Skip to main content
PMC home page
Biophysical Journal logoLink to Biophysical Journal
. 2002 Dec;83(6):3626–3636. doi: 10.1016/S0006-3495(02)75363-0

In situ analysis of spatial relationships between proteins of the nuclear pore complex.

Marc Damelin 1, Pamela A Silver 1
PMCID: PMC1302438  PMID: 12496130

Abstract

Macromolecular transport between the nucleus and cytoplasm occurs through the nuclear pore complexes (NPCs). The NPC in the budding yeast Saccharomyces cerevisiae is a 60-MDa structure embedded in the nuclear envelope and composed of ~30 proteins, termed nucleoporins or nups. Here we present a large-scale analysis of spatial relationships between nucleoporins using fluorescence resonance energy transfer (FRET) in living yeast cells. Energy transfer was measured in a panel of strains, each of which coexpresses the enhanced cyan and yellow fluorescent proteins as fusions to distinct nucleoporins. With this approach, we have determined 13 nucleoporin pairs yielding FRET signals. Independent experiments are consistent with the FRET results: Nup120 localization is perturbed in the nic96-1 mutant, as is Nup82 localization in the nup116Delta mutant. To better understand the spatial relationship represented by an in vivo FRET signal, we have investigated the requirements of these signals. We demonstrate that in one case FRET signal is lost upon insertion of a short spacer between the nucleoporin and its enhanced yellow fluorescent protein label. We also show that the Nup120 FRET signals depend on whether the fluorescent moiety is fused to the N- or C-terminus of Nup120. Combined with existing data on NPC structure, the FRET pairs identified in this study allow us to propose a refined molecular model of the NPC. We suggest that the approach may serve as a prototype for the in situ study of other large macromolecular complexes.

Full Text

The Full Text of this article is available as a PDF (358.6 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aitchison J. D., Blobel G., Rout M. P. Nup120p: a yeast nucleoporin required for NPC distribution and mRNA transport. J Cell Biol. 1995 Dec;131(6 Pt 2):1659–1675. doi: 10.1083/jcb.131.6.1659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bailer S. M., Balduf C., Katahira J., Podtelejnikov A., Rollenhagen C., Mann M., Pante N., Hurt E. Nup116p associates with the Nup82p-Nsp1p-Nup159p nucleoporin complex. J Biol Chem. 2000 Aug 4;275(31):23540–23548. doi: 10.1074/jbc.M001963200. [DOI] [PubMed] [Google Scholar]
  3. Belgareh N., Snay-Hodge C., Pasteau F., Dagher S., Cole C. N., Doye V. Functional characterization of a Nup159p-containing nuclear pore subcomplex. Mol Biol Cell. 1998 Dec;9(12):3475–3492. doi: 10.1091/mbc.9.12.3475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Corbett A. H., Silver P. A. Nucleocytoplasmic transport of macromolecules. Microbiol Mol Biol Rev. 1997 Jun;61(2):193–211. doi: 10.1128/mmbr.61.2.193-211.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Damelin M., Silver P. A. Mapping interactions between nuclear transport factors in living cells reveals pathways through the nuclear pore complex. Mol Cell. 2000 Jan;5(1):133–140. doi: 10.1016/s1097-2765(00)80409-8. [DOI] [PubMed] [Google Scholar]
  6. Davis L. I. The nuclear pore complex. Annu Rev Biochem. 1995;64:865–896. doi: 10.1146/annurev.bi.64.070195.004245. [DOI] [PubMed] [Google Scholar]
  7. Day R. N. Visualization of Pit-1 transcription factor interactions in the living cell nucleus by fluorescence resonance energy transfer microscopy. Mol Endocrinol. 1998 Sep;12(9):1410–1419. doi: 10.1210/mend.12.9.0168. [DOI] [PubMed] [Google Scholar]
  8. Del Priore V., Snay C. A., Bahr A., Cole C. N. The product of the Saccharomyces cerevisiae RSS1 gene, identified as a high-copy suppressor of the rat7-1 temperature-sensitive allele of the RAT7/NUP159 nucleoporin, is required for efficient mRNA export. Mol Biol Cell. 1996 Oct;7(10):1601–1621. doi: 10.1091/mbc.7.10.1601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fahrenkrog B., Hurt E. C., Aebi U., Panté N. Molecular architecture of the yeast nuclear pore complex: localization of Nsp1p subcomplexes. J Cell Biol. 1998 Nov 2;143(3):577–588. doi: 10.1083/jcb.143.3.577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fahrenkrog B., Hübner W., Mandinova A., Panté N., Keller W., Aebi U. The yeast nucleoporin Nup53p specifically interacts with Nic96p and is directly involved in nuclear protein import. Mol Biol Cell. 2000 Nov;11(11):3885–3896. doi: 10.1091/mbc.11.11.3885. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gordon G. W., Berry G., Liang X. H., Levine B., Herman B. Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy. Biophys J. 1998 May;74(5):2702–2713. doi: 10.1016/S0006-3495(98)77976-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gorsch L. C., Dockendorff T. C., Cole C. N. A conditional allele of the novel repeat-containing yeast nucleoporin RAT7/NUP159 causes both rapid cessation of mRNA export and reversible clustering of nuclear pore complexes. J Cell Biol. 1995 May;129(4):939–955. doi: 10.1083/jcb.129.4.939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Grandi P., Doye V., Hurt E. C. Purification of NSP1 reveals complex formation with 'GLFG' nucleoporins and a novel nuclear pore protein NIC96. EMBO J. 1993 Aug;12(8):3061–3071. doi: 10.1002/j.1460-2075.1993.tb05975.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Grandi P., Emig S., Weise C., Hucho F., Pohl T., Hurt E. C. A novel nuclear pore protein Nup82p which specifically binds to a fraction of Nsp1p. J Cell Biol. 1995 Sep;130(6):1263–1273. doi: 10.1083/jcb.130.6.1263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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. 1995 Jan 3;14(1):76–87. doi: 10.1002/j.1460-2075.1995.tb06977.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hailey Dale W., Davis Trisha N., Muller Eric G. D. Fluorescence resonance energy transfer using color variants of green fluorescent protein. Methods Enzymol. 2002;351:34–49. doi: 10.1016/s0076-6879(02)51840-1. [DOI] [PubMed] [Google Scholar]
  17. Heath C. V., Copeland C. S., Amberg D. C., Del Priore V., Snyder M., Cole C. N. Nuclear pore complex clustering and nuclear accumulation of poly(A)+ RNA associated with mutation of the Saccharomyces cerevisiae RAT2/NUP120 gene. J Cell Biol. 1995 Dec;131(6 Pt 2):1677–1697. doi: 10.1083/jcb.131.6.1677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Heim R., Tsien R. Y. Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr Biol. 1996 Feb 1;6(2):178–182. doi: 10.1016/s0960-9822(02)00450-5. [DOI] [PubMed] [Google Scholar]
  19. Ho A. K., Shen T. X., Ryan K. J., Kiseleva E., Levy M. A., Allen T. D., Wente S. R. Assembly and preferential localization of Nup116p on the cytoplasmic face of the nuclear pore complex by interaction with Nup82p. Mol Cell Biol. 2000 Aug;20(15):5736–5748. doi: 10.1128/mcb.20.15.5736-5748.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hurwitz M. E., Blobel G. NUP82 is an essential yeast nucleoporin required for poly(A)+ RNA export. J Cell Biol. 1995 Sep;130(6):1275–1281. doi: 10.1083/jcb.130.6.1275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Immink Richard G. H., Gadella Theodorus W. J., Jr, Ferrario Silvia, Busscher Marco, Angenent Gerco C. Analysis of MADS box protein-protein interactions in living plant cells. Proc Natl Acad Sci U S A. 2002 Feb 19;99(4):2416–2421. doi: 10.1073/pnas.042677699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Jiang Xuejun, Sorkin Alexander. Coordinated traffic of Grb2 and Ras during epidermal growth factor receptor endocytosis visualized in living cells. Mol Biol Cell. 2002 May;13(5):1522–1535. doi: 10.1091/mbc.01-11-0552. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kenna M. A., Petranka J. G., Reilly J. L., Davis L. I. Yeast N1e3p/Nup170p is required for normal stoichiometry of FG nucleoporins within the nuclear pore complex. Mol Cell Biol. 1996 May;16(5):2025–2036. doi: 10.1128/mcb.16.5.2025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lutzmann Malik, Kunze Ruth, Buerer Andrea, Aebi Ueli, Hurt Ed. Modular self-assembly of a Y-shaped multiprotein complex from seven nucleoporins. EMBO J. 2002 Feb 1;21(3):387–397. doi: 10.1093/emboj/21.3.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mahajan N. P., Linder K., Berry G., Gordon G. W., Heim R., Herman B. Bcl-2 and Bax interactions in mitochondria probed with green fluorescent protein and fluorescence resonance energy transfer. Nat Biotechnol. 1998 Jun;16(6):547–552. doi: 10.1038/nbt0698-547. [DOI] [PubMed] [Google Scholar]
  26. Majoul I., Straub M., Hell S. W., Duden R., Söling H. D. KDEL-cargo regulates interactions between proteins involved in COPI vesicle traffic: measurements in living cells using FRET. Dev Cell. 2001 Jul;1(1):139–153. doi: 10.1016/s1534-5807(01)00004-1. [DOI] [PubMed] [Google Scholar]
  27. Majoul Irina, Straub Martin, Duden Rainer, Hell Stefan W., Söling Hans-Dieter. Fluorescence resonance energy transfer analysis of protein-protein interactions in single living cells by multifocal multiphoton microscopy. J Biotechnol. 2002 Jan;82(3):267–277. doi: 10.1016/s1389-0352(01)00042-3. [DOI] [PubMed] [Google Scholar]
  28. Marelli M., Aitchison J. D., Wozniak R. W. Specific binding of the karyopherin Kap121p to a subunit of the nuclear pore complex containing Nup53p, Nup59p, and Nup170p. J Cell Biol. 1998 Dec 28;143(7):1813–1830. doi: 10.1083/jcb.143.7.1813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mattaj I. W., Englmeier L. Nucleocytoplasmic transport: the soluble phase. Annu Rev Biochem. 1998;67:265–306. doi: 10.1146/annurev.biochem.67.1.265. [DOI] [PubMed] [Google Scholar]
  30. Miyawaki A., Llopis J., Heim R., McCaffery J. M., Adams J. A., Ikura M., Tsien R. Y. Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature. 1997 Aug 28;388(6645):882–887. doi: 10.1038/42264. [DOI] [PubMed] [Google Scholar]
  31. Mochizuki N., Yamashita S., Kurokawa K., Ohba Y., Nagai T., Miyawaki A., Matsuda M. Spatio-temporal images of growth-factor-induced activation of Ras and Rap1. Nature. 2001 Jun 28;411(6841):1065–1068. doi: 10.1038/35082594. [DOI] [PubMed] [Google Scholar]
  32. Murphy R., Wente S. R. An RNA-export mediator with an essential nuclear export signal. Nature. 1996 Sep 26;383(6598):357–360. doi: 10.1038/383357a0. [DOI] [PubMed] [Google Scholar]
  33. Nakielny S., Shaikh S., Burke B., Dreyfuss G. Nup153 is an M9-containing mobile nucleoporin with a novel Ran-binding domain. EMBO J. 1999 Apr 1;18(7):1982–1995. doi: 10.1093/emboj/18.7.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Nehrbass U., Rout M. P., Maguire S., Blobel G., Wozniak R. W. The yeast nucleoporin Nup188p interacts genetically and physically with the core structures of the nuclear pore complex. J Cell Biol. 1996 Jun;133(6):1153–1162. doi: 10.1083/jcb.133.6.1153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Nigg E. A. Nucleocytoplasmic transport: signals, mechanisms and regulation. Nature. 1997 Apr 24;386(6627):779–787. doi: 10.1038/386779a0. [DOI] [PubMed] [Google Scholar]
  36. Ohno M., Fornerod M., Mattaj I. W. Nucleocytoplasmic transport: the last 200 nanometers. Cell. 1998 Feb 6;92(3):327–336. doi: 10.1016/s0092-8674(00)80926-5. [DOI] [PubMed] [Google Scholar]
  37. Rappsilber J., Siniossoglou S., Hurt E. C., Mann M. A generic strategy to analyze the spatial organization of multi-protein complexes by cross-linking and mass spectrometry. Anal Chem. 2000 Jan 15;72(2):267–275. doi: 10.1021/ac991081o. [DOI] [PubMed] [Google Scholar]
  38. Rout M. P., Aitchison J. D., Suprapto A., Hjertaas K., Zhao Y., Chait B. T. The yeast nuclear pore complex: composition, architecture, and transport mechanism. J Cell Biol. 2000 Feb 21;148(4):635–651. doi: 10.1083/jcb.148.4.635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Ruiz-Velasco V., Ikeda S. R. Functional expression and FRET analysis of green fluorescent proteins fused to G-protein subunits in rat sympathetic neurons. J Physiol. 2001 Dec 15;537(Pt 3):679–692. doi: 10.1111/j.1469-7793.2001.00679.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sato Moritoshi, Ozawa Takeaki, Inukai Kouichi, Asano Tomoichiro, Umezawa Yoshio. Fluorescent indicators for imaging protein phosphorylation in single living cells. Nat Biotechnol. 2002 Mar;20(3):287–294. doi: 10.1038/nbt0302-287. [DOI] [PubMed] [Google Scholar]
  41. 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. 1997 Jan;8(1):33–46. doi: 10.1091/mbc.8.1.33. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Siniossoglou S., Lutzmann M., Santos-Rosa H., Leonard K., Mueller S., Aebi U., Hurt E. Structure and assembly of the Nup84p complex. J Cell Biol. 2000 Apr 3;149(1):41–54. doi: 10.1083/jcb.149.1.41. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Siniossoglou S., Wimmer C., Rieger M., Doye V., Tekotte H., Weise C., Emig S., Segref A., Hurt E. C. A novel complex of nucleoporins, which includes Sec13p and a Sec13p homolog, is essential for normal nuclear pores. Cell. 1996 Jan 26;84(2):265–275. doi: 10.1016/s0092-8674(00)80981-2. [DOI] [PubMed] [Google Scholar]
  44. Speicher D. W., Marchesi V. T. Erythrocyte spectrin is comprised of many homologous triple helical segments. Nature. 1984 Sep 13;311(5982):177–180. doi: 10.1038/311177a0. [DOI] [PubMed] [Google Scholar]
  45. Stoffler D., Fahrenkrog B., Aebi U. The nuclear pore complex: from molecular architecture to functional dynamics. Curr Opin Cell Biol. 1999 Jun;11(3):391–401. doi: 10.1016/S0955-0674(99)80055-6. [DOI] [PubMed] [Google Scholar]
  46. Stryer L. Fluorescence energy transfer as a spectroscopic ruler. Annu Rev Biochem. 1978;47:819–846. doi: 10.1146/annurev.bi.47.070178.004131. [DOI] [PubMed] [Google Scholar]
  47. Ting A. Y., Kain K. H., Klemke R. L., Tsien R. Y. Genetically encoded fluorescent reporters of protein tyrosine kinase activities in living cells. Proc Natl Acad Sci U S A. 2001 Dec 18;98(26):15003–15008. doi: 10.1073/pnas.211564598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Truong K., Sawano A., Mizuno H., Hama H., Tong K. I., Mal T. K., Miyawaki A., Ikura M. FRET-based in vivo Ca2+ imaging by a new calmodulin-GFP fusion molecule. Nat Struct Biol. 2001 Dec;8(12):1069–1073. doi: 10.1038/nsb728. [DOI] [PubMed] [Google Scholar]
  49. Tsien R. Y. The green fluorescent protein. Annu Rev Biochem. 1998;67:509–544. doi: 10.1146/annurev.biochem.67.1.509. [DOI] [PubMed] [Google Scholar]
  50. Warren Derek T., Andrews Paul D., Gourlay Campbell W., Ayscough Kathryn R. Sla1p couples the yeast endocytic machinery to proteins regulating actin dynamics. J Cell Sci. 2002 Apr 15;115(Pt 8):1703–1715. doi: 10.1242/jcs.115.8.1703. [DOI] [PubMed] [Google Scholar]
  51. Weiss T. S., Chamberlain C. E., Takeda T., Lin P., Hahn K. M., Farquhar M. G. Galpha i3 binding to calnuc on Golgi membranes in living cells monitored by fluorescence resonance energy transfer of green fluorescent protein fusion proteins. Proc Natl Acad Sci U S A. 2001 Dec 18;98(26):14961–14966. doi: 10.1073/pnas.261572098. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Wente S. R., Blobel G. A temperature-sensitive NUP116 null mutant forms a nuclear envelope seal over the yeast nuclear pore complex thereby blocking nucleocytoplasmic traffic. J Cell Biol. 1993 Oct;123(2):275–284. doi: 10.1083/jcb.123.2.275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Wilson Marieangela C., Meredith David, Halestrap Andrew P. Fluorescence resonance energy transfer studies on the interaction between the lactate transporter MCT1 and CD147 provide information on the topology and stoichiometry of the complex in situ. J Biol Chem. 2001 Nov 21;277(5):3666–3672. doi: 10.1074/jbc.M109658200. [DOI] [PubMed] [Google Scholar]
  54. Winston F., Dollard C., Ricupero-Hovasse S. L. Construction of a set of convenient Saccharomyces cerevisiae strains that are isogenic to S288C. Yeast. 1995 Jan;11(1):53–55. doi: 10.1002/yea.320110107. [DOI] [PubMed] [Google Scholar]
  55. Yan Y., Winograd E., Viel A., Cronin T., Harrison S. C., Branton D. Crystal structure of the repetitive segments of spectrin. Science. 1993 Dec 24;262(5142):2027–2030. doi: 10.1126/science.8266097. [DOI] [PubMed] [Google Scholar]
  56. Yang Q., Rout M. P., Akey C. W. Three-dimensional architecture of the isolated yeast nuclear pore complex: functional and evolutionary implications. Mol Cell. 1998 Jan;1(2):223–234. doi: 10.1016/s1097-2765(00)80023-4. [DOI] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES