. 1993; 4: 569-581Crossref PubMed Scopus (78) Google Scholar, 22Matunis M.J. Coutavas E. Blobel G. J. Cell Biol. 1996; 135: 1457-1470Crossref PubMed Scopus (961) Google Scholar), suggesting that the majority of cytoplasmic Ran is GDP-bound. Thus, the nucleotide bound state of Ran may act as a cellular marker that allows the transport machinery to distinguish between the nuclear and cytoplasmic compartments of the cell. This strict compartmentalization of the Ran effectors also suggests that Ran must shuttle between the nucleus and cytoplasm to undergo a complete round of GTP hydrolysis. It is hypothesized that RanGTP exits the nucleus complexed with importin-β-like transport receptors, and RanGDP is then re-imported into the nucleus by the small homodimeric protein NTF2 (23Ribbeck K. Lippowsky G. Kent H.M. Stewart M. Görlich D. EMBO J. 1998; 17: 6587-6598Crossref PubMed Scopus (360) Google Scholar, 24Smith A. Brownawell A. Macara I.G. Curr. Biol. 1998; 8: 1403-1406Abstract Full Text Full Text PDF PubMed Google Scholar, 25Quimby B.B. Lamtina T. L'Hernault S. Corbett A.H. J. Biol. Chem. 2000; 275: 28575-28582Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar) to replenish the nuclear stores of Ran. NTF2 was first identified as a factor required for efficient import of proteins into the nucleus (26Moore M.S. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10212-10216Crossref PubMed Scopus (291) Google Scholar). Consistent with the role of NTF2 in importing Ran into the nucleus, NTF2 has been shown to interact at non-overlapping sites with both Ran and a subset of nuclear pore proteins containing FxFG repeats (27Clarkson W.D. Kent H.M. Stewart M. J. Mol. Biol. 1996; 263: 517-524Crossref PubMed Scopus (108) Google Scholar). NTF2 specifically interacts with the GDP-bound form of Ran, and this interaction has been extensively characterized through mutational analysis of both NTF2 and Ran (27Clarkson W.D. Kent H.M. Stewart M. J. Mol. Biol. 1996; 263: 517-524Crossref PubMed Scopus (108) Google Scholar, 28Paschal B.M. Gerace L. J. Cell Biol. 1995; 129: 925-937Crossref PubMed Scopus (342) Google Scholar, 29Clarkson W.D. Corbett A.H. Paschal B.M. Kent H.M. McCoy A.J. Gerace L. Silver P.A. Stewart M. J. Mol. Biol. 1997; 272: 716-730Crossref PubMed Scopus (65) Google Scholar, 30Kent H.M. Moore M.S. Quimby B.B. Baker A.M. McCoy A.J. Murphy G.A. Corbett A.H. Stewart M. J. Mol. Biol. 1999; 289: 565-577Crossref PubMed Scopus (15) Google Scholar, 31Quimby B.B. Wilson C.A. Corbett A.H. Mol. Biol. Cell. 2000; 11: 2617-2629Crossref PubMed Scopus (31) Google Scholar) as well as analysis of the NTF2-Ran co-crystal structure (32Stewart M. Kent H. McCoy A. J. Mol. Biol. 1998; 277: 635-646Crossref PubMed Scopus (137) Google Scholar). These studies indicate that the interaction between NTF2 and Ran is required to concentrate Ran in the nucleus and consequently for protein transport between the nucleus and the cytoplasm (33Stewart M. Cell Struct. Funct. 2000; 25: 217-225Crossref PubMed Scopus (35) Google Scholar). The interaction between NTF2 and nucleoporins is predicted to be more complex than the Ran-NTF2 interaction because NTF2 is capable of interacting with multiple FxFG nucleoporins that line the central channel of the pore (34Grote M. Kubitscheck U. Reichelt R. Peters R. J. Cell Sci. 1995; 108: 2963-2972Crossref PubMed Google Scholar). Recent in vitro binding studies indicate that the interaction between NTF2 and nucleoporins is relatively weak (35Bayliss R. Ribbeck K. Akin D. Kent H.M. Feldherr C.M. Görlich D. Stewart M. J. Mol. Biol. 1999; 293: 579-593Crossref PubMed Scopus (151) Google Scholar, 36Chaillan-Huntington C. Braslavsky C.V. Kuhlmann J. Stewart M. J. Biol. Chem. 2000; 275: 5874-5879Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). This suggests a model in which NTF2 transiently interacts with nucleoporins enabling the NTF2-RanGDP complex to move through the pore by hopping from one repeat to another (37Bayliss R. Littlewood T. Stewart M. Cell. 2000; 102: 99-108Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar). If this is the case, one would predict that decreasing or increasing the affinity of NTF2 for FxFG nucleoporins could have a detrimental effect on NTF2 function. However, the lack of an NTF2-FxFG co-crystal structure has made it difficult to engineer mutants crucial to testing this hypothesis. Bayliss et al. (35Bayliss R. Ribbeck K. Akin D. Kent H.M. Feldherr C.M. Görlich D. Stewart M. J. Mol. Biol. 1999; 293: 579-593Crossref PubMed Scopus (151) Google Scholar) utilized the crystal structure of NTF2 bound to RanGDP to predict which residues in NTF2 might be involved in binding to nucleoporins. They identified residue Trp-7 as a potential site for FxFG binding and engineered a mutation in the rat NTF2 protein, W7A NTF2, with a reduced affinity for FxFG nucleoporins. They went on to demonstrate that the W7A NTF2 protein only weakly stimulates nuclear import of RanGDP in vitro. However, a recent study by Ribbeck and Görlich (38Ribbeck K. Görlich D. EMBO J. 2001; 20: 1320-1330Crossref PubMed Scopus (579) Google Scholar) that examined the rate of translocation of both NTF2 and W7R NTF2 showed that W7R NTF2 enters the nucleus quite rapidly compared with a control green fluorescent protein (GFP). This led these authors (38Ribbeck K. Görlich D. EMBO J. 2001; 20: 1320-1330Crossref PubMed Scopus (579) Google Scholar) to suggest that there are other residues in NTF2 in addition to Trp-7 that are critical for mediating the interaction with NPCs. The transport receptor importin-β also interacts with FxFG nucleoporins (8Radu A. Blobel G. Moore M.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1769-1773Crossref PubMed Scopus (386) Google Scholar, 9Rexach M. Blobel G. Cell. 1995; 83: 683-692Abstract Full Text PDF PubMed Scopus (665) Google Scholar, 10Chi N.C. Adam J.H.E. Adam S.A. J. Cell Biol. 1995; 130: 265-274Crossref PubMed Scopus (247) Google Scholar, 11Hu T. Guan T. Gerace L. J. Cell Biol. 1996; 134: 589-601Crossref PubMed Scopus (152) Google Scholar, 12Shah S. Forbes D.J. Curr. Biol. 1998; 8: 1376-1386Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 13Kose S. Imamoto N. Yoneda Y. FEBS Lett. 1999; 463: 327-330Crossref PubMed Scopus (13) Google Scholar, 14Seedorf M. Damelin M. Kahana J. Taura T. Silver P.A. Mol. Cell. Biol. 1999; 19: 1547-1557Crossref PubMed Scopus (118) Google Scholar, 15Kehlenbach R.H. Dickmanns A. Kehlenbach A. Guan T. Gerace L. J. Cell Biol. 1999; 145: 645-657Crossref PubMed Scopus (177) Google Scholar, 16Damelin M. Silver P.A. Mol. Cell. 2000; 5: 133-140Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar) presumably to allow the translocation of importin-β-cargo complexes through the NPC. One could envision that the import of RanGDP by NTF2 via the ability of NTF2 to interact with nucleoporins is analogous to the import of protein cargo by importin-β. If so, the recent co-crystal structure of importin-β bound to five FxFG nucleoporin repeats from Nsp1p (37Bayliss R. Littlewood T. Stewart M. Cell. 2000; 102: 99-108Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar) might give some clues about how NTF2 and FxFG nucleoporins interact. The importin-β-FxFG structure revealed that the phenylalanines of the FxFG repeat cores are buried in a hydrophobic pocket on importin-β, with the phenylalanines of the FxFG repeat core forming stacking interactions with themselves and hydrophobic residues in importin-β. This observation is consistent with the finding that a hydrophobic residue, Trp-7, is involved in the NTF2-FxFG nucleoporin interaction. However, mutations in Trp-7 appear only to weaken and not eliminate the interaction between NTF2 and nucleoporins (35Bayliss R. Ribbeck K. Akin D. Kent H.M. Feldherr C.M. Görlich D. Stewart M. J. Mol. Biol. 1999; 293: 579-593Crossref PubMed Scopus (151) Google Scholar, 38Ribbeck K. Görlich D. EMBO J. 2001; 20: 1320-1330Crossref PubMed Scopus (579) Google Scholar), suggesting that other hydrophobic residues in NTF2 are also involved in this interaction. To fully understand the function of NTF2, it is imperative to define all residues that are critical for NTF2-nucleoporin interactions in vivo. In this study, we have used mutational analysis to probe the interaction between NTF2 and FxFG nucleoporins in vivo. First, we engineered a series of mutant NTF2 proteins that disrupt the interaction between NTF2 and FxFG nucleoporins to varying extents. Our data show that the strength of NTF2 binding to nucleoporins can be reduced significantly without reducing cell viability. However, if this interaction is abolished completely, cells can no longer survive. Second, we isolated a mutant of NTF2 that has an increased affinity for FxFG nucleoporins. This mutation renders the NTF2 protein nonfunctionalin vivo, and overexpression of this mutant protein blocks NLS-mediated protein import. Together, these results support a model in which the interaction between NTF2 and FxFG nucleoporins occurs at a sufficiently low affinity to enable NTF2 to move freely from one FxFG repeat to another as it translocates through the pore complex. Furthermore, our data demonstrate that the fine-tuned interaction between NTF2 and FxFG nucleoporins is critical in vivo. All chemicals were obtained from Sigma or United States Biological unless otherwise noted. All DNA manipulations were performed according to standard methods (39Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar), and all media were prepared by standard procedures (40Rose M.D. Winston F. Hieter P. Methods in Yeast Genetics: A Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1990Google Scholar). All plasmids used in this study are described in Table I. The wild-type (PSY580) and NTF2 deletion strains (ACY114) used in this study have been described (41Corbett A.H. Silver P.A. J. Biol. Chem. 1996; 271: 18477-18484Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar).Table IPlasmids usedPlasmidDescriptionpAC18GAL1–10, 2µ, URA3, amppAC78GSP1,CEN, TRP1, amppAC82NTF2, GAL1–10, 2µ, URA3, amppAC117NTF2,CEN, LEU2, amppAC130ntf2D21A, BluescriptpAC149ntf2D21A, GAL1–10, 2µ, URA3, amppAC150ntf2D21A, CEN, LEU2, amppAC163ntf2N77Y,CEN, LEU2, amppAC197ntf2N77Y-GFP, 2µ, URA3, amppAC240NTF2, BluescriptpAC253ntf2N77Y, GAL1–10, 2µ, URA3, amppAC267ntf2F5A, BluescriptpAC345ntf2F5A, CEN, LEU2, amppAC410GSP1-GFP,2µ, URA3, amppAC411ntf2F5A-GFP, 2µ, URA3, amppAC420S. cerevisiaeNtf2pF5A bacterial expression vectorpAC626NTF2, CEN, URA3, amppAC627myc-GSP1, CEN, TRP1, amppAC628S. cerevisiaeNtf2p bacterial expression vector (55Wong D.H. Corbett A.H. Kent H.M. Stewart M. Silver P.A. Mol. Cell Biol. 1997; 17: 3755-3767Crossref PubMed Scopus (90) Google Scholar)pAC697pADH-NLS-GAL4AD-GFP, 2µ, LEU2 (42)pAC709NTF2-GFP, 2µ, URA3, amppAC715YRB, CEN, TRP, amppAC738ntf2Y112A, CEN, LEU2, amppAC739ntf2Y112D, CEN, LEU2, amppAC744ntf2Y112A, BluescriptpAC745ntf2Y112D, BluescriptpAC746Δ6ntf2, CEN, LEU2, amppAC750S. cerevisiaeNtf2pY112A bacterial expression vectorpAC751S. cerevisiae Ntf2pY112D bacterial expression vectorpAC760ntf2F5A/Y112A, CEN, LEU2, amppAC792pADH-NLS-GAL4AD-GFP, 2µ, LEU2(42)pAC797ntf2Y112A-GFP, 2µ, URA3, amppAC798ntf2Y112D-GFP, 2µ, URA3, amppAC799ntf2F5A/Y112A-GFP, 2µ, URA3, amppAC812S. cerevisiaeNtf2pF5A/Y112A bacterial expression vectorpAC814ntf2D21A-GFP, 2µ, URA3, amppAC821S. cerevisiaeNtf2pN77Y bacterial expression vectorpAC913S. cerevisiae Δ6Ntf2p bacterial expression vectorpAC914Δ6Ntf2p-GFP, 2µ, URA3, amp Open table in a new tab Site-directed mutagenesis was performed on pBS-NTF2 (pAC240) using the QuickChange PCR-based mutagenesis kit from Stratagene (La Jolla, CA). Mutated sequences were confirmed by DNA sequencing. Δ6NTF2 was made using a PCR-based strategy. The NTF2 promoter was amplified from pAC117 with 5′-GGC ACC GGT CAT TAT AAA GAT AAT AGT ATT AAA ACC-3′ and 5′-GCG AGC TCC CCT TTC ATA TTG TTC GGC TA-3′ as primers. The resulting PCR product was digested with AgeI and SacI and subcloned into pAC715 (pRS314YRB1) digested with AgeI andSacI. The Ntf2p coding region from Gln-10 to the stop codon including the 3′-untranslated region was amplified from pAC117 with 5′-GCG ACC GGT CAA AAC TTC ACC CAG TTT TAC TA-3′ and 5′-CCC TCG AGC GCT ATC GCC TTA TAC ATC G-3′ as primers. The resulting PCR product was digested with AgeI and XhoI and subcloned into pAC715 containing the NTF2 promoter digested withAgeI and XhoI. The six-amino acid deletion was confirmed by DNA sequencing then subcloned into the SacI andXhoI sites of pPS315 (CEN, LEUplasmid). Wild-type and mutant Ntf2p-GFP fusion proteins were transformed into the NTF2 deletion strain, ACY114, maintained by a wild-type copy of GSP1 (scRan) (pAC78). scRan-GFP was transformed into ACY114 expressing each of the mutant alleles of NTF2 as the only copy of NTF2. The GFP fusion proteins were localized by viewing the GFP signal directly in living cells through a GFP-optimized filter (Chroma Technology) using an Olympus BX60 epifluorescence microscope equipped with a Photometrics Quantix digital camera. The NLS-GFP import assay was performed as described previously (42Shulga N. Roberts P. Gu Z. Spitz L. Tabb M.M. Nomura M. Goldfarb D.S. J. Cell Biol. 1996; 135: 329-339Crossref PubMed Scopus (186) Google Scholar). Briefly, cells were grown to early mid-log phase in synthetic media containing 2% glucose (w/v) at 25 °C, pelleted, resuspended in 1 ml of 10 mm sodium azide, 10 mm 2-deoxy-d-glucose in glucose-free synthetic media, and incubated at 25 °C for 45 min. The cells were then pelleted, washed with 1 ml of ice-cold ddH20, repelleted, resuspended in 100 µl of glucose-containing synthetic media prewarmed to 37 °C, and incubated at 37 °C. For scoring, 2-µl samples were removed every 2.5 min, and cells were observed and counted through a GFP-optimized filter (Chroma Technology) using an Olympus BX60 epifluorescence microscope. Cells were scored as “nuclear” if the nucleus was both brighter than the surrounding cytoplasm and a nuclear-cytoplasmic boundary was visible. At least 100 cells were counted at each time point. The in vivo function of each of the Ntf2 mutant proteins was tested by using a plasmid shuffle technique. The NTF2deletion strain, ACY114, was transformed with plasmids encoding each of the Ntf2 mutant proteins. Single transformants were grown in liquid culture to saturation. The saturated cultures were serially diluted (1:10) and spotted on fluoroorotic acid (5-FOA) plates to eliminate the URA3 plasmid-encoded wild-type Ntf2p (43Boeke J.D. Truehart J. Natsoulis G. Fink G. Methods Enzymol. 1987; 154: 164-175Crossref PubMed Scopus (1083) Google Scholar). This results in cells that express each of the mutant Ntf2 proteins on a low copy centromeric plasmid as the sole copy of Ntf2p. All Ntf2 proteins were purified from Escherichia coliessentially as described (25Quimby B.B. Lamtina T. L'Hernault S. Corbett A.H. J. Biol. Chem. 2000; 275: 28575-28582Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar) with the following changes. After lysis and clarification, E. coli lysates were applied to DEAE ion exchange column pre-equilibrated with 10 mm Tris-HCl, pH 8.0. Ntf2p was eluted from the column with a gradient of 0 to 1m NaCl. Fractions containing Ntf2p were pooled, concentrated using a Centriprep-10 (Amicon) concentrator, and applied to a column of Sephacryl SR100 pre-equilibrated in 20 mmTris-HCl, pH 7.5. Fractions containing purified Ntf2p were collected, pooled, and stored at −80 °C in 10% glycerol. Purified Ntf2 proteins were cross-linked to cyanogen bromide-Sepharose beads as described previously (25Quimby B.B. Lamtina T. L'Hernault S. Corbett A.H. J. Biol. Chem. 2000; 275: 28575-28582Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Yeast cell extracts were prepared as described (25Quimby B.B. Lamtina T. L'Hernault S. Corbett A.H. J. Biol. Chem. 2000; 275: 28575-28582Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Two mg of BQY65 (MATα ura3–52 trp1Δ63 leu2Δ1 his3Δ200 GSP1Δ::HIS3 GSP2Δ::HIS3 myc-GSP1) cell extract was incubated with 50 µl of Ntf2p-Sepharose beads. For the competition experiment, either 300 µg of wild-type Ntf2p or 300 µg of bovine serum albumin was added in addition to the yeast cell extract. Binding was carried out in PBSM (PBS, 2.5 mmMgCl2) in a total volume of 500 µl at 4 °C for 1 h. Following the binding, the beads were pelleted, and unbound lysate was removed from the beads. This fraction was designated the unbound sample, and 20 µg of total protein, corresponding to 1% of the total unbound volume, was analyzed. After the unbound sample was removed, beads were washed once for 10 min in 1 ml of PBSM and twice for 10 min in PBSMT (PBS, 2.5 mm MgCl2, 0.5% Triton X-100). The beads that contained the bound fraction were then boiled in sample loading buffer (100 µl total volume), 10% of the total bound proteins (10 µl) was loaded onto an SDS-polyacrylamide gel. Because the bound proteins were eluted directly into sample buffer, it was impossible to quantitate the total amount of bound sample. However, within each experiment the percentage of total bound protein loaded was equal, so direct comparisons could be made between each of the bound lanes within each experiment. This is also the case for the unbound samples. Samples (bound and unbound) were resolved by polyacrylamide gel electrophoresis, and transferred to nitrocellulose for immunoblotting. For many of the experiments some low level of nonspecific or low affinity binding of Nup2p to the beads (whether they were charged with Ntf2 protein or not) was observed. This Nup2p was readily washed from the beads by the PBSM/PBSMT washes used in our protocol and could be recovered in a wash fraction by trichloroacetic acid precipitation (data not shown). This means that in some experiments, the amount of total protein (summing the unbound and bound lanes) is not equal despite the fact that the same lysates with identical amounts of Nup2p were used for each sample within the experiment. For all experiments the amount of unbound (1% of total unbound volume) and bound (10% of total bound volume) sample loaded in each lane was identical, and therefore direct comparisons can be made between the bound lanes shown for all samples and between the unbound lanes shown for all samples. Immunoblot analysis was performed essentially as described (44Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44939) Google Scholar). Ntf2p-GFP was detected by incubation with 1:10,000 dilution of rabbit anti-GFP polyclonal antibody. Nup2p was detected by incubation with 1:3000 dilution of mAb414 (Babco). Myc-Gsp1p was detected with 1:500 dilution of mouse anti-Myc monoclonal antibody (Santa Cruz Biotechnology). Dominant negative alleles ofNTF2 were generated using random PCR mutagenesis. A 4.0-kilobase pair PCR product that included the entireNTF2 open reading frame was generated using Taqpolymerase under mutagenic conditions (7 mmMgCl2) and purified on a Qiaquick PCR column from Qiagen (Chatsworth, CA). The plasmid pAC82 was linearized withBamHI and SalI to remove a 475-base pair insert containing the coding region of NTF2. Gap repair ligation (56Orr-Weaver T.L. Szostak J.W. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 4417-4421Crossref PubMed Scopus (307) Google Scholar) was achieved by co-transforming the wild-type strain PSY580 with the PCR product and linearized the GAL1-NTF2 plasmid (pAC82). Colonies were selected on synthetic complete media plus 2% (w/v) glucose lacking leucine (SD −Leu). The plates were then replica plated to synthetic complete media plus 2% galactose (w/v) lacking leucine (Sgal −Leu) supplemented with 7.5 mg of the vital dye erythrocin B/liter and incubated at 30 °C. Addition of galactose to the media initiates the overexpression of the plasmid encoded Ntf2 protein. Uptake of the erythrocin B dye by dead cells, which results in a pink colony, was used to identify colonies that died upon overexpression of the Ntf2 protein. Plasmids were rescued from dominant negative colonies and retransformed into wild-type cells (PSY580) to confirm plasmid linkage and the dominant negative phenotype. Plasmids that retested were sequenced to identify the mutations. Cells were grown in SD −Leu overnight at 30 °C, diluted 1:200 into synthetic complete media supplemented with 2% raffinose (w/v) lacking leucine, grown at 30 °C to ∼107 cells/ml, and induced with 2% galactose (w/v). Cells were counted every 2 h. Two hundred cells were plated at each time point on SD −Leu media and incubated for 2 days at 30 °C. Viability was determined by counting the number of colonies that arose from the 200 cells plated. As the two phenylalanines in the FxFG repeat core have a very hydrophobic nature, one would anticipate that their binding site on NTF2 would also be hydrophobic, analogous to the hydrophobic FxFG binding site identified on importin-β (37Bayliss R. Littlewood T. Stewart M. Cell. 2000; 102: 99-108Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar). Previous work consistent with this hypothesis has shown that residue Trp-7 of rNTF2 contributes to the interaction between rNTF2 and FxFG nucleoporins (35Bayliss R. Ribbeck K. Akin D. Kent H.M. Feldherr C.M. Görlich D. Stewart M. J. Mol. Biol. 1999; 293: 579-593Crossref PubMed Scopus (151) Google Scholar) and that mutation of Trp-7 of rNTF2 to alanine reduces binding to FxFG nucleoporins (35Bayliss R. Ribbeck K. Akin D. Kent H.M. Feldherr C.M. Görlich D. Stewart M. J. Mol. Biol. 1999; 293: 579-593Crossref PubMed Scopus (151) Google Scholar). Moreover, in crystal structures of both wild-type and mutant rNTF2 (35Bayliss R. Ribbeck K. Akin D. Kent H.M. Feldherr C.M. Görlich D. Stewart M. J. Mol. Biol. 1999; 293: 579-593Crossref PubMed Scopus (151) Google Scholar, 45Bullock T.L. Clarkson W.D. Kent H.M. Stewart M. J. Mol. Biol. 1996; 260: 422-431Crossref PubMed Scopus (119) Google Scholar), the hydrophobic patch surrounding Trp-7 is often involved in hydrophobic crystal contacts between adjacent molecules in the crystal lattice (29Clarkson W.D. Corbett A.H. Paschal B.M. Kent H.M. McCoy A.J. Gerace L. Silver P.A. Stewart M. J. Mol. Biol. 1997; 272: 716-730Crossref PubMed Scopus (65) Google Scholar, 45Bullock T.L. Clarkson W.D. Kent H.M. Stewart M. J. Mol. Biol. 1996; 260: 422-431Crossref PubMed Scopus (119) Google Scholar). In particular, close interactions between the aromatic ring of Phe-126 and both Trp-7 and Trp-112 are found in the crystal packing of the W7A-rNTF2 mutant (35Bayliss R. Ribbeck K. Akin D. Kent H.M. Feldherr C.M. Görlich D. Stewart M. J. Mol. Biol. 1999; 293: 579-593Crossref PubMed Scopus (151) Google Scholar). Although these two residues are no" @default.
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• W2091386365 date "2001-10-01" @default.
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• W2091386365 title "Functional Analysis of the Hydrophobic Patch on Nuclear Transport Factor 2 Involved in Interactions with the Nuclear Porein Vivo" @default.
• W2091386365 cites W1559808711 @default.
• W2091386365 cites W1588701970 @default.
• W2091386365 cites W1830127169 @default.
• W2091386365 cites W1963846953 @default.
• W2091386365 cites W1968433691 @default.
• W2091386365 cites W1968863167 @default.
• W2091386365 cites W1968872346 @default.
• W2091386365 cites W1985732160 @default.
• W2091386365 cites W1987945169 @default.
• W2091386365 cites W1994512535 @default.
• W2091386365 cites W1996381709 @default.
• W2091386365 cites W1998883267 @default.
• W2091386365 cites W1999921567 @default.
• W2091386365 cites W2002017977 @default.
• W2091386365 cites W2008984155 @default.
• W2091386365 cites W2011428210 @default.
• W2091386365 cites W2021384128 @default.
• W2091386365 cites W2022864103 @default.
• W2091386365 cites W2026340100 @default.
• W2091386365 cites W2026388719 @default.
• W2091386365 cites W2027810400 @default.
• W2091386365 cites W2048912550 @default.
• W2091386365 cites W2053520952 @default.
• W2091386365 cites W2057414105 @default.
• W2091386365 cites W2058041522 @default.
• W2091386365 cites W2061365312 @default.
• W2091386365 cites W2063812024 @default.
• W2091386365 cites W2071957053 @default.
• W2091386365 cites W2072142876 @default.
• W2091386365 cites W2074819879 @default.
• W2091386365 cites W2075791686 @default.
• W2091386365 cites W2079990775 @default.
• W2091386365 cites W2081343860 @default.
• W2091386365 cites W2082072187 @default.
• W2091386365 cites W2082809022 @default.
• W2091386365 cites W2083964225 @default.
• W2091386365 cites W2084316792 @default.
• W2091386365 cites W2094420612 @default.
• W2091386365 cites W2099282845 @default.
• W2091386365 cites W2101108802 @default.
• W2091386365 cites W2105987719 @default.
• W2091386365 cites W2107057369 @default.
• W2091386365 cites W2111674631 @default.
• W2091386365 cites W2125761409 @default.
• W2091386365 cites W2132320427 @default.
• W2091386365 cites W2132672808 @default.
• W2091386365 cites W2133201628 @default.
• W2091386365 cites W2136710801 @default.
• W2091386365 cites W2146399969 @default.
• W2091386365 cites W2162226086 @default.
• W2091386365 cites W2165306451 @default.
• W2091386365 cites W2171788412 @default.
• W2091386365 cites W2172298521 @default.
• W2091386365 cites W4211020343 @default.
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