Skip to main content
PMC home page
Molecular Biology of the Cell logoLink to Molecular Biology of the Cell
. 1997 Jan;8(1):33–46. doi: 10.1091/mbc.8.1.33

In vitro reconstitution of a heterotrimeric nucleoporin complex consisting of recombinant Nsp1p, Nup49p, and Nup57p.

N L Schlaich 1, M Häner 1, A Lustig 1, U Aebi 1, E C Hurt 1
PMCID: PMC276057  PMID: 9017593

Abstract

The yeast nucleoporins Nsp1p, Nup49p, and Nup57p form a complex at the nuclear pores which is involved in nucleocytoplasmic transport. To investigate the molecular basis underlying complex formation, recombinant full-length Nup49p and Nup57p and the carboxyl-terminal domain of Nsp1p, which lacks the FXFG repeat domain, were expressed in Escherichia coli. When the three purified proteins were mixed together, they spontaneously associated to form a 150-kDa complex of 1:1:1 stoichiometry. In this trimeric complex, Nup57p fulfills the role of an organizing center, to which Nup49p and Nsp1p individually bind. For this interaction to occur, only two heptad repeat regions of the Nsp1p carboxyl-terminal domain are required, each region being about 50 amino acids in length. Finally, the reconstituted complex has the capability to bind to full-length Nic96p but not to mutant forms which also do not interact in vivo. When added to permeabilized yeast cells, the complex associates with the nuclear envelope and the nuclear pores. We conclude that Nsp1p, Nup49p, and Nup57p can reconstitute a complex in vitro which is competent for further assembly with other components of nuclear pores.

Full text

PDF
35

Images in this article

36Image
on p.36
37Image
on p.37
37Image
on p.37
39Image
on p.39
39Image
on p.39
40Image
on p.40
41Image
on p.41
42Image
on p.42
43Image
on p.43

Selected References

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

  1. Akey C. W. Structural plasticity of the nuclear pore complex. J Mol Biol. 1995 Apr 28;248(2):273–293. doi: 10.1016/s0022-2836(95)80050-6. [DOI] [PubMed] [Google Scholar]
  2. Barlowe C. COPII: a membrane coat that forms endoplasmic reticulum-derived vesicles. FEBS Lett. 1995 Aug 1;369(1):93–96. doi: 10.1016/0014-5793(95)00618-j. [DOI] [PubMed] [Google Scholar]
  3. Bischoff F. R., Krebber H., Kempf T., Hermes I., Ponstingl H. Human RanGTPase-activating protein RanGAP1 is a homologue of yeast Rna1p involved in mRNA processing and transport. Proc Natl Acad Sci U S A. 1995 Feb 28;92(5):1749–1753. doi: 10.1073/pnas.92.5.1749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bogerd H. P., Fridell R. A., Madore S., Cullen B. R. Identification of a novel cellular cofactor for the Rev/Rex class of retroviral regulatory proteins. Cell. 1995 Aug 11;82(3):485–494. doi: 10.1016/0092-8674(95)90437-9. [DOI] [PubMed] [Google Scholar]
  5. Buss F., Kent H., Stewart M., Bailer S. M., Hanover J. A. Role of different domains in the self-association of rat nucleoporin p62. J Cell Sci. 1994 Feb;107(Pt 2):631–638. doi: 10.1242/jcs.107.2.631. [DOI] [PubMed] [Google Scholar]
  6. Buss F., Stewart M. Macromolecular interactions in the nucleoporin p62 complex of rat nuclear pores: binding of nucleoporin p54 to the rod domain of p62. J Cell Biol. 1995 Feb;128(3):251–261. doi: 10.1083/jcb.128.3.251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Carmo-Fonseca M., Kern H., Hurt E. C. Human nucleoporin p62 and the essential yeast nuclear pore protein NSP1 show sequence homology and a similar domain organization. Eur J Cell Biol. 1991 Jun;55(1):17–30. [PubMed] [Google Scholar]
  8. Carmo-Fonseca M., Kern H., Hurt E. C. Human nucleoporin p62 and the essential yeast nuclear pore protein NSP1 show sequence homology and a similar domain organization. Eur J Cell Biol. 1991 Jun;55(1):17–30. [PubMed] [Google Scholar]
  9. Dabauvalle M. C., Benavente R., Chaly N. Monoclonal antibodies to a Mr 68,000 pore complex glycoprotein interfere with nuclear protein uptake in Xenopus oocytes. Chromosoma. 1988 Nov;97(3):193–197. doi: 10.1007/BF00292960. [DOI] [PubMed] [Google Scholar]
  10. Dabauvalle M. C., Loos K., Scheer U. Identification of a soluble precursor complex essential for nuclear pore assembly in vitro. Chromosoma. 1990 Dec;100(1):56–66. doi: 10.1007/BF00337603. [DOI] [PubMed] [Google Scholar]
  11. Davis L. I., Blobel G. Identification and characterization of a nuclear pore complex protein. Cell. 1986 Jun 6;45(5):699–709. doi: 10.1016/0092-8674(86)90784-1. [DOI] [PubMed] [Google Scholar]
  12. Davis L. I., Fink G. R. The NUP1 gene encodes an essential component of the yeast nuclear pore complex. Cell. 1990 Jun 15;61(6):965–978. doi: 10.1016/0092-8674(90)90062-j. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Dingwall C., Laskey R. A. Nuclear targeting sequences--a consensus? Trends Biochem Sci. 1991 Dec;16(12):478–481. doi: 10.1016/0968-0004(91)90184-w. [DOI] [PubMed] [Google Scholar]
  15. Doye V., Hurt E. C. Genetic approaches to nuclear pore structure and function. Trends Genet. 1995 Jun;11(6):235–241. doi: 10.1016/s0168-9525(00)89057-5. [DOI] [PubMed] [Google Scholar]
  16. Finlay D. R., Meier E., Bradley P., Horecka J., Forbes D. J. A complex of nuclear pore proteins required for pore function. J Cell Biol. 1991 Jul;114(1):169–183. doi: 10.1083/jcb.114.1.169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Fischer U., Huber J., Boelens W. C., Mattaj I. W., Lührmann R. The HIV-1 Rev activation domain is a nuclear export signal that accesses an export pathway used by specific cellular RNAs. Cell. 1995 Aug 11;82(3):475–483. doi: 10.1016/0092-8674(95)90436-0. [DOI] [PubMed] [Google Scholar]
  18. Gerace L. Nuclear export signals and the fast track to the cytoplasm. Cell. 1995 Aug 11;82(3):341–344. doi: 10.1016/0092-8674(95)90420-4. [DOI] [PubMed] [Google Scholar]
  19. Goldberg M. W., Allen T. D. Structural and functional organization of the nuclear envelope. Curr Opin Cell Biol. 1995 Jun;7(3):301–309. doi: 10.1016/0955-0674(95)80083-2. [DOI] [PubMed] [Google Scholar]
  20. Goldfarb D. S. Protein translocation. GTPase cycle for nuclear transport. Curr Biol. 1994 Jan 1;4(1):57–60. doi: 10.1016/s0960-9822(00)00013-0. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. 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]
  24. Grote M., Kubitscheck U., Reichelt R., Peters R. Mapping of nucleoporins to the center of the nuclear pore complex by post-embedding immunogold electron microscopy. J Cell Sci. 1995 Sep;108(Pt 9):2963–2972. doi: 10.1242/jcs.108.9.2963. [DOI] [PubMed] [Google Scholar]
  25. Guan T., Müller S., Klier G., Panté N., Blevitt J. M., Haner M., Paschal B., Aebi U., Gerace L. Structural analysis of the p62 complex, an assembly of O-linked glycoproteins that localizes near the central gated channel of the nuclear pore complex. Mol Biol Cell. 1995 Nov;6(11):1591–1603. doi: 10.1091/mbc.6.11.1591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Görlich D., Kostka S., Kraft R., Dingwall C., Laskey R. A., Hartmann E., Prehn S. Two different subunits of importin cooperate to recognize nuclear localization signals and bind them to the nuclear envelope. Curr Biol. 1995 Apr 1;5(4):383–392. doi: 10.1016/s0960-9822(95)00079-0. [DOI] [PubMed] [Google Scholar]
  27. Görlich D., Prehn S., Laskey R. A., Hartmann E. Isolation of a protein that is essential for the first step of nuclear protein import. Cell. 1994 Dec 2;79(5):767–778. doi: 10.1016/0092-8674(94)90067-1. [DOI] [PubMed] [Google Scholar]
  28. Hu T., Guan T., Gerace L. Molecular and functional characterization of the p62 complex, an assembly of nuclear pore complex glycoproteins. J Cell Biol. 1996 Aug;134(3):589–601. doi: 10.1083/jcb.134.3.589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Imamoto N., Tachibana T., Matsubae M., Yoneda Y. A karyophilic protein forms a stable complex with cytoplasmic components prior to nuclear pore binding. J Biol Chem. 1995 Apr 14;270(15):8559–8565. doi: 10.1074/jbc.270.15.8559. [DOI] [PubMed] [Google Scholar]
  30. Izaurralde E., Mattaj I. W. RNA export. Cell. 1995 Apr 21;81(2):153–159. doi: 10.1016/0092-8674(95)90323-2. [DOI] [PubMed] [Google Scholar]
  31. Kadowaki T., Goldfarb D., Spitz L. M., Tartakoff A. M., Ohno M. Regulation of RNA processing and transport by a nuclear guanine nucleotide release protein and members of the Ras superfamily. EMBO J. 1993 Jul;12(7):2929–2937. doi: 10.1002/j.1460-2075.1993.tb05955.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kita K., Omata S., Horigome T. Purification and characterization of a nuclear pore glycoprotein complex containing p62. J Biochem. 1993 Mar;113(3):377–382. doi: 10.1093/oxfordjournals.jbchem.a124054. [DOI] [PubMed] [Google Scholar]
  33. Melchior F., Gerace L. Mechanisms of nuclear protein import. Curr Opin Cell Biol. 1995 Jun;7(3):310–318. doi: 10.1016/0955-0674(95)80084-0. [DOI] [PubMed] [Google Scholar]
  34. Moore M. S., Blobel G. A G protein involved in nucleocytoplasmic transport: the role of Ran. Trends Biochem Sci. 1994 May;19(5):211–216. doi: 10.1016/0968-0004(94)90024-8. [DOI] [PubMed] [Google Scholar]
  35. Moroianu J., Blobel G., Radu A. Previously identified protein of uncertain function is karyopherin alpha and together with karyopherin beta docks import substrate at nuclear pore complexes. Proc Natl Acad Sci U S A. 1995 Mar 14;92(6):2008–2011. doi: 10.1073/pnas.92.6.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Nehrbass U., Fabre E., Dihlmann S., Herth W., Hurt E. C. Analysis of nucleo-cytoplasmic transport in a thermosensitive mutant of nuclear pore protein NSP1. Eur J Cell Biol. 1993 Oct;62(1):1–12. [PubMed] [Google Scholar]
  37. Nehrbass U., Kern H., Mutvei A., Horstmann H., Marshallsay B., Hurt E. C. NSP1: a yeast nuclear envelope protein localized at the nuclear pores exerts its essential function by its carboxy-terminal domain. Cell. 1990 Jun 15;61(6):979–989. doi: 10.1016/0092-8674(90)90063-k. [DOI] [PubMed] [Google Scholar]
  38. Paschal B. M., Gerace L. Identification of NTF2, a cytosolic factor for nuclear import that interacts with nuclear pore complex protein p62. J Cell Biol. 1995 May;129(4):925–937. doi: 10.1083/jcb.129.4.925. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Radu A., Blobel G., Moore M. S. Identification of a protein complex that is required for nuclear protein import and mediates docking of import substrate to distinct nucleoporins. Proc Natl Acad Sci U S A. 1995 Feb 28;92(5):1769–1773. doi: 10.1073/pnas.92.5.1769. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Radu A., Moore M. S., Blobel G. The peptide repeat domain of nucleoporin Nup98 functions as a docking site in transport across the nuclear pore complex. Cell. 1995 Apr 21;81(2):215–222. doi: 10.1016/0092-8674(95)90331-3. [DOI] [PubMed] [Google Scholar]
  41. Reichelt R., Holzenburg A., Buhle E. L., Jr, Jarnik M., Engel A., Aebi U. Correlation between structure and mass distribution of the nuclear pore complex and of distinct pore complex components. J Cell Biol. 1990 Apr;110(4):883–894. doi: 10.1083/jcb.110.4.883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Rexach M., Blobel G. Protein import into nuclei: association and dissociation reactions involving transport substrate, transport factors, and nucleoporins. Cell. 1995 Dec 1;83(5):683–692. doi: 10.1016/0092-8674(95)90181-7. [DOI] [PubMed] [Google Scholar]
  43. Rout M. P., Blobel G. Isolation of the yeast nuclear pore complex. J Cell Biol. 1993 Nov;123(4):771–783. doi: 10.1083/jcb.123.4.771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Schlenstedt G., Hurt E., Doye V., Silver P. A. Reconstitution of nuclear protein transport with semi-intact yeast cells. J Cell Biol. 1993 Nov;123(4):785–798. doi: 10.1083/jcb.123.4.785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Simos G., Hurt E. C. Nucleocytoplasmic transport: factors and mechanisms. FEBS Lett. 1995 Aug 1;369(1):107–112. doi: 10.1016/0014-5793(95)00674-x. [DOI] [PubMed] [Google Scholar]
  46. 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]
  47. Starr C. M., D'Onofrio M., Park M. K., Hanover J. A. Primary sequence and heterologous expression of nuclear pore glycoprotein p62. J Cell Biol. 1990 Jun;110(6):1861–1871. doi: 10.1083/jcb.110.6.1861. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  49. Tartakoff A. M., Schneiter R. The nuclear GTPase cycle: promoting peripheralization? Trends Cell Biol. 1995 Jan;5(1):5–8. doi: 10.1016/s0962-8924(00)88925-4. [DOI] [PubMed] [Google Scholar]
  50. Weis K., Mattaj I. W., Lamond A. I. Identification of hSRP1 alpha as a functional receptor for nuclear localization sequences. Science. 1995 May 19;268(5213):1049–1053. doi: 10.1126/science.7754385. [DOI] [PubMed] [Google Scholar]
  51. Wen W., Meinkoth J. L., Tsien R. Y., Taylor S. S. Identification of a signal for rapid export of proteins from the nucleus. Cell. 1995 Aug 11;82(3):463–473. doi: 10.1016/0092-8674(95)90435-2. [DOI] [PubMed] [Google Scholar]
  52. Wimmer C., Doye V., Grandi P., Nehrbass U., Hurt E. C. A new subclass of nucleoporins that functionally interact with nuclear pore protein NSP1. EMBO J. 1992 Dec;11(13):5051–5061. doi: 10.1002/j.1460-2075.1992.tb05612.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Zapp M. L. The ins and outs of RNA nucleocytoplasmic transport. Curr Opin Genet Dev. 1995 Apr;5(2):229–233. doi: 10.1016/0959-437x(95)80013-1. [DOI] [PubMed] [Google Scholar]

Articles from Molecular Biology of the Cell are provided here courtesy of American Society for Cell Biology

RESOURCES