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Linker Nups connect the nuclear pore complex inner ring with the outer ring and transport channel

Nature Structural & Molecular Biology volume 22, pages 774–781 (2015)Cite this article

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Abstract

Nuclear pore complexes (NPCs) mediate transport between the nucleus and cytoplasm. NPCs are composed of ∼30 nucleoporins (Nups), most of which are organized in stable subcomplexes. How these modules are interconnected within the large NPC framework has been unknown. Here we show a mechanism of how supercomplexes can form between NPC modules. The Nup192 inner-pore-ring complex serves as a seed to which the Nup82 outer-ring complex and Nsp1 channel complex are tethered. The linkage between these subcomplexes is generated by short sequences within linker Nups. The conserved Nup145N is the most versatile connector of NPC modules because it has three discrete binding sites for Nup192, Nup170 and Nup82. We assembled a large part of a Chaetomium thermophilum NPC protomer in vitro, providing a step forward toward the reconstitution of the entire NPC.

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Figure 1: CtNup145N binds Nups of different NPC modules.
Figure 2: Binding of reconstituted C. thermophilum Nup82–Nup159C–Nsp1C complex to the IRC via CtNup145NΔFG.
Figure 3: Conserved binding motifs for Nup192 and Nup170 in CtNup145N, ScNup145 and ScNup116.
Figure 4: Reconstitution of the CtNsp1 complex and binding to CtNic96 and CtNup192.
Figure 5: Reconstitution of the C. thermophilum Nup192–Nic96–Nsp1C–Nup57C–Nup49C supercomplex.
Figure 6: Reconstitution of the C. thermophilum inner-ring complex with the Nup82–Nup159C–Nsp1C outer-ring complex and Nsp1C–Nup49C–Nup57C channel complex.

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References

  1. Ptak, C., Aitchison, J.D. & Wozniak, R.W. The multifunctional nuclear pore complex: a platform for controlling gene expression. Curr. Opin. Cell Biol. 28, 46–53 (2014).

    Article  CAS  Google Scholar 

  2. Bilokapic, S. & Schwartz, T.U. 3D ultrastructure of the nuclear pore complex. Curr. Opin. Cell Biol. 24, 86–91 (2012).

    Article  CAS  Google Scholar 

  3. Wente, S.R. & Rout, M.P. The nuclear pore complex and nuclear transport. Cold Spring Harb. Perspect. Biol. 2, a000562 (2010).

    Article  CAS  Google Scholar 

  4. Fahrenkrog, B. & Aebi, U. The nuclear pore complex: nucleocytoplasmic transport and beyond. Nat. Rev. Mol. Cell Biol. 4, 757–766 (2003).

    Article  CAS  Google Scholar 

  5. Frey, S., Richter, R.P. & Gorlich, D. FG-rich repeats of nuclear pore proteins form a three-dimensional meshwork with hydrogel-like properties. Science 314, 815–817 (2006).

    Article  CAS  Google Scholar 

  6. Lim, R.Y. et al. Nanomechanical basis of selective gating by the nuclear pore complex. Science 318, 640–643 (2007).

    Article  CAS  Google Scholar 

  7. Rexach, M. & Blobel, G. Protein import into nuclei: association and dissociation reactions involving transport substrate, transport factors, and nucleoporins. Cell 83, 683–692 (1995).

    Article  CAS  Google Scholar 

  8. Schwartz, T.U. Modularity within the architecture of the nuclear pore complex. Curr. Opin. Struct. Biol. 15, 221–226 (2005).

    Article  CAS  Google Scholar 

  9. Siniossoglou, S. et al. A novel complex of nucleoporins, which includes Sec13p and a Sec13p homolog, is essential for normal nuclear pores. Cell 84, 265–275 (1996).

    Article  CAS  Google Scholar 

  10. Walther, T.C. et al. The conserved Nup107–160 complex is critical for nuclear pore complex assembly. Cell 113, 195–206 (2003).

    Article  CAS  Google Scholar 

  11. Stuwe, T. et al. Nuclear pores: architecture of the nuclear pore complex coat. Science 347, 1148–1152 (2015).

    Article  CAS  Google Scholar 

  12. Kelley, K., Knockenhauer, K.E., Kabachinski, G. & Schwartz, T.U. Atomic structure of the Y complex of the nuclear pore. Nat. Struct. Mol. Biol. 22, 425–431 (2015).

    Article  CAS  Google Scholar 

  13. Bui, K.H. et al. Integrated structural analysis of the human nuclear pore complex scaffold. Cell 155, 1233–1243 (2013).

    Article  CAS  Google Scholar 

  14. Belgareh, N. et al. Functional characterization of a Nup159p-containing nuclear pore subcomplex. Mol. Biol. Cell 9, 3475–3492 (1998).

    Article  CAS  Google Scholar 

  15. Grandi, P. et al. A novel nuclear pore protein Nup82p which specifically binds to a fraction of Nsp1p. J. Cell Biol. 130, 1263–1273 (1995).

    Article  CAS  Google Scholar 

  16. Tieg, B. & Krebber, H. Dbp5: from nuclear export to translation. Biochim. Biophys. Acta 1829, 791–798 (2013).

    Article  CAS  Google Scholar 

  17. Weirich, C.S., Erzberger, J.P., Berger, J.M. & Weis, K. The N-terminal domain of Nup159 forms a beta-propeller that functions in mRNA export by tethering the helicase Dbp5 to the nuclear pore. Mol. Cell 16, 749–760 (2004).

    Article  CAS  Google Scholar 

  18. Gaik, M. et al. Structural basis for assembly and function of the Nup82 complex in the nuclear pore scaffold. J. Cell Biol. 208, 283–297 (2015).

    Article  Google Scholar 

  19. Stelter, P. et al. Molecular basis for the functional interaction of dynein light chain with the nuclear-pore complex. Nat. Cell Biol. 9, 788–796 (2007).

    Article  CAS  Google Scholar 

  20. Grandi, P., Schlaich, N., Tekotte, H. & Hurt, E.C. Functional interaction of Nic96p with a core nucleoporin complex consisting of Nsp1p, Nup49p and a novel protein Nup57p. EMBO J. 14, 76–87 (1995).

    Article  CAS  Google Scholar 

  21. Amlacher, S. et al. Insight into structure and assembly of the nuclear pore complex by utilizing the genome of a eukaryotic thermophile. Cell 146, 277–289 (2011).

    Article  CAS  Google Scholar 

  22. Vollmer, B. & Antonin, W. The diverse roles of the Nup93/Nic96 complex proteins - structural scaffolds of the nuclear pore complex with additional cellular functions. Biol. Chem. 395, 515–528 (2014).

    Article  CAS  Google Scholar 

  23. Eisenhardt, N., Redolfi, J. & Antonin, W. Interaction of Nup53 with Ndc1 and Nup155 is required for nuclear pore complex assembly. J. Cell Sci. 127, 908–921 (2014).

    Article  CAS  Google Scholar 

  24. Mansfeld, J. et al. The conserved transmembrane nucleoporin NDC1 is required for nuclear pore complex assembly in vertebrate cells. Mol. Cell 22, 93–103 (2006).

    Article  CAS  Google Scholar 

  25. Onischenko, E., Stanton, L.H., Madrid, A.S., Kieselbach, T. & Weis, K. Role of the Ndc1 interaction network in yeast nuclear pore complex assembly and maintenance. J. Cell Biol. 185, 475–491 (2009).

    Article  CAS  Google Scholar 

  26. Rothballer, A. & Kutay, U. Poring over pores: nuclear pore complex insertion into the nuclear envelope. Trends Biochem. Sci. 38, 292–301 (2013).

    Article  CAS  Google Scholar 

  27. Solmaz, S.R., Chauhan, R., Blobel, G. & Melcak, I. Molecular architecture of the transport channel of the nuclear pore complex. Cell 147, 590–602 (2011).

    Article  CAS  Google Scholar 

  28. Chatel, G., Desai, S.H., Mattheyses, A.L., Powers, M.A. & Fahrenkrog, B. Domain topology of nucleoporin Nup98 within the nuclear pore complex. J. Struct. Biol. 177, 81–89 (2012).

    Article  CAS  Google Scholar 

  29. Fabre, E., Boelens, W.C., Wimmer, C., Mattaj, I.W. & Hurt, E.C. Nup145p is required for nuclear export of mRNA and binds homopolymeric RNA in vitro via a novel conserved motif. Cell 78, 275–289 (1994).

    Article  CAS  Google Scholar 

  30. Fontoura, B.M., Blobel, G. & Matunis, M.J. A conserved biogenesis pathway for nucleoporins: proteolytic processing of a 186-kilodalton precursor generates Nup98 and the novel nucleoporin, Nup96. J. Cell Biol. 144, 1097–1112 (1999).

    Article  CAS  Google Scholar 

  31. Rosenblum, J.S. & Blobel, G. Autoproteolysis in nucleoporin biogenesis. Proc. Natl. Acad. Sci. USA 96, 11370–11375 (1999).

    Article  CAS  Google Scholar 

  32. Teixeira, M.T. et al. Two functionally distinct domains generated by in vivo cleavage of Nup145p: a novel biogenesis pathway for nucleoporins. EMBO J. 16, 5086–5097 (1997).

    Article  CAS  Google Scholar 

  33. Stelter, P. et al. Monitoring spatiotemporal biogenesis of macromolecular assemblies by pulse-chase epitope labeling. Mol. Cell 47, 788–796 (2012).

    Article  CAS  Google Scholar 

  34. Wente, S.R. & Blobel, G. NUP145 encodes a novel yeast glycine-leucine-phenylalanine-glycine (GLFG) nucleoporin required for nuclear envelope structure. J. Cell Biol. 125, 955–969 (1994).

    Article  CAS  Google Scholar 

  35. Wente, S.R., Rout, M.P. & Blobel, G. A new family of yeast nuclear pore complex proteins. J. Cell Biol. 119, 705–723 (1992).

    Article  CAS  Google Scholar 

  36. Bailer, S.M. et al. Nup116p and nup100p are interchangeable through a conserved motif which constitutes a docking site for the mRNA transport factor gle2p. EMBO J. 17, 1107–1119 (1998).

    Article  CAS  Google Scholar 

  37. Bailer, S.M. et al. Nup116p associates with the Nup82p-Nsp1p-Nup159p nucleoporin complex. J. Biol. Chem. 275, 23540–23548 (2000).

    Article  CAS  Google Scholar 

  38. Ho, A.K. et al. Assembly and preferential localization of Nup116p on the cytoplasmic face of the nuclear pore complex by interaction with Nup82p. Mol. Cell. Biol. 20, 5736–5748 (2000).

    Article  CAS  Google Scholar 

  39. Yoshida, K., Seo, H.S., Debler, E.W., Blobel, G. & Hoelz, A. Structural and functional analysis of an essential nucleoporin heterotrimer on the cytoplasmic face of the nuclear pore complex. Proc. Natl. Acad. Sci. USA 108, 16571–16576 (2011).

    Article  CAS  Google Scholar 

  40. Stuwe, T., von Borzyskowski, L.S., Davenport, A.M. & Hoelz, A. Molecular basis for the anchoring of proto-oncoprotein Nup98 to the cytoplasmic face of the nuclear pore complex. J. Mol. Biol. 419, 330–346 (2012).

    Article  CAS  Google Scholar 

  41. Lutzmann, M. et al. Reconstitution of Nup157 and Nup145N into the Nup84 complex. J. Biol. Chem. 280, 18442–18451 (2005).

    Article  CAS  Google Scholar 

  42. Laurell, E. et al. Phosphorylation of Nup98 by multiple kinases is crucial for NPC disassembly during mitotic entry. Cell 144, 539–550 (2011).

    Article  CAS  Google Scholar 

  43. Schlaich, N.L., Haner, M., Lustig, A., Aebi, U. & Hurt, E.C. In vitro reconstitution of a heterotrimeric nucleoporin complex consisting of recombinant Nsp1p, Nup49p, and Nup57p. Mol. Biol. Cell 8, 33–46 (1997).

    Article  CAS  Google Scholar 

  44. Ulrich, A., Partridge, J.R. & Schwartz, T.U. The stoichiometry of the nucleoporin 62 subcomplex of the nuclear pore in solution. Mol. Biol. Cell 25, 1484–1492 (2014).

    Article  Google Scholar 

  45. Schlaich, N.L., Häner, M., Lustig, A., Aebi, U. & Hurt, E.C. In vitro reconstitution of a heterotrimeric nucleoporin complex consisting of recombinant Nsp1p, Nup49p and Nup57p. Mol. Biol. Cell 8, 33–46 (1997).

    Article  CAS  Google Scholar 

  46. Bailer, S.M., Balduf, C. & Hurt, E. The Nsp1p carboxy-terminal domain is organized into functionally distinct coiled-coil regions required for assembly of nucleoporin subcomplexes and nucleocytoplasmic transport. Mol. Cell. Biol. 21, 7944–7955 (2001).

    Article  CAS  Google Scholar 

  47. Andersen, K.R. et al. Scaffold nucleoporins Nup188 and Nup192 share structural and functional properties with nuclear transport receptors. eLife 2, e00745 (2013).

    Article  Google Scholar 

  48. Schrader, N. et al. Structural basis of the nic96 subcomplex organization in the nuclear pore channel. Mol. Cell 29, 46–55 (2008).

    Article  CAS  Google Scholar 

  49. Rabut, G., Doye, V. & Ellenberg, J. Mapping the dynamic organization of the nuclear pore complex inside single living cells. Nat. Cell Biol. 6, 1114–1121 (2004).

    Article  CAS  Google Scholar 

  50. Rout, M.P. et al. The yeast nuclear pore complex: composition, architecture, and transport mechanism. J. Cell Biol. 148, 635–651 (2000).

    Article  CAS  Google Scholar 

  51. Bock, T. et al. An integrated approach for genome annotation of the eukaryotic thermophile Chaetomium thermophilum. Nucleic Acids Res. 42, 13525–13533 (2014).

    Article  CAS  Google Scholar 

  52. Nissan, T.A., Bassler, J., Petfalski, E., Tollervey, D. & Hurt, E. 60S pre-ribosome formation viewed from assembly in the nucleolus until export to the cytoplasm. EMBO J. 21, 5539–5547 (2002).

    Article  CAS  Google Scholar 

  53. Thierbach, K. et al. Protein interfaces of the conserved Nup84 complex from Chaetomium thermophilum shown by crosslinking mass spectrometry and electron microscopy. StruCture 21, 1672–1682 (2013).

    Article  CAS  Google Scholar 

  54. Waterhouse, A.M., Procter, J.B., Martin, D.M., Clamp, M. & Barton, G.J. Jalview Version 2: a multiple sequence alignment editor and analysis workbench. Bioinformatics 25, 1189–1191 (2009).

    Article  CAS  Google Scholar 

  55. Cole, C., Barber, J.D. & Barton, G.J. The Jpred 3 secondary structure prediction server. Nucleic Acids Res. 36, W197–W201 (2008).

    Article  CAS  Google Scholar 

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Acknowledgements

Plasmids YEplac112-ProtA-TEV-CtMlp1N and Yep351-ProtA-TEV-ScNup170 were kindly provided by L. Dimitrova and P. Sarges (in E.H.'s laboratory), and CtMlp1N and CtNup84 were purified by L. Dimitrova and J. Schwarz (in E.H.'s laboratory). This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 638/B2 to E.H.).

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  1. Jessica Fischer and Roman Teimer: These authors contributed equally to this work.

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  1. Biochemistry Center, University of Heidelberg, Heidelberg, Germany

    Jessica Fischer, Roman Teimer, Stefan Amlacher, Ruth Kunze & Ed Hurt

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Contributions

J.F. and R.T. designed and performed the experiments. J.F. performed biochemical purifications and reconstitution of the CtNup82 and CtNsp1 complex. S.A. and R.K. identified the A and B binding motifs in CtNup145. R.T. further restricted the A and B motifs in CtNup145 and performed biochemical purifications and growth analysis related to CtNup145N, ScNup116, ScNup100 and ScNup145N. J.F. and R.T. conducted the reconstitution of supercomplexes. E.H. directed the project and, together with J.F. and R.T., wrote the manuscript.

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Correspondence to Ed Hurt.

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Integrated supplementary information

Supplementary Figure 1 CtNup145N bridges CtNup192 and CtNup170.

In vitro binding assay using immobilized IgG–ctNup192–Nup145NΔFG complex and ctNup170 in ~2.5x or ~5x molar excess (input, lanes 1–2). Samples are TEV eluates analyzed by SDS–PAGE and Coomassie staining (lanes 3–8). MW, molecular weight. Uncropped images are shown in Supplementary Data Set 1.

Supplementary Figure 2 Multiple sequence alignment of Nup145N, Nup116, Nup100 and Nup98 orthologs.

Multiple sequence alignment of Pezizomycotina Nup145N orthologs including Chaetomium thermophilum (c.t.), Chaetomium globosum (c.g.), Neurospora crassa (n.c.), Aspergillus fumigatus (a.f.), Aspergillus nidulans (a.n.) and Penicillium marnefeii (p.m.). Saccharomycetes Nup145N, Nup100 and Nup116 orthologs including Kazachstania naganishii (k.n), Kazachstania africana (k.a.), Zygosaccharomyces bailii (z.b.), Zygosaccharomyces rouxii (z.r.), Saccharomyces cerevisiae (s.c.), Kluyveromyces marxianus (k.m.), Kluyveromyces rouxii (k.r.), Candida glabrata (c.gl.). Vertebrate Nup98 orthologs including Homo sapiens (h.s.), Danio rerio (d.r.) and Sarcophilus harrisii (s.h.).

Supplementary Figure 3 ScNup192 binds to ScNup145NMD-A.

In vitro binding assay using immobilized ProtA-tagged scNup145N constructs and scNup192. Samples are SDS eluates analyzed by SDS–PAGE and Coomassie staining (lanes 1–4). MW, molecular weight. Uncropped images are shown in Supplementary Data Set 1.

Supplementary Figure 4 Growth analysis of Saccharomyces cerevisiae strain nup116Δ complemented by nup116ΔMD-A.

Growth analysis of Saccharomyces cerevisiae strain nup116Δ after transformation with empty plasmids (YCplac22) or plasmids containing wild-type scNUP116 or mutant nup116ΔMD–A, respectively. Shown are 12 individual transformants per strain.

Supplementary Figure 5 Multiple sequence alignments of Nic96 and Nsp1 and growth analysis of S. cerevisiae nic96 nsp1 double mutants.

(a) Multiple sequence alignment of Nic96 orthologs including Chaetomium thermophilum (C.t.), Saccharomyces cerevisiae (S.c.), Pichia stipitis (P.s.), Candida glabrata (C.g.), Neurospora crassa (N.c.), Xenopus laevis (X.l.), Drosophila melanogaster (D.m.), and Homo sapiens (H.s.). IM–1, interaction motif 1. (b) Multiple sequence alignment of Nsp1 orthologs including Saccharomyces cerevisiae (S.c.), Chaetomium thermophilum (C.t.), Rattus norvegicus (R.n.), Homo sapiens (H.s.), and Thielavia heterothallica (T.h.). The nsp1–L640S mutation is marked by a green arrow. The S759P mutation and the C-terminal truncation starting at T775 of the nsp1 ts18 allele are marked by orange arrows. See also Bailer, S.M. et al., Mol Cell Biol. 21, 7944–55, 2001 and Supplementary Table 1. (c) Synthetic lethal interaction between nic96 and nsp1 mutant alleles. Shown are 3 individual transformants per strain grown on 5-FOA for 6 days. In the case of nsp1 ts18, one of the three tested transformants showed growth on 5-FOA, which we assume to be a false-positive (e.g. a ura3− suppressor).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 and Supplementary Tables 1–2 (PDF 1242 kb)

Supplementary Data Set 1

Uncropped gels and blots (PDF 4183 kb)

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Fischer, J., Teimer, R., Amlacher, S. et al. Linker Nups connect the nuclear pore complex inner ring with the outer ring and transport channel. Nat Struct Mol Biol 22, 774–781 (2015). https://doi.org/10.1038/nsmb.3084

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