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• W2110580557 abstract "Nup116p is a GLFG nucleoporin involved in RNA export processes. We show here that Nup116p physically interacts with the Nup82p-Nsp1p-Nup159p nuclear pore subcomplex, which plays a central role in nuclear mRNA export. For this association, a sequence within the C-terminal domain of Nup116p that includes the conserved nucleoporin RNA-binding motif was sufficient and necessary. Consistent with this biochemical interaction, protein A-Nup116p and the protein A-tagged Nup116p C-terminal domain, like the members of the Nup82p complex, localized to the cytoplasmic side of the nuclear pore complex, as revealed by immunogold labeling. Finally, synthetic lethal interactions were found between mutant alleles of NUP116and all members of the Nup82p complex. Thus, Nup116p consists of three independent functional domains: 1) the C-terminal part interacts with the Nup82p complex; 2) the Gle2p-binding sequence interacts with Gle2p/Rae1p; and 3) the GLFG domain interacts with shuttling transport receptors such as karyopherin-β family members. Nup116p is a GLFG nucleoporin involved in RNA export processes. We show here that Nup116p physically interacts with the Nup82p-Nsp1p-Nup159p nuclear pore subcomplex, which plays a central role in nuclear mRNA export. For this association, a sequence within the C-terminal domain of Nup116p that includes the conserved nucleoporin RNA-binding motif was sufficient and necessary. Consistent with this biochemical interaction, protein A-Nup116p and the protein A-tagged Nup116p C-terminal domain, like the members of the Nup82p complex, localized to the cytoplasmic side of the nuclear pore complex, as revealed by immunogold labeling. Finally, synthetic lethal interactions were found between mutant alleles of NUP116and all members of the Nup82p complex. Thus, Nup116p consists of three independent functional domains: 1) the C-terminal part interacts with the Nup82p complex; 2) the Gle2p-binding sequence interacts with Gle2p/Rae1p; and 3) the GLFG domain interacts with shuttling transport receptors such as karyopherin-β family members. nuclear pore complex N- and C-terminal domains, respectively nucleoporin RNA-binding motif Gle2p-binding sequence polymerase chain reaction protein A green fluorescent protein polyacrylamide gel electrophoresis The nuclear pore complex (NPC)1 is a huge organelle with an intricate structure of octagonal symmetry (for review, see Ref.1Stoffler D. Fahrenkrog B. Aebi U. Curr. Opin. Cell Biol. 1999; 11: 391-401Crossref PubMed Scopus (296) Google Scholar). It allows passive diffusion of small molecules and controls active transport of macromolecules in and out of the nucleus. Of the estimated proteins constituting the NPC, almost all have been identified in the case of the yeast Saccharomyces cerevisiae (2Doye V. Hurt E. Curr. Opin. Cell Biol. 1997; 9: 401-411Crossref PubMed Scopus (212) Google Scholar). In subsequent studies, their nearest neighborhood was analyzed by characterizing their biochemical organization into subcomplexes or by immunolocalizing them to distinct sites within the structural framework of the NPC (for review, see Ref. 1Stoffler D. Fahrenkrog B. Aebi U. Curr. Opin. Cell Biol. 1999; 11: 391-401Crossref PubMed Scopus (296) Google Scholar). Following the elucidation of the Ran cycle, which drives vectorial nucleocytoplasmic transport, and the discovery of the shuttling importin/karyopherin-β transport receptor family together with their cargoes, interest is now focusing on the understanding of the actual transport mechanism through the NPCs (for review, see Ref. 3Gorlich D. Kutay U. Annu. Rev. Cell Dev. Biol. 1999; 15: 607-660Crossref PubMed Scopus (1651) Google Scholar) In yeast, several NPC subcomplexes are known. The Nup84p complex has functions in both NPC biogenesis and nuclear mRNA export (4Aitchison J.D. Blobel G. Rout M.P. J. Cell Biol. 1995; 131: 1659-1675Crossref PubMed Scopus (117) Google Scholar, 5Goldstein A.L. Snay C.A. Heath C.V. Cole C.N. Mol. Biol. Cell. 1996; 7: 917-934Crossref PubMed Scopus (65) Google Scholar, 6Siniossoglou S. Wimmer C. Rieger M. Doye V. Tekotte H. Weise C. Emig S. Segref A. Hurt E.C. Cell. 1996; 84: 265-275Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 7Teixeira M.T. Siniossoglou S. Podtelejnikov S. Benichou J.C. Mann M. Dujon B. Hurt E. Fabre E. EMBO J. 1997; 16: 5086-5097Crossref PubMed Scopus (88) Google Scholar). The Nup170p-Nup157p-Nup188p complex may functionally contribute to transport processes and structural integrity of the NPC as well as to cell cycle control (8Nehrbass U. Rout M.P. Maguire S. Blobel G. Wozniak R.W. J. Cell Biol. 1996; 133: 1153-1162Crossref PubMed Scopus (75) Google Scholar, 9Zabel U. Doye V. Tekotte H. Wepf R. Grandi P. Hurt E.C. J. Cell Biol. 1996; 133: 1141-1152Crossref PubMed Scopus (85) Google Scholar, 10Marelli M. Aitchison J.D. Wozniak R.W. J. Cell Biol. 1998; 143: 1813-1830Crossref PubMed Scopus (133) Google Scholar). Nsp1p is unique compared with other nucleoporins in that it forms two distinct NPC subcomplexes. The first one isolated is the Nsp1p-Nup49p-Nup57p-Nic96p complex involved in nuclear protein import and localized to the nucleoplasmic and cytoplasmic face of the central gated channel and to the nuclear basket (11Grandi P. Schlaich N. Tekotte H. Hurt E.C. EMBO J. 1995; 14: 76-87Crossref PubMed Scopus (133) Google Scholar, 12Bucci M. Wente S.R. J. Cell Biol. 1997; 136: 1185-1199Crossref PubMed Scopus (93) Google Scholar, 13Fahrenkrog B. Hurt E.C. Aebi U. Pante N. J. Cell Biol. 1998; 143: 577Crossref PubMed Scopus (93) Google Scholar). The higher eucaryotic NPC subcomplex p62-p54-p58/p45 with its associated NUP93 shows striking homology to this Nsp1p subcomplex (14Dabauvalle M.C. Loos K. Scheer U. Chromosoma (Berl.). 1990; 100: 56-66Crossref PubMed Scopus (71) Google Scholar, 15Finlay D.R. Meier E. Bradley P. Horecka J. Forbes D.J. J. Cell Biol. 1991; 114: 169-183Crossref PubMed Scopus (174) Google Scholar, 16Hu T. Guan T. Gerace L. J. Cell Biol. 1996; 134: 589-601Crossref PubMed Scopus (151) Google Scholar, 17Grandi P. Dang T. Pante N. Shevchenko A. Mann M. Forbes D. Hurt E. Mol. Biol. Cell. 1997; 8: 2017-2038Crossref PubMed Scopus (125) Google Scholar). The second Nsp1p complex is formed by Nup82p and Nup159p (18Grandi P. Emig S. Weise C. Hucho F. Pohl T. Hurt E.C. J. Cell Biol. 1995; 130: 1263-1273Crossref PubMed Scopus (89) Google Scholar, 19Belgareh N. Snay-Hodge C. Pasteau F. Dagher S. Cole C.N. Doye V. Mol. Biol. Cell. 1998; 9: 3475-3492Crossref PubMed Scopus (79) Google Scholar, 20Hurwitz M.E. Strambio-de-Castillia C. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11241-11245Crossref PubMed Scopus (51) Google Scholar). As with the Nic96p complex, all components are essential and tethered to each other via their C-terminal coiled-coil domains. Temperature-sensitive mutants of Nup82p and Nup159p display severe defects in nuclear mRNA export, but not in nuclear protein import (18Grandi P. Emig S. Weise C. Hucho F. Pohl T. Hurt E.C. J. Cell Biol. 1995; 130: 1263-1273Crossref PubMed Scopus (89) Google Scholar, 20Hurwitz M.E. Strambio-de-Castillia C. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11241-11245Crossref PubMed Scopus (51) Google Scholar, 21Hurwitz M.E. Blobel G. J. Cell Biol. 1995; 130: 1275-1281Crossref PubMed Scopus (56) Google Scholar, 22Del Priore V. Heath C. Snay C. MacMillan A. Gorsch L. Dagher S. Cole C. J. Cell Sci. 1997; 110: 2987-2999Crossref PubMed Google Scholar). While Nup159p-N seems to be involved in mRNA export, the C-terminal domains of Nup159p and Nup82p are required for stable subcomplex formation and their integration into the NPC (19Belgareh N. Snay-Hodge C. Pasteau F. Dagher S. Cole C.N. Doye V. Mol. Biol. Cell. 1998; 9: 3475-3492Crossref PubMed Scopus (79) Google Scholar, 20Hurwitz M.E. Strambio-de-Castillia C. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11241-11245Crossref PubMed Scopus (51) Google Scholar, 23Kraemer D.M. Strambio-de-Castillia C. Blobel G. Rout M.P. J. Biol. Chem. 1995; 270: 19017-19021Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). The Nup82p complex most likely represents the yeast counterpart of the higher eucaryotic CAN/NUP214-NUP88/84-p66-CRM1 complex (24Bastos R. Ribas de Pouplana L. Enarson M. Bodoor K. Burke B. J. Cell Biol. 1997; 137: 989-1000Crossref PubMed Scopus (86) Google Scholar, 25Fornerod M. van Deursen J. van Baal S. Reynolds A. Davis D. Murti K.G. Fransen J. Grosveld G. EMBO J. 1997; 16: 807-816Crossref PubMed Scopus (396) Google Scholar). Several proteins involved in nuclear mRNA export have been shown or are likely to interact with the Nup82p or CAN/NUP214 complex. TAP, the higher eucaryotic homologue of yeast Mex67p, a transport factor essential for mRNA export, interacts with the CAN/NUP214 FG repeat region (26Segref A. Sharma K. Doye V. Hellwig A. Huber J. Luhrmann R. Hurt E. EMBO J. 1997; 16: 3256-3271Crossref PubMed Scopus (432) Google Scholar, 27Santos-Rosa H. Moreno H. Simos G. Segref A. Fahrenkrog B. Pante N. Hurt E. Mol. Cell. Biol. 1998; 18: 6826-6838Crossref PubMed Scopus (220) Google Scholar, 28Katahira J. Sträßer K. Podtelejnikov A. Mann M. Jung J.U. Hurt E. EMBO J. 1999; 18: 2593-2609Crossref PubMed Scopus (339) Google Scholar). Recently, the N-terminal part of Nup159p was found to directly interact with Dbp5p, an ATP-driven RNA helicase (29Hodge C.A. Colot H.V. Stafford P. Cole C.N. EMBO J. 1999; 18: 5778-5788Crossref PubMed Scopus (161) Google Scholar, 30Schmitt 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 (219) Google Scholar). Gle1p, an essential nuclear export signal containing protein involved in mRNA export, acts as a high copy suppressor of C-terminal temperature-sensitive mutations in Nup159p and Nup82p (20Hurwitz M.E. Strambio-de-Castillia C. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11241-11245Crossref PubMed Scopus (51) Google Scholar, 31Murphy R. Wente S.R. Nature. 1996; 383: 357-360Crossref PubMed Scopus (202) Google Scholar, 32Del Priore V. Snay C.A. Bahr A. Cole C.N. Mol. Biol. Cell. 1996; 7: 1601-1621Crossref PubMed Scopus (52) Google Scholar) and is a good candidate to bind to the Nup82p complex via Dbp5p (29Hodge C.A. Colot H.V. Stafford P. Cole C.N. EMBO J. 1999; 18: 5778-5788Crossref PubMed Scopus (161) Google Scholar, 33Strahm Y. Fahrenkrog B. Zenklusen D. Rychner E. Kantor J. Rosbach M. Stutz F. EMBO J. 1999; 18: 5761-5777Crossref PubMed Scopus (129) Google Scholar). In addition, GLE1 is synthetically lethal with NUP116 and NUP100 (31Murphy R. Wente S.R. Nature. 1996; 383: 357-360Crossref PubMed Scopus (202) Google Scholar). Taken together, since the Nup82p complex is exclusively localized to the cytoplasmic side of the NPC and interacts with the above-mentioned proteins involved in mRNA transport, its components are likely to play a crucial role in mRNA export steps through the NPCs and possibly release of the transport cargo into the cytoplasm. Nup116p is a nucleoporin that was initially found to be genetically linked to Nsp1p (34Wimmer C. Doye V. Grandi P. Nehrbass U. Hurt E.C. EMBO J. 1992; 11: 5051-5061Crossref PubMed Scopus (133) Google Scholar) and that shows homology to Nup100p and Nup145p-N over its entire length (35Wente S.R. Rout M.P. Blobel G. J. Cell Biol. 1992; 119: 705-723Crossref PubMed Scopus (194) Google Scholar, 36Wente S.R. Blobel G. J. Cell Biol. 1993; 123: 275-284Crossref PubMed Scopus (162) Google Scholar, 37Wente S.R. Blobel G. J. Cell Biol. 1994; 125: 955-969Crossref PubMed Scopus (111) Google Scholar, 38Fabre E. Boelens W.C. Wimmer C. Mattaj I.W. Hurt E.C. Cell. 1994; 78: 275-289Abstract Full Text PDF PubMed Scopus (115) Google Scholar). Nup116p, Nup100p, and Nup145p-N share (i) an N-terminally located GLFG repeat domain, previously shown to bind karyopherin-β-like transport factors (39Iovine M.K. Watkins J.L. Wente S.R. J. Cell Biol. 1995; 131: 1699-1713Crossref PubMed Scopus (165) Google Scholar, 40Iovine M.K. Wente S.R. J. Cell Biol. 1997; 137: 797-811Crossref PubMed Scopus (72) Google Scholar), and (ii) a conserved C-terminal domain that includes the nucleoporin RNA-binding motif (NRM) shown to bind to homopolymeric RNA in vitro and to perform a redundant function (38Fabre E. Boelens W.C. Wimmer C. Mattaj I.W. Hurt E.C. Cell. 1994; 78: 275-289Abstract Full Text PDF PubMed Scopus (115) Google Scholar). Nup116p, however, differs from Nup100p and Nup145p-N by harboring an evolutionarily conserved sequence of ∼60 amino acids called the Gle2p-binding sequence (GLEBS), which mediates stable complex formation with Gle2p (41Bailer S.M. Siniossoglou S. Podtelejnikov A. Hellwig A. Mann M. Hurt E. EMBO J. 1998; 17: 1107-1119Crossref PubMed Scopus (117) Google Scholar, 42Ho A.K. Raczniak G.A. Ives E.B. Wente S.R. Mol. Biol. Cell. 1998; 9: 355-373Crossref PubMed Scopus (35) Google Scholar). Disruption of either GLE2 or NUP116 leads to temperature sensitivity and a concomitant defect in nuclear poly(A)+RNA export (36Wente S.R. Blobel G. J. Cell Biol. 1993; 123: 275-284Crossref PubMed Scopus (162) Google Scholar, 43Murphy R. Watkins J.L. Wente S.R. Mol. Biol. Cell. 1996; 7: 1921-1937Crossref PubMed Scopus (147) Google Scholar). For nup116Δ cells, an additional defect in tRNA export was reported (44Sarkar S. Hopper A.K. Mol. Biol. Cell. 1998; 9: 3041-3055Crossref PubMed Scopus (150) Google Scholar); it is not clear, however, whether these RNA transport defects are direct or merely due to sealed nuclear pores observed in these mutants (36Wente S.R. Blobel G. J. Cell Biol. 1993; 123: 275-284Crossref PubMed Scopus (162) Google Scholar, 41Bailer S.M. Siniossoglou S. Podtelejnikov A. Hellwig A. Mann M. Hurt E. EMBO J. 1998; 17: 1107-1119Crossref PubMed Scopus (117) Google Scholar, 43Murphy R. Watkins J.L. Wente S.R. Mol. Biol. Cell. 1996; 7: 1921-1937Crossref PubMed Scopus (147) Google Scholar). The fact that the Gle2p homologue in Schizosaccharomyces pombe, Rae1p, is essential for mRNA export (45Brown J.A. Bharathi A. Ghosh A. Whalen W. Fitzgerald E. Dhar R. J. Biol. Chem. 1995; 270: 7411-7419Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 46Whalen W.A. Bharathi A. Danielewicz D. Dhar R. Yeast. 1997; 13: 1167-1179Crossref PubMed Scopus (35) Google Scholar) strongly points to a direct role of this protein in RNA transport processes. NUP98, the putative higher eucaryotic homologue of Nup116p, also carries a GLEBS in its N-terminal half where GLE2/RAE1 docks and a C-terminally located NRM (47Powers M.A. Macaulay C. Masiarz F.R. Forbes D.J. J. Cell Biol. 1995; 128: 721-736Crossref PubMed Scopus (106) Google Scholar, 48Radu A. Moore M.S. Blobel G. Cell. 1995; 81: 215-222Abstract Full Text PDF PubMed Scopus (386) Google Scholar, 49Powers M.A. Forbes D.J. Dahlberg J.E. Lund E. J. Cell Biol. 1997; 136: 241-250Crossref PubMed Scopus (179) Google Scholar, 50Pritchard C.E. Fornerod M. Kasper L.H. van Deursen J.M. J. Cell Biol. 1999; 145: 237-254Crossref PubMed Scopus (193) Google Scholar). Microinjection of polyclonal anti-NUP98 antibodies intoXenopus oocytes blocks nuclear export of several types of RNAs (49Powers M.A. Forbes D.J. Dahlberg J.E. Lund E. J. Cell Biol. 1997; 136: 241-250Crossref PubMed Scopus (179) Google Scholar), whereas overexpression of the NUP98 GLEBS in tissue culture cells results in nuclear retention of poly(A)+ RNA (50Pritchard C.E. Fornerod M. Kasper L.H. van Deursen J.M. J. Cell Biol. 1999; 145: 237-254Crossref PubMed Scopus (193) Google Scholar). Finally, a role of NUP98 in nuclear export of human immunodeficiency virus-1 Rev was also suggested (51Zolotukhin A.S. Felber B.K. J. Virol. 1999; 73: 120-127Crossref PubMed Google Scholar). NUP98, which is preferentially located on the nucleoplasmic side of the NPC, could represent a more mobile nucleoporin that shuttles between the nucleus and cytoplasm together with its associated GLE2/RAE1 (48Radu A. Moore M.S. Blobel G. Cell. 1995; 81: 215-222Abstract Full Text PDF PubMed Scopus (386) Google Scholar, 50Pritchard C.E. Fornerod M. Kasper L.H. van Deursen J.M. J. Cell Biol. 1999; 145: 237-254Crossref PubMed Scopus (193) Google Scholar, 51Zolotukhin A.S. Felber B.K. J. Virol. 1999; 73: 120-127Crossref PubMed Google Scholar, 52Kraemer D. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9119-9124Crossref PubMed Scopus (79) Google Scholar). To further understand the function of Nup116p, Nup100p, and Nup145p-N within the structural framework of the NPCs, we aimed to define their physical and functional interaction with other nucleoporins. Previously, we showed that Nup116p is targeted to the NPC via its C-terminal domain (41Bailer S.M. Siniossoglou S. Podtelejnikov A. Hellwig A. Mann M. Hurt E. EMBO J. 1998; 17: 1107-1119Crossref PubMed Scopus (117) Google Scholar). We demonstrate here that Nup116p is predominantly localized to the cytoplasmic side of the NPC, where it physically and genetically interacts with the Nup82p-Nsp1p-Nup159p complex via its C-terminal domain including the NRM. Our genetic data support the idea that Nup116p consists of at least three independent functional domains, of which the C-terminal part interacts with the Nup82p complex, the GLEBS with Gle2p/Rae1p, and the GLFG domain with shuttling transport receptors such as the karyopherin-β family. The yeast strains used in this work are listed in TableI. Microbiological techniques, plasmid transformation, mating, sporulation of diploids, and tetrad analysis were done essentially as described (6Siniossoglou S. Wimmer C. Rieger M. Doye V. Tekotte H. Weise C. Emig S. Segref A. Hurt E.C. Cell. 1996; 84: 265-275Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). DNA manipulations (restriction analysis, end-filling reactions, ligations, PCR amplifications, etc.) were carried out essentially as described (58Maniatis T. Fritsch E.F. Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1982Google Scholar).Table IYeast strainsStrainGenotypeRef.RS453Mat a/α, ade2/ade2, his3/his3, leu2/leu2, trp1/trp1, ura3/ura3Segrefet al. (26Segref A. Sharma K. Doye V. Hellwig A. Huber J. Luhrmann R. Hurt E. EMBO J. 1997; 16: 3256-3271Crossref PubMed Scopus (432) Google Scholar)BJ2168Mat a, leu2, trp1, ura3-52, pep4-3, pre1-407, prb1-1122Aris and Blobel (59Aris J.P. Blobel G. J. Cell Biol. 1988; 107: 17-31Crossref PubMed Scopus (189) Google Scholar)nup116Δ(URA3)Matα, ade2, his3, leu2, trp1, ura3, nup116∷URA3Fabre et al. (38Fabre E. Boelens W.C. Wimmer C. Mattaj I.W. Hurt E.C. Cell. 1994; 78: 275-289Abstract Full Text PDF PubMed Scopus (115) Google Scholar)nup116Δ(HIS3)Matα, ade2, his3, leu2, trp1, ura3, nup116∷HIS3This studyLGY106Mat a, ura3–52, leu2Δ1, his3Δ200, nup159∷HIS3Gorsch et al. (55Gorsch L.C. Dockendorff T.C. Cole C.N. J. Cell Biol. 1995; 129: 939-955Crossref PubMed Scopus (166) Google Scholar)(pLG4-URA3-NUP159 CEN)nup100ΔMat a, ade2, his3, leu2, trp1, ura3, nup100∷LEU2Fabre et al. (38Fabre E. Boelens W.C. Wimmer C. Mattaj I.W. Hurt E.C. Cell. 1994; 78: 275-289Abstract Full Text PDF PubMed Scopus (115) Google Scholar)nup82ΔMat a, ade2, his3, leu2, trp1, ura3, nup82∷HIS3Grandi et al. (18Grandi P. Emig S. Weise C. Hucho F. Pohl T. Hurt E.C. J. Cell Biol. 1995; 130: 1263-1273Crossref PubMed Scopus (89) Google Scholar)(pRS316-URA3-NUP82)nup145ΔMat a, ura3-Δ851, trp1 Δ63, leu2 Δ1, nup145∷HIS3Teixeira et al.(7Teixeira M.T. Siniossoglou S. Podtelejnikov S. Benichou J.C. Mann M. Dujon B. Hurt E. Fabre E. EMBO J. 1997; 16: 5086-5097Crossref PubMed Scopus (88) Google Scholar)(pRS316-URA3-NUP145)nup82Δ/nup116ΔMatα, ade2, his3, leu2, trp1, ura3, nup116∷HIS3 nup82∷HIS3This study(pRS316-URA3-NUP82)nsp1Δ/nup116ΔMatα, ade2, his3, leu2, trp1, ura3, nup116∷HIS3 nsp1∷HIS3This study(pRS316-URA3-NSP1)nup159Δ/nup116ΔMat a, his3, leu2, trp1, ura3, nup116∷HIS3 nup159∷HIS3This study(pLG4-URA3-NUP159 CEN)nup82Δ/nup100ΔMatα, ade2, his3, leu2, trp1, ura3, nup100∷LEU2 nup82∷HIS3This study(pRS316-URA3-NUP82)JU4-2xJR26-19B/ProtA-DHFR1-aDHFR, dihydrofolate reductase.Mat a/α,ade2-1/ade2-1, ade8/ADE8, can1-100/can1-100, his4/HIS4,Nehrbass et al. (60Nehrbass U. Fabre E. Dihlmann S. Herth W. Hurt E.C. Eur. J. Cell Biol. 1993; 62: 1-12PubMed Google Scholar)his3/HIS3, leu2-3/leu2-3, lys1-1/lys1-1, ura3-52/ura3-52(pYep13-ProtA-DHFR)1-a DHFR, dihydrofolate reductase. Open table in a new tab All fusion constructs, which were tagged amino-terminally with either two IgG-binding domains derived fromStaphylococcus aureus ProtA, green fluorescent protein (GFP) or Myc, were expressed under the control of the NOP1promoter. The NSP1 construct is exceptional since it is expressed under the control of the alcohol dehydrogenase promoter and does not carry a tag. All constructs contained authentic 3′-noncoding sequences except for pRS315-ProtA-NUP98-(498–920), which contains a NUP116 3′-noncoding region, and pRS414-GFP-NUP159-(2–1460), which contains an alcohol dehydrogenase 3′-noncoding region. Expression of all fusion proteins was verified by Western blot analysis using commercially available anti-ProtA, anti-GFP, or anti-Myc antibodies. The constructs are listed in TableII.Table IIPlasmidsPlasmidCommentsRef.pRS414-ProtA-NUP116-(Δ1–58)The construct is called NUP116 in Fig. 5.Bailer et al. (41Bailer S.M. Siniossoglou S. Podtelejnikov A. Hellwig A. Mann M. Hurt E. EMBO J. 1998; 17: 1107-1119Crossref PubMed Scopus (117) Google Scholar)pRS414-ProtA-NUP116-(706–1113)The construct encodes ProtA-Nup116p-C.Bailer et al. (41Bailer S.M. Siniossoglou S. Podtelejnikov A. Hellwig A. Mann M. Hurt E. EMBO J. 1998; 17: 1107-1119Crossref PubMed Scopus (117) Google Scholar)pRS414-ProtA-NUP100-(559–959)The construct encodes ProtA-Nup100p-C.This studypRS315-ProtA-NUP145-(247–605)The construct encodes ProtA-Nup145p-NΔGLFG.Teixeira et al. (7Teixeira M.T. Siniossoglou S. Podtelejnikov S. Benichou J.C. Mann M. Dujon B. Hurt E. Fabre E. EMBO J. 1997; 16: 5086-5097Crossref PubMed Scopus (88) Google Scholar)pRS315-ProtA-NUP116-(706–854)This studypRS315-ProtA-NUP116-(855–966)This studypRS315-ProtA-NUP116-(967–1113)This studypRS315-ProtA-NUP116-(855–1113)The 5th–8th constructs encode parts of Nup116p-C.This studypRS314-GFP-NUP116-(706–1113)The construct is calledNUP116-C in Fig. 5.Bailer et al. (41Bailer S.M. Siniossoglou S. Podtelejnikov A. Hellwig A. Mann M. Hurt E. EMBO J. 1998; 17: 1107-1119Crossref PubMed Scopus (117) Google Scholar)pRS414-GFP-NUP100-(559–959)The construct encodes GFP-Nup100p-C.This studypRS414-ProtA-NUP116-(59–705)The construct is called nup116ΔC in Fig. 5.Bailer et al. (41Bailer S.M. Siniossoglou S. Podtelejnikov A. Hellwig A. Mann M. Hurt E. EMBO J. 1998; 17: 1107-1119Crossref PubMed Scopus (117) Google Scholar)pRS414-ProtA-NUP116-(Δ110–166)The construct isnup116Δ GLEBS in Fig. 5.Bailer et al. (41Bailer S.M. Siniossoglou S. Podtelejnikov A. Hellwig A. Mann M. Hurt E. EMBO J. 1998; 17: 1107-1119Crossref PubMed Scopus (117) Google Scholar)pRS414-ProtA-NUP116-(Δ923–1113)The construct is callednup116Δ NRM in Fig. 5.This studypRS316-NUP82Grandi et al. (18Grandi P. Emig S. Weise C. Hucho F. Pohl T. Hurt E.C. J. Cell Biol. 1995; 130: 1263-1273Crossref PubMed Scopus (89) Google Scholar)pCH1122-URA3-ADE3-NSP1Wimmer et al. (34Wimmer C. Doye V. Grandi P. Nehrbass U. Hurt E.C. EMBO J. 1992; 11: 5051-5061Crossref PubMed Scopus (133) Google Scholar)YCplac33-URA3-NUP159 (pLG4)Del Priore et al.(22Del Priore V. Heath C. Snay C. MacMillan A. Gorsch L. Dagher S. Cole C. J. Cell Sci. 1997; 110: 2987-2999Crossref PubMed Google Scholar)pSB32-pADH-NSP1-L640SThe construct is callednsp1-L640S in Fig. 5.Wimmer et al. (34Wimmer C. Doye V. Grandi P. Nehrbass U. Hurt E.C. EMBO J. 1992; 11: 5051-5061Crossref PubMed Scopus (133) Google Scholar)pUN100-ProtA-TEV-NUP82This construct is called NUP82in Fig. 5 B.This studypRS315-ProtA-nup82-27This construct is callednup82-27 in Fig. 5 B.This studypRS315-GFP-NUP82-(2-713)This studyYCplac111-LEU2-NUP159 (pLG1)This construct is calledNUP159 in Fig. 5.Gorsch et al. (55)YCplac111-LEU2-rat7-1/nup159-1This construct is called nup159-1 in Fig. 5.Del Priore et al.(22Del Priore V. Heath C. Snay C. MacMillan A. Gorsch L. Dagher S. Cole C. J. Cell Sci. 1997; 110: 2987-2999Crossref PubMed Google Scholar)(pSD3)YCplac111-LEU2-NUP159ΔRep (pAM1)This construct is called nup159ΔRep in Fig. 5.Del Prioreet al. (22Del Priore V. Heath C. Snay C. MacMillan A. Gorsch L. Dagher S. Cole C. J. Cell Sci. 1997; 110: 2987-2999Crossref PubMed Google Scholar)YCplac111-LEU2-NUP159ΔN (pVDP16)This construct is called nup159ΔN in Fig. 5.Del Prioreet al. (22Del Priore V. Heath C. Snay C. MacMillan A. Gorsch L. Dagher S. Cole C. J. Cell Sci. 1997; 110: 2987-2999Crossref PubMed Google Scholar)YCplac111-LEU2-NUP159-C (pVDP17)This construct is called nup159-C in Fig. 5.Del Prioreet al. (22Del Priore V. Heath C. Snay C. MacMillan A. Gorsch L. Dagher S. Cole C. J. Cell Sci. 1997; 110: 2987-2999Crossref PubMed Google Scholar)pRS315-GFP-nup82-27Like the 19th construct; ProtA was replaced by GFP.This studypRS314-GFP-NUP82-(2–713)This construct is calledNUP82 in Fig. 5 D.This studypRS314-myc-NUP116-(706–1113)This studypRS414-GFP-NUP116-(2–1113)Bailer et al. (41Bailer S.M. Siniossoglou S. Podtelejnikov A. Hellwig A. Mann M. Hurt E. EMBO J. 1998; 17: 1107-1119Crossref PubMed Scopus (117) Google Scholar)pRS414-GFP-NUP159-(2–1460)This studypRS314-GFP-NIC96-(2–838)This studypRS315-ProtA-NUP98-(498–920)Using human cDNA of NUP98 as a template, a PCR fragment (HindIII-ApaI) was generated and fused in frame to a pNOP1-ProtA cassette. ANUP116 3′-noncoding region was added.This studypRS314-Myc-nup82–27This construct is callednup82-27 in Fig. 5 D.This study Open table in a new tab For complete disruption of the NUP116 open reading frame, a disruption cassette was designed where two PCR fragments consisting of 300 base pairs of NUP116 5′-untranslated region including the NUP116 ATG codon (XbaI-BamHI) and the NUP116 stop codon followed by 300 base pairs of 3′-untranslated region (BamHI-ApaI) were ligated into pBluescript. The HIS3 gene was inserted at the BamHI site. pBluescript-nup116Δ(HIS3) was cut usingXbaI-ApaI to releasenup116Δ(HIS3), which was used to transform the diploid strain RS453. Heterozygous HIS+ transformants were analyzed for correct integration at the NUP116 locus by PCR. After sporulation, haploid nup116Δ(HIS3)progeny were isolated that were temperature-sensitive for growth at 37 °C. Temperature-sensitive alleles of NUP82 mapping in the N-terminal region were generated according to published methods (56Muhlrad D. Hunter R. Parker R. Yeast. 1992; 8: 79-82Crossref PubMed Scopus (416) Google Scholar). A unique NheI site was introduced by PCR just before the putative coiled-coil domain of the NUP82 gene (at codon 521 by changing agt to agc), and the resulting DNA fragments were subcloned into pRS315-pNOP-ProtA. The resulting plasmid was cut at the unique NsiI and NheI sites, which releases a DNA fragment encoding amino acids 108–519 of Nup82p. The gapped vector was transformed into the NUP82 shuffle strain along with PCR fragments encoding amino acids 2–626 of Nup82p that had been amplified under mutagenic conditions (57Vartanian J.P. Henry M. Wain-Hobson S. Nucleic Acids Res. 1996; 24: 2627-2631Crossref PubMed Scopus (119) Google Scholar). Transformants were selected on SDC-Leu; subsequently, the wild-type plasmid was shuffled out on 5-fluoroorotic acid. 5-Fluoroorotic acid survivors were restreaked on YPD plates and selected for temperature-sensitive growth at 37 °C. Plasmids were isolated from such temperature-sensitive mutants and analyzed further. To detect the Myc tag, the monoclonal anti-c-Myc antibody-2 (9E10.3, culture supernatant; Neomarkers, Fremont, CA) was used. To visualize ProtA-tagged proteins, rabbit peroxidase-anti-peroxidase procedure (Dako A/S, Glostrup, Denmark) was used. Ascites fluid of monoclonal antibody 32D6 (a kind gift of J. Aris, University of Florida) was used to detect the C-terminal part of Nsp1p. A rabbit anti-Nup82p antibody 2E. Hurt, unpublished data. was used to visualize Nup82p. The rabbit anti-Nic96p antiserum was as described (11Grandi P. Schlaich N. Tekotte H. Hurt E.C. EMBO J. 1995; 14: 76-87Crossref PubMed Scopus (133) Google Scholar). The anti-Nup159p (No. 4) antiserum raised in guinea pig against the repeat region (55Gorsch L.C. Dockendorff T.C. Cole C.N. J. Cell Biol. 1995; 129: 939-955Crossref PubMed Scopus (166) Google Scholar) was kindly provided by C. Cole (Dartmouth Medical School, Hanover NH). Purification of ProtA fusion proteins from yeast, SDS-PAGE, Western blotting, expression and localization of GFP fusion proteins in yeast, and mass spectrometric protein identification were done as described earlier (26Segref A. Sharma K. Doye V. Hellwig A. Huber J. Luhrmann R. Hurt E. EMBO J. 1997; 16: 3256-3271Crossref PubMed Scopus (432) Google Scholar). The procedure used for immunodetection of ProtA-tagged proteins using electron microscopy was described previously (13Fahrenkrog B. Hurt E.C. Aebi U. Pante N. J. Cell Biol. 1998; 143: 577Crossref PubMed Scopus (93) Google Scholar). Minor modifications included spheroplasting using zymolyase for 15 min. To permeabilize the cells, 0.025% Triton X-100 was added to the buffer. The mechanism by which the three related nucleoporins Nup116p, Nup100p, and Nup145p-N (for a schematic drawing of the domain organization, see Fig.1 A), which all share a conserved C-terminal domain, interact with other nucleoporins and thereby perform their function in nucleocytoplasmic transport is unknown. We have previously shown that the C-terminal domain of Nup116p can target GFP to the nuclear pores (41Bailer S.M. Siniossoglou S. Podtelejnikov A. Hellwig A. Mann M. Hur" @default.
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• W2110580557 title "Nup116p Associates with the Nup82p-Nsp1p-Nup159p Nucleoporin Complex" @default.
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