FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2023521597> ?p ?o ?g. }

• W2023521597 endingPage "6452" @default.
• W2023521597 startingPage "6445" @default.
• W2023521597 abstract "Our previous studies have focused on a family ofSaccharomyces cerevisiae nuclear pore complex (NPC) proteins that contain domains composed of repetitive tetrapeptide glycine-leucine-phenylalanine-glycine (GLFG) motifs. We have previously shown that the GLFG regions of Nup116p and Nup100p directly bind the karyopherin transport factor Kap95p during nuclear protein import. In this report, we have further investigated potential roles for the GLFG region in mRNA export. The subcellular localizations of green fluorescent protein (GFP)-tagged mRNA transport factors were individually examined in yeast cells overexpressing the Nup116-GLFG region. The essential mRNA export factors Mex67-GFP, Mtr2-GFP, and Dbp5-GFP accumulated in the nucleus. In contrast, the localizations of Gle1-GFP and Gle2-GFP remained predominantly associated with the NPC, as in wild type cells. The localization of Kap95p was also not perturbed with GLFG overexpression. Coimmunoprecipitation experiments from yeast cell lysates resulted in the isolation of a Mex67p-Nup116p complex. Soluble binding assays with bacterially expressed recombinant proteins confirmed a direct interaction between Mex67p and the Nup116-GLFG or Nup100-GLFG regions. Mtr2p was not required for in vitro binding of Mex67p to the GLFG region. To map the Nup116-GLFG subregion(s) required for Kap95p and/or Mex67p association, yeast two-hybrid analysis was used. Of the 33 Nup116-GLFG repeats that compose the domain, a central subregion of nine GLFG repeats was sufficient for binding either Kap95p or Mex67p. Interestingly, the first 12 repeats from the full-length region only had a positive interaction with Mex67p, whereas the last 12 were only positive with Kap95p. Thus, the GLFG domain may have the capacity to bind both karyopherins and an mRNA export factor simultaneously. Taken together, our in vivo and in vitro results define an essential role for a direct Mex67p-GLFG interaction during mRNA export. Our previous studies have focused on a family ofSaccharomyces cerevisiae nuclear pore complex (NPC) proteins that contain domains composed of repetitive tetrapeptide glycine-leucine-phenylalanine-glycine (GLFG) motifs. We have previously shown that the GLFG regions of Nup116p and Nup100p directly bind the karyopherin transport factor Kap95p during nuclear protein import. In this report, we have further investigated potential roles for the GLFG region in mRNA export. The subcellular localizations of green fluorescent protein (GFP)-tagged mRNA transport factors were individually examined in yeast cells overexpressing the Nup116-GLFG region. The essential mRNA export factors Mex67-GFP, Mtr2-GFP, and Dbp5-GFP accumulated in the nucleus. In contrast, the localizations of Gle1-GFP and Gle2-GFP remained predominantly associated with the NPC, as in wild type cells. The localization of Kap95p was also not perturbed with GLFG overexpression. Coimmunoprecipitation experiments from yeast cell lysates resulted in the isolation of a Mex67p-Nup116p complex. Soluble binding assays with bacterially expressed recombinant proteins confirmed a direct interaction between Mex67p and the Nup116-GLFG or Nup100-GLFG regions. Mtr2p was not required for in vitro binding of Mex67p to the GLFG region. To map the Nup116-GLFG subregion(s) required for Kap95p and/or Mex67p association, yeast two-hybrid analysis was used. Of the 33 Nup116-GLFG repeats that compose the domain, a central subregion of nine GLFG repeats was sufficient for binding either Kap95p or Mex67p. Interestingly, the first 12 repeats from the full-length region only had a positive interaction with Mex67p, whereas the last 12 were only positive with Kap95p. Thus, the GLFG domain may have the capacity to bind both karyopherins and an mRNA export factor simultaneously. Taken together, our in vivo and in vitro results define an essential role for a direct Mex67p-GLFG interaction during mRNA export. nuclear pore complex phenylalanine-glycine GAL4transcriptional activation domain GAL4DNA binding domain green fluorescent protein glycine-leucine-phenylalanine-glycine glutathioneS-transferase LexA DNA binding domain, MBP, maltose-binding protein synthetic complete polyacrylamide gel electrophoresis dithiothreitol To move between the nuclear and cytoplasmic compartments of a eukaryotic cell, all molecules must pass through nuclear pore complexes (NPCs)1 embedded in the nuclear envelope. Ions, metabolites, and small proteins may diffuse through an ∼9-nm aqueous channel in the NPC. In contrast, the movement of large macromolecules, including proteins and RNA, is energy-dependent and facilitated (reviewed in Refs. 1Talcott B. Moore M.S. Trends Cell Biol. 1999; 9: 312-318Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 2Nakielny S. Dreyfuss G. Cell. 1999; 99: 677-690Abstract Full Text Full Text PDF PubMed Scopus (655) Google Scholar, 3Mattaj I.W. Englmeier L. Annu. Rev. Biochem. 1998; 67: 265-306Crossref PubMed Scopus (1011) Google Scholar). The central channel of the proteinaceous NPC is formed by a symmetrical, 8-fold assembly of spoke-like structures sandwiched between nuclear and cytoplasmic rings. Distinct filamentous structures extend from these rings on both the nuclear and cytoplasmic faces, with the filaments on the nuclear side culminating in a basket-like structure. Overall, a vertebrate NPC measures ∼200 nm from the tips of the cytoplasmic filaments to the base of the nucleoplasmic basket (4Stoffler D. Fahrenkrog B. Aebi U. Curr. Opin. Cell Biol. 1999; 11: 391-401Crossref PubMed Scopus (299) Google Scholar, 5Yang Q. Rout M.P. Akey C.W. Mol. Cell. 1998; 1: 223-234Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar). Unraveling the mechanism for active transport between the cytoplasmic and nucleoplasmic NPC faces will require an in-depth understanding of both the NPC itself and soluble transport factors. The Saccharomyces cerevisiae NPC is built by the oligomerization of over 30 different polypeptides collectively referred to as nucleoporins (4Stoffler D. Fahrenkrog B. Aebi U. Curr. Opin. Cell Biol. 1999; 11: 391-401Crossref PubMed Scopus (299) Google Scholar, 6Rout M.P. Aitchison J.D. Suprapto A. Hjertaas K. Zhao Y. Chait B.T. J. Cell Biol. 2000; 148: 635-651Crossref PubMed Scopus (1155) Google Scholar, 7Doye V. Hurt E. Curr. Opin. Cell Biol. 1997; 9: 401-411Crossref PubMed Scopus (213) Google Scholar). This includes three integral membrane proteins, a subset of proteins with predicted coiled-coil domains, several proteins with novel primary structure, and a family of 13 nucleoporins with phenylalanine-glycine (FG) repeat domains. All FG repeat domains share the common feature of multiple FG dipeptide repeats with variable length spacers (8Rout M.P. Wente S.R. Trends Cell Biol. 1994; 4: 357-365Abstract Full Text PDF PubMed Scopus (247) Google Scholar). However, there are at least two distinct subclasses: 1) glycine-leucine-phenylalanine-glycine (GLFG) repeat domains, which are separated by spacers that lack acidic residues and are enriched in serine, threonine, glutamine, and asparagine residues, and 2) phenylalanine-any amino acid-phenylalanine-glycine (FXFG) repeat domains, which have charged spacers. In vertebrates and yeast, different FG nucleoporins reside in each of the NPC substructures, and there is significant evidence for a direct involvement of FG nucleoporins in both nuclear import and export (4Stoffler D. Fahrenkrog B. Aebi U. Curr. Opin. Cell Biol. 1999; 11: 391-401Crossref PubMed Scopus (299) Google Scholar, 6Rout M.P. Aitchison J.D. Suprapto A. Hjertaas K. Zhao Y. Chait B.T. J. Cell Biol. 2000; 148: 635-651Crossref PubMed Scopus (1155) Google Scholar, 9Ryan K.J. Wente S.R. Curr. Opin. Cell Biol. 2000; 12: 361-371Crossref PubMed Scopus (214) Google Scholar). Recently, several reports have documented in vitrobiochemical interactions between shuttling transport factors and individual components of the NPC (reviewed in Ref. 9Ryan K.J. Wente S.R. Curr. Opin. Cell Biol. 2000; 12: 361-371Crossref PubMed Scopus (214) Google Scholar). In particular, there has been considerable focus on the interactions between FG repeat nucleoporins and karyopherins (also known as importins/exportins). Karyopherins are a family of shuttling transport factors that recognize specific nuclear import or export signals in their respective cargo (reviewed in Refs. 2Nakielny S. Dreyfuss G. Cell. 1999; 99: 677-690Abstract Full Text Full Text PDF PubMed Scopus (655) Google Scholar, 3Mattaj I.W. Englmeier L. Annu. Rev. Biochem. 1998; 67: 265-306Crossref PubMed Scopus (1011) Google Scholar, 10Pemberton L.F. Blobel G. Rosenblum J.S. Curr. Opin. Cell Biol. 1998; 10: 392-399Crossref PubMed Scopus (213) Google Scholar, and 11Adam S.A. Curr. Opin. Cell Biol. 1999; 11: 402-406Crossref PubMed Scopus (77) Google Scholar). All yeast FG nucleoporins have been shown to bind at least one karyopherin in vitro(reviewed in Ref. 9Ryan K.J. Wente S.R. Curr. Opin. Cell Biol. 2000; 12: 361-371Crossref PubMed Scopus (214) Google Scholar). Current models for the mechanism of docking and translocation through the NPC are based on direct karyopherin-nucleoporin binding. Karyopherins also interact with the small GTPase Ran, which acts as a molecular switch regulating the association of karyopherins with their cargo and the NPC (2Nakielny S. Dreyfuss G. Cell. 1999; 99: 677-690Abstract Full Text Full Text PDF PubMed Scopus (655) Google Scholar, 12Gorlich D. Kutay U. Annu. Rev. Cell Dev. Biol. 1999; 15: 607-660Crossref PubMed Scopus (1676) Google Scholar). The crystal structures of different karyopherins (or karyopherin domains) complexed with either RanGTP or a FXFG nucleoporin were recently reported (13Bayliss R. Littlewood T. Stewart M. Cell. 2000; 102: 99-108Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar, 14Chook Y.M. Blobel G. Nature. 1999; 399: 230-237Crossref PubMed Scopus (294) Google Scholar, 15Vetter I.R. Arndt A. Kutay U. Gorlich D. Wittinghofer A. Cell. 1999; 97: 635-646Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar). A comparison of the structure of importin-β when complexed with either RanGTP or an FXFG repeat region has suggested binding of RanGTP to importin-β may induce a conformational change in importin-β that occludes the FXFG repeat binding site. This would release importin-β from the nucleoporin. However, it is unclear if such a mechanism contributes to vectoral movement along the central axis of the NPC or to a terminal release step from the NPC. Several factors with specific roles in mRNA export have also been shown to physically or genetically interact with nucleoporins. For example, in S. cerevisiae, the RNA helicase Dbp5p directly associates with Nup159p (16Schmitt C. Von Kobbe C. Bachi A. Pante N. Rodrigues J.P. Boscheron C. Rigaut G. Wilm M. Seraphin B. Carmo-Fonseca M. Izaurralde E. EMBO J. 1999; 18: 4332-4347Crossref PubMed Scopus (225) Google Scholar). The mRNA export factor Gle2p physically associates with Nup116p (17Ho A.K. Raczniak G.A. Ives E.B. Wente S.R. Mol. Biol. Cell. 1998; 9: 355-373Crossref PubMed Scopus (35) Google Scholar, 18Bailer S.M. Siniossoglou S. Podtelejnikov A. Hellwig A. Mann M. Hurt E. EMBO J. 1998; 17: 1107-1119Crossref PubMed Scopus (117) Google Scholar), and gle2mutants are synthetically lethal with a nup100 null mutant (19Murphy R. Watkins J.L. Wente S.R. Mol. Biol. Cell. 1996; 7: 1921-1937Crossref PubMed Scopus (151) Google Scholar). The essential factor Gle1p interacts with Dbp5p (20Hodge C.A. Colot H.V. Stafford P. Cole C.N. EMBO J. 1999; 18: 5778-5788Crossref PubMed Scopus (162) Google Scholar), andgle1 mutants are synthetically lethal with eitherrip1/nup42 or nup100 null mutants (19Murphy R. Watkins J.L. Wente S.R. Mol. Biol. Cell. 1996; 7: 1921-1937Crossref PubMed Scopus (151) Google Scholar, 21Stutz F. Kantor J. Zhang D. McCarthy T. Neville M. Rosbash M. Genes Dev. 1997; 11: 2857-2868Crossref PubMed Scopus (84) Google Scholar). The essential mRNA export factor Mex67p forms a heterodimeric complex with Mtr2p, and Mtr2p mediates association with Nup85p (22Santos-Rosa H. Moreno H. Simos G. Segref A. Fahrenkrog B. Pante N. Hurt E. Mol. Cell. Biol. 1998; 18: 6826-6838Crossref PubMed Scopus (223) Google Scholar). The vertebrate Mex67p homologue, TAP, directly associates with vertebrate Nup214p, Nup98p, and hCG1 (23Bachi A. Braun I.C. Rodriques J.P. Pante N. Ribbeck K. Von Kobbe C. Kutay U. Wilm M. Görlich D. Carmo-Fonseca M. Izaurralde E. RNA (NY). 2000; 6: 136-158Crossref PubMed Scopus (275) Google Scholar, 24Katahira J. Sträßer K. Podtelejnikov A. Mann M. Jung J.U. Hurt E. EMBO J. 1999; 18: 2593-2609Crossref PubMed Scopus (344) Google Scholar). Interestingly, all of these nucleoporins, except Nup85p, are FG family members. Taken together, it is intriguing that both karyopherins and mRNA export factors interact with FG nucleoporins. Although much progress has been made in defining interactions between soluble transport factors and nucleoporins, the exact molecular mechanisms for translocation through the NPC are still unknown. To reveal critical events that mediate NPC translocation, we have analyzed the interface between dynamic transport factors and two homologous components of the S. cerevisiae NPC, the GLFG nucleoporins Nup116p and Nup100p. Nup116p and Nup100p are localized on both sides of the NPC (6Rout M.P. Aitchison J.D. Suprapto A. Hjertaas K. Zhao Y. Chait B.T. J. Cell Biol. 2000; 148: 635-651Crossref PubMed Scopus (1155) Google Scholar, 25Ho A.K. Shen T. Ryan K.J. Kiseleva E. Aach Levy M. Allen T.D. Wente S.R. Mol. Cell. Biol. 2000; 20: 5736-5748Crossref PubMed Scopus (40) Google Scholar), and their C-terminal regions interact with Nup82p (25Ho A.K. Shen T. Ryan K.J. Kiseleva E. Aach Levy M. Allen T.D. Wente S.R. Mol. Cell. Biol. 2000; 20: 5736-5748Crossref PubMed Scopus (40) Google Scholar). Nup82p plays a key role in the localization of Nup116p at the cytoplasmic face of the NPC. Our previous studies have documented anin vivo requirement for Nup116p and Nup100p in nuclear transport (19Murphy R. Watkins J.L. Wente S.R. Mol. Biol. Cell. 1996; 7: 1921-1937Crossref PubMed Scopus (151) Google Scholar, 26Wente S.R. Blobel G. J. Cell Biol. 1993; 123: 275-284Crossref PubMed Scopus (169) Google Scholar, 27Iovine M.K. Watkins J.L. Wente S.R. J. Cell Biol. 1995; 131: 1699-1713Crossref PubMed Scopus (169) Google Scholar, 28Iovine M.K. Wente S.R. J. Cell Biol. 1997; 137: 797-811Crossref PubMed Scopus (72) Google Scholar, 29Murphy R. Wente S. Nature. 1996; 383: 357-360Crossref PubMed Scopus (204) Google Scholar). The unique N-terminal region of Nup116p binds the mRNA export factor Gle2p (17Ho A.K. Raczniak G.A. Ives E.B. Wente S.R. Mol. Biol. Cell. 1998; 9: 355-373Crossref PubMed Scopus (35) Google Scholar, 18Bailer S.M. Siniossoglou S. Podtelejnikov A. Hellwig A. Mann M. Hurt E. EMBO J. 1998; 17: 1107-1119Crossref PubMed Scopus (117) Google Scholar), whereas the GLFG regions of Nup116p and Nup100p directly bind Kap95p (27Iovine M.K. Watkins J.L. Wente S.R. J. Cell Biol. 1995; 131: 1699-1713Crossref PubMed Scopus (169) Google Scholar, 28Iovine M.K. Wente S.R. J. Cell Biol. 1997; 137: 797-811Crossref PubMed Scopus (72) Google Scholar) and other karyopherins (20Hodge C.A. Colot H.V. Stafford P. Cole C.N. EMBO J. 1999; 18: 5778-5788Crossref PubMed Scopus (162) Google Scholar, 30Aitchison J.D. Blobel G. Rout M.P. Science. 1996; 274: 624-627Crossref PubMed Scopus (271) Google Scholar, 31Hellmuth K. Lau D.M. Bischoff F.R. Künzler M. Hurt E. Simos G. Mol. Cell. Biol. 1998; 18: 6374-6386Crossref PubMed Scopus (206) Google Scholar, 32Rout M.P. Blobel G. Aitchison J.D. Cell. 1997; 89: 715-725Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, 33Marelli M. Aitchison J.D. Wozniak R.W. J. Cell Biol. 1998; 143: 1813-1830Crossref PubMed Scopus (133) Google Scholar, 34Damelin M. Silver P.A. Mol. Cell. 2000; 5: 133-140Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 35Seedorf M. Damelin M. Kahana J. Taura T. Silver P.A. Mol. Cell. Biol. 1999; 19: 1547-1557Crossref PubMed Scopus (118) Google Scholar). Interestingly, overexpression of the Nup116-GLFG region inhibits mRNA export and cell growth (27Iovine M.K. Watkins J.L. Wente S.R. J. Cell Biol. 1995; 131: 1699-1713Crossref PubMed Scopus (169) Google Scholar). This suggests an essential role for the GLFG region in mRNA export. However, a direct role for karyopherins in mRNA export has not been documented. We have found that the Nup116p and Nup100p GLFG regions interact in vivo and in vitro with both the karyopherin Kap95p (27Iovine M.K. Watkins J.L. Wente S.R. J. Cell Biol. 1995; 131: 1699-1713Crossref PubMed Scopus (169) Google Scholar, 28Iovine M.K. Wente S.R. J. Cell Biol. 1997; 137: 797-811Crossref PubMed Scopus (72) Google Scholar) and the mRNA export factor Mex67p (this work). Furthermore, Kap95p and Mex67p bind to both common and distinct aspects of the Nup116-GLFG region. All yeast strains used in this study are listed in Table I. The sequence encoding the green fluorescent protein (GFP) was fused in frame before the stop codons for the chromosomal MEX67, DBP5, GLE1, andMTR2 genes. This was achieved using the gene integration method of Baudin et al. (36Baudin A. Ozier K.O. Denouel A. Lacroute F. Cullin C. Nucleic Acids Res. 1993; 21: 3329-3330Crossref PubMed Scopus (1127) Google Scholar). Respective polymerase chain reaction products were generated with oligonucleotides and a template containing genes for GFP and the Schizosaccharomyces pombe HIS5 (pGFP-HIS5; gift of J. Aitchison). The resulting DNA fragments were introduced into SWY518 by standard transformation methods and colonies selected on media lacking histidine. Correct integration was confirmed by immunoblot and direct fluorescence microscopy. The resulting strains were back-crossed to a wild type yeast strain, and the GFP-tagged progeny was used in this study. For GFP tagging the genomic copy of GLE2, a similar strategy was used except the S. cerevisiae HIS3 gene served as the selectable marker, and the polymerase chain reaction fragment was transformed into the diploid SWY595. The resulting diploid was sporulated and dissected to obtain SWY1920.Table IYeast strainsStrainGenotypeSourceSWY518MATaade2–1::ADE2 ura3–1 leu2–3,112 trp1–1 his3–11,15 can1–10051SWY595MATa/α ade2–1::ADE2/ade2–1::ADE2 ura3–1/ura3–1 leu2–3,112/leu2–3,112 trp1–1/trp1–1 his3–11,15/his3–11,15 can1–100/can1–10051SWY1920MATaGLE2-GFP:HIS3 ade2–1::ADE2 ura3–1 leu2–3,112 trp1–1 his3–11,15 can1–100This studySWY2131MATa MEX67-GFP:HIS5 ade2–1::ADE2 ura3–1 leu2–3,112 trp1–1 his3–11,15 can1–100This studySWY2154MATα MEX67-GFP:HIS5 ade2–1::ADE2 ura3–1 leu2–3,112 trp1–1 his3–11,15 can1–100This studySWY2253MATaGLE1-GFP:HIS5 ade2–1::ADE2 ura3–1 leu2–3,112 trp1–1 his3–11,15 can1–100This studySWY2257MATa MTR2-GFP:HIS5 ade2–1::ADE2 ura3–1 leu2–3,112 trp1–1 his3–11,15 can1–100This studySWY2261MATaDBP5-GFP:HIS5 ade2–1::ADE2 ura3–1 leu2–3,112 trp1–1 his3–11,15 can1–100This studyL40MATa his3Δ200 trp1–901 leu2–3,112 ade2 LYS2::(LexAop)4-HIS3 URA3::(LexAop)8-lacZS. HollenbergPJ69–4AMATa trp1–901 leu2–3,112 ura3–52 his3–200 gal4Δ gal80Δ LYS2::GAL1-HIS3 GAL2-ADE2 met2::GAL7-lacZ42 Open table in a new tab The plasmids used in this study are listed in TableII and were maintained in either BL21 (pSW156, pSW304, and pSW329) or DH5α (all others).Table IIPlasmidsPlasmidConstruction2-aAll nucleotide positions given begin with the initiation codon (bp = base pairs). Vector backbone references: pNLS-E1 (52); pGEX-3X (Amersham Pharmacia Biotech); pQE-32 (Qiagen); pMAL-cRI (New England Biolabs); pCH432 for LexA binding domain fusions (41); pGBD-C1 (42); pSW291 (27).SourcepNLS-E1 backbonepSW163GLFG region ofNUP116 (bp 532–2145) inBamHI/SacI27pSW384GLFG region ofNUP100 (bp 4–1782) inBamHI/SacI27pGEX-3X backbonepSW304GLFG region of NUP116 (bp 481–2190) inBamHI28pSW433GLFG region of NUP100(bp 4–1830) in BamHIThis studypQE-32 backbonepSW1261KAP95 in BamHIThis studypMAL-cRIpSW156NUP116 in BamHI2pSW1237MEX67 inEcoRI/SalIThis studypCH432 backbonepSW310/332KAP95 in BamHI27pGBD-C1 backbonepSW1254MEX67 inEcoRI/SalIThis studypSW291 backbonepSW1232Full-length NUP116 GLFG region (bp 478–2196) in NcoI/XhoIThis studypSW1233Repeats 1–25 of NUP116 GLFG region (bp 478–1809) in NcoI/XhoIThis studypSW1234Repeats 9–33 of NUP116 GLFG region (bp 925–2196) in NcoI/XhoIThis studypSW1235Repeats 9–25 of NUP116 GLFG region (bp 925–1809) in NcoI/XhoIThis studypSW1246Repeats 9–21 of NUP116 GLFG region (bp 925–1605) in NcoI/XhoIThis studypSW1247Repeats 13–25 of NUP116 GLFG region (bp 1084–1809) in NcoI/XhoIThis studypSW1248Repeats 13–21 of NUP116 GLFG region (bp 1084–1605) in NcoI/XhoIThis studypSW1290Repeats 1–12 of NUP116 GLFG region (bp 478–1086) in NcoI/XhoIThis studypSW1291Repeats 22–33 of NUP116 GLFG region (bp 1606–2196) in NcoI/XhoIThis study2-a All nucleotide positions given begin with the initiation codon (bp = base pairs). Vector backbone references: pNLS-E1 (52Guarente L. Yocum R.R. Gifford P. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 7410-7414Crossref PubMed Scopus (367) Google Scholar); pGEX-3X (Amersham Pharmacia Biotech); pQE-32 (Qiagen); pMAL-cRI (New England Biolabs); pCH432 for LexA binding domain fusions (41Hardy C.F.J. Mol. Cell. Biol. 1996; 16: 1832-1841Crossref PubMed Scopus (60) Google Scholar); pGBD-C1 (42James P. Halladay J. Craig E.A. Genetics. 1996; 144: 1425-1436Crossref PubMed Google Scholar); pSW291 (27Iovine M.K. Watkins J.L. Wente S.R. J. Cell Biol. 1995; 131: 1699-1713Crossref PubMed Scopus (169) Google Scholar). Open table in a new tab pSW163 (Nup116-GLFG under a galactose-inducible promoter), pSW384 (Nup100-GLFG under a galactose-inducible promoter), or pNLS-E1 (vector) were transformed into the respective GFP-tagged strains by standard methods. Stationary phase cultures grown in synthetic complete (SC) media lacking uracil (−ura) with 2% glucose were pelleted, washed once in an equal volume of SC−ura, 2% raffinose and used to inoculate SC−ura, 2% raffinose at an initialA600 of 0.015. When theA600 reached 0.1 to 0.2, galactose (J. T. Baker Inc.) was added to one-half of the culture at a final concentration of 2%. The raffinose and galactose cultures were incubated 5 h at 30 °C. A small portion of the live cells was removed for viewing direct fluorescence, and the remaining cells were prepared for indirect immunofluorescence microscopy as described previously (27Iovine M.K. Watkins J.L. Wente S.R. J. Cell Biol. 1995; 131: 1699-1713Crossref PubMed Scopus (169) Google Scholar, 37Wente S.R. Rout M.P. Blobel G. J. Cell Biol. 1992; 119: 705-723Crossref PubMed Scopus (198) Google Scholar). To test for nucleolar fragmentation in the GLFG-overexpressing strains, fixed cells were incubated overnight with tissue culture supernatant monoclonal antibody D77 (38Henriquez R. Blobel G. Aris J.P. J. Biol. Chem. 1990; 265: 2209-2215Abstract Full Text PDF PubMed Google Scholar) (1:10) for detection of Nop1p. Bound antibody was detected with a rhodamine donkey anti-mouse antibody (Chemicon; 1:400). For detection of Kap95p, a rabbit polyclonal antibody (28Iovine M.K. Wente S.R. J. Cell Biol. 1997; 137: 797-811Crossref PubMed Scopus (72) Google Scholar) raised against Kap95p was used at a 1:20 dilution (16 h, 4 °C). Bound antibody was detected with a Texas Red-conjugated donkey anti-rabbit antibody (Jackson Immunoresearch; 1:200). Images were collected with a × 100 objective on an Olympus BX50 microscope using a Dage-MTI CCD-300-RC camera (Dage MTI, Michigan City, IN). Whole cell lysates of SWY518 (MEX67) and SWY2131 (MEX67-GFP) were prepared in 20 mm Tris-HCl, pH 6.5, 150 mmNaCl, 5 mm MgCl2, 2% Triton X-100 by glass bead lysis. Immunoprecipitations were conducted as described previously (25Ho A.K. Shen T. Ryan K.J. Kiseleva E. Aach Levy M. Allen T.D. Wente S.R. Mol. Cell. Biol. 2000; 20: 5736-5748Crossref PubMed Scopus (40) Google Scholar). For immunoprecipitations, 1 μl of affinity-purified rabbit polyclonal anti-GFP antibody (gift from M. Linder) was used per 100 μl of cell extract. Blots were probed with an anti-GFP antibody at a 1:1000 dilution (16 h, 4 °C), affinity-purified rabbit polyclonal anti-Nup116-GLFG antibody WU956 (39Bucci M. Wente S.R. Mol. Biol. Cell. 1998; 9: 2439-2461Crossref PubMed Scopus (42) Google Scholar) at a 1:2000 dilution (16 h, 4 °C), or affinity-purified rabbit polyclonal antibody WU600 (27Iovine M.K. Watkins J.L. Wente S.R. J. Cell Biol. 1995; 131: 1699-1713Crossref PubMed Scopus (169) Google Scholar) raised against the Nup116 C-terminal region at a 1:1000 dilution (16 h, 4 °C). Bound antibodies were detected with alkaline phosphatase-conjugated anti-rabbit IgG (Promega) diluted 1:7500 (1 h, 23 °C). After incubation with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl-1-phosphate (Promega), the blots were developed for color visualization. For purification of the glutathioneS-transferase (GST) fusion proteins GST-Nup116-GLFG or GST-Nup100-GLFG, Escherichia coli strains containing pSW304 or pSW433, respectively, were grown in 1 liter of LB-rich media containing 100 μg/ml carbenicillin at 37 °C until theA600 was between 0.9 and 1.0. Cultures were induced for 3.5 h with 0.3 mmisopropyl-1-thio-β-d-galactopyranoside and cell pellets frozen at −70 °C. Thawed pellets were resuspended in ice-cold 20 mm sodium phosphate, pH 7.3, 150 mm NaCl, 10 mm EDTA, 0.1 mm DTT, 0.1 mmphenylmethylsulfonyl fluoride, 0.1 μm pepstatin A, 0.1 μm leupeptin, 0.1 mm benzamidine, and 0.01% sodium azide. 50 mg of lysozyme was added, and the suspensions were incubated on ice for 30 min before sonication (15 s pulses, 15 s rest for a total of 3 min). The suspension was clarified in a Sorvall SS-34 rotor at 9000 × g for 30 min, and Triton X-100 was added to a final concentration of 1%. The clarified lysate was loaded onto 2.5 ml of packed Glutathione-Sepharose 4B (Amersham Pharmacia) equilibrated in 20 mm sodium phosphate, pH 7.3, 150 mm NaCl, 10 mm EDTA, 0.1 mmDTT, 1% Triton X-100. After washing the column with 15 ml of 20 mm sodium phosphate, pH 7.3, 150 mm NaCl, 10 mm EDTA, 0.1 mm DTT, 1% Triton X-100, the same buffer lacking Triton X-100 was used until theA280 of the flow-through was 0. Finally, the protein was eluted in 50 mm Tris, pH 9.0, 20 mmglutathione. The maltose-binding protein (MBP) fusions with Mex67p (MBP-Mex67) and full-length Nup116p (MBP-Nup116) were purified as above except the thawed pellets were resuspended in 100 mm Tris, pH 7.5, 50 mm NaCl, 10 mm EDTA, 10 mmβ-mercaptoethanol, 5 μg/ml leupeptin, 1% aprotinin, 0.1 mg/ml Pefabloc. After sonication, NaCl was added to a final concentration of 0.5 m, and the clarified lysate was loaded onto 4 ml of packed amylose resin (New England Biolabs) previously equilibrated in column buffer (10 mm sodium phosphate, 0.5 mNaCl, pH 7.0, 0.25% Tween 20). The column was washed with 15 ml of column buffer, followed by column buffer without detergent until theA280 of the flow-through was 0. Finally, the protein was eluted in the latter buffer containing 10 mmmaltose. The expression of polyhistidine (His6)-tagged Kap95p was induced in bacteria as described above, and the protein was subsequently purified on nickel-nitrilotriacetic acid-agarose (Qiagen) as described by the manufacturer. Proteins were dialyzed into either 20 mm Hepes-KOH, pH 7.0, 100 mm potassium acetate, 2 mm magnesium acetate, 0.1% Tween 20, 5 mm β-mercaptoethanol, 10% glycerol (GST-Nup116-GLFG) or 20 mm Hepes-KOH, pH 6.8, 150 mm potassium acetate, 2 mm magnesium acetate, 0.1% Tween 20, 2 mm DTT, 0.1% casamino acids (GST-Nup100-GLFG). Binding assays were conducted as described previously (40Rexach M. Blobel G. Cell. 1995; 83: 683-692Abstract Full Text PDF PubMed Scopus (665) Google Scholar) except 2 μg of protein was added per 10 μl of packed Glutathione-Sepharose. Bound and unbound fractions were separated by electrophoresis on 7.5 or 9% SDS-polyacrylamide gels and detected by Coomassie staining. pSW332 and the various pSW291 constructs (Table II) were cotransformed into the yeast strain L40 (Table I), and pSW1254 and the various pSW291 constructs (Table II) were cotransformed into the yeast strain PJ69-4A (Table I). Transformants were selected on SC-leu-trp and assayed for two-hybrid interaction by growth on SC-leu-trp-his (for L40) or SC-leu-trp-his-ade (for PJ69–4A). All plasmids were checked for specificity and ability to self-activate with pCH428 (pLexA-Orc2) (41Hardy C.F.J. Mol. Cell. Biol. 1996; 16: 1832-1841Crossref PubMed Scopus (60) Google Scholar), pGBD-C1 (42James P. Halladay J. Craig E.A. Genetics. 1996; 144: 1425-1436Crossref PubMed Google Scholar), or pSE1111 (pGAD-Snf4) (43Yang X. Hubbard E.J. Carlson M. Science. 1992; 257: 680-682Crossref PubMed Scopus (173) Google Scholar). Previous studies have shown that overexpression of the Nup116-GLFG region in yeast cells results in nucleolar fragmentation, accumulation of polyadenylated RNA within the nucleus, and cell lethality (27Iovine M.K. Watkins J.L. Wente S.R. J. Cell Biol. 1995; 131: 1699-1713Crossref PubMed Scopus (169) Google Scholar). The GLFG overexpression phenotype does not result in any detectable NPC or nuclear envelope structural perturbations, and the overexpressed Nup116-GLFG region localizes in both the cytoplasm and nucleus (27Iovine M.K. Watkins J.L. Wente S.R. J. Cell Biol. 1995; 131: 1699-1713Crossref PubMed Scopus (169) Google Scholar). We previously suggested this phenotype may be due to titration of an essential GLFG-interacting factor away from the NPC. Recently, several S. cerevisiaefactors have been identified that are specifically required for mRNA export and dispensable for the transport of proteins and other classes of RNA (44Cole C.N. Hammell C.M. Curr. Biol. 1998; 8: 368-372Abstract Full Text Full Te" @default.
• W2023521597 created "2016-06-24" @default.
• W2023521597 creator A5017446481 @default.
• W2023521597 creator A5029863922 @default.
• W2023521597 creator A5071077154 @default.
• W2023521597 date "2001-03-01" @default.
• W2023521597 modified "2023-10-14" @default.
• W2023521597 title "The GLFG Regions of Nup116p and Nup100p Serve as Binding Sites for Both Kap95p and Mex67p at the Nuclear Pore Complex" @default.
• W2023521597 cites W1599868278 @default.
• W2023521597 cites W1675243257 @default.
• W2023521597 cites W1903792504 @default.
• W2023521597 cites W1963846953 @default.
• W2023521597 cites W1968863167 @default.
• W2023521597 cites W1968872346 @default.
• W2023521597 cites W1971531897 @default.
• W2023521597 cites W1973052124 @default.
• W2023521597 cites W1984013070 @default.
• W2023521597 cites W1985732160 @default.
• W2023521597 cites W1994234356 @default.
• W2023521597 cites W1996364283 @default.
• W2023521597 cites W1996381709 @default.
• W2023521597 cites W1997160430 @default.
• W2023521597 cites W2005855998 @default.
• W2023521597 cites W2012428415 @default.
• W2023521597 cites W2015506926 @default.
• W2023521597 cites W2015851068 @default.
• W2023521597 cites W2023893656 @default.
• W2023521597 cites W2026117440 @default.
• W2023521597 cites W2045845081 @default.
• W2023521597 cites W2050230359 @default.
• W2023521597 cites W2051567211 @default.
• W2023521597 cites W2058949200 @default.
• W2023521597 cites W2059296589 @default.
• W2023521597 cites W2067415570 @default.
• W2023521597 cites W2080408951 @default.
• W2023521597 cites W2082809022 @default.
• W2023521597 cites W2087202439 @default.
• W2023521597 cites W2094095762 @default.
• W2023521597 cites W2105657024 @default.
• W2023521597 cites W2108084815 @default.
• W2023521597 cites W2110580557 @default.
• W2023521597 cites W2113927132 @default.
• W2023521597 cites W2115109190 @default.
• W2023521597 cites W2123898323 @default.
• W2023521597 cites W2127901995 @default.
• W2023521597 cites W2133201628 @default.
• W2023521597 cites W2138459463 @default.
• W2023521597 cites W2140673402 @default.
• W2023521597 cites W2142371650 @default.
• W2023521597 cites W2145885644 @default.
• W2023521597 cites W2146498183 @default.
• W2023521597 cites W2147543837 @default.
• W2023521597 cites W2151298994 @default.
• W2023521597 cites W2156202665 @default.
• W2023521597 cites W2158254431 @default.
• W2023521597 cites W2162226086 @default.
• W2023521597 cites W2162882160 @default.
• W2023521597 cites W2164405703 @default.
• W2023521597 cites W2165306451 @default.
• W2023521597 cites W2427117587 @default.
• W2023521597 doi "https://doi.org/10.1074/jbc.m008311200" @default.
• W2023521597 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11104765" @default.
• W2023521597 hasPublicationYear "2001" @default.
• W2023521597 type Work @default.
• W2023521597 sameAs 2023521597 @default.
• W2023521597 citedByCount "112" @default.
• W2023521597 countsByYear W20235215972012 @default.
• W2023521597 countsByYear W20235215972013 @default.
• W2023521597 countsByYear W20235215972014 @default.
• W2023521597 countsByYear W20235215972015 @default.
• W2023521597 countsByYear W20235215972016 @default.
• W2023521597 countsByYear W20235215972017 @default.
• W2023521597 countsByYear W20235215972018 @default.
• W2023521597 countsByYear W20235215972019 @default.
• W2023521597 countsByYear W20235215972020 @default.
• W2023521597 countsByYear W20235215972021 @default.
• W2023521597 countsByYear W20235215972022 @default.
• W2023521597 countsByYear W20235215972023 @default.
• W2023521597 crossrefType "journal-article" @default.
• W2023521597 hasAuthorship W2023521597A5017446481 @default.
• W2023521597 hasAuthorship W2023521597A5029863922 @default.
• W2023521597 hasAuthorship W2023521597A5071077154 @default.
• W2023521597 hasBestOaLocation W20235215971 @default.
• W2023521597 hasConcept C12554922 @default.
• W2023521597 hasConcept C126725582 @default.
• W2023521597 hasConcept C185592680 @default.
• W2023521597 hasConcept C190062978 @default.
• W2023521597 hasConcept C55493867 @default.
• W2023521597 hasConcept C86803240 @default.
• W2023521597 hasConceptScore W2023521597C12554922 @default.
• W2023521597 hasConceptScore W2023521597C126725582 @default.
• W2023521597 hasConceptScore W2023521597C185592680 @default.
• W2023521597 hasConceptScore W2023521597C190062978 @default.
• W2023521597 hasConceptScore W2023521597C55493867 @default.
• W2023521597 hasConceptScore W2023521597C86803240 @default.
• W2023521597 hasIssue "9" @default.
• W2023521597 hasLocation W20235215971 @default.
• W2023521597 hasOpenAccess W2023521597 @default.

• next