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. 1995 Jan 3;14(1):76–87. doi: 10.1002/j.1460-2075.1995.tb06977.x

Functional interaction of Nic96p with a core nucleoporin complex consisting of Nsp1p, Nup49p and a novel protein Nup57p.

P Grandi 1, N Schlaich 1, H Tekotte 1, E C Hurt 1
PMCID: PMC398054  PMID: 7828598

Abstract

Nic96p has been isolated previously in a complex together with the nuclear pore proteins Nsp1p, Nup49p and a p54 polypeptide. In a genetic screen for Nsp1p-interacting components, we now find NIC96, as well as a novel gene NUP57 which encodes the p54 protein (called Nup57p). Nup57p which is essential for cell growth contains GLFG repeats in the N-terminal half and heptad repeats in the C-terminal half. The domain organization of Nic96p is more complex: N-terminally located heptad repeats mediate binding to a trimeric Nsp1p-Nup49p-Nup57p complex, but are not required for the formation of this core complex; single amino acid substitutions in the central domain yield thermosensitive mutants, which do not impair interaction with the Nsp1 complex; the C-terminal domain is neither essential nor required for binding to the nucleoporin complex, but strikingly mutations in this part cause synthetic lethality with nsp1 and nup57 mutant alleles. Since a strain in which the Nic96p heptad repeats were deleted shows, similar to nsp1 and nup49 mutants, cytoplasmic mislocalization of a nuclear reporter protein, we propose that the interaction of the heterotrimeric Nsp1p-Nup49p-Nup57p core complex with Nic96p is required for protein transport into the nucleus.

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Selected References

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

  1. Akey C. W. Interactions and structure of the nuclear pore complex revealed by cryo-electron microscopy. J Cell Biol. 1989 Sep;109(3):955–970. doi: 10.1083/jcb.109.3.955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Akey C. W., Radermacher M. Architecture of the Xenopus nuclear pore complex revealed by three-dimensional cryo-electron microscopy. J Cell Biol. 1993 Jul;122(1):1–19. doi: 10.1083/jcb.122.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Akey C. W. Visualization of transport-related configurations of the nuclear pore transporter. Biophys J. 1990 Aug;58(2):341–355. doi: 10.1016/S0006-3495(90)82381-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Belanger K. D., Kenna M. A., Wei S., Davis L. I. Genetic and physical interactions between Srp1p and nuclear pore complex proteins Nup1p and Nup2p. J Cell Biol. 1994 Aug;126(3):619–630. doi: 10.1083/jcb.126.3.619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bergès T., Petfalski E., Tollervey D., Hurt E. C. Synthetic lethality with fibrillarin identifies NOP77p, a nucleolar protein required for pre-rRNA processing and modification. EMBO J. 1994 Jul 1;13(13):3136–3148. doi: 10.1002/j.1460-2075.1994.tb06612.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bonner W. M. Protein migration into nuclei. I. Frog oocyte nuclei in vivo accumulate microinjected histones, allow entry to small proteins, and exclude large proteins. J Cell Biol. 1975 Feb;64(2):421–430. doi: 10.1083/jcb.64.2.421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  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. Cordes V. C., Reidenbach S., Köhler A., Stuurman N., van Driel R., Franke W. W. Intranuclear filaments containing a nuclear pore complex protein. J Cell Biol. 1993 Dec;123(6 Pt 1):1333–1344. doi: 10.1083/jcb.123.6.1333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cordes V., Waizenegger I., Krohne G. Nuclear pore complex glycoprotein p62 of Xenopus laevis and mouse: cDNA cloning and identification of its glycosylated region. Eur J Cell Biol. 1991 Jun;55(1):31–47. [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. 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]
  14. Doye V., Wepf R., Hurt E. C. A novel nuclear pore protein Nup133p with distinct roles in poly(A)+ RNA transport and nuclear pore distribution. EMBO J. 1994 Dec 15;13(24):6062–6075. doi: 10.1002/j.1460-2075.1994.tb06953.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dwyer N., Blobel G. A modified procedure for the isolation of a pore complex-lamina fraction from rat liver nuclei. J Cell Biol. 1976 Sep;70(3):581–591. doi: 10.1083/jcb.70.3.581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Elledge S. J., Davis R. W. A family of versatile centromeric vectors designed for use in the sectoring-shuffle mutagenesis assay in Saccharomyces cerevisiae. Gene. 1988 Oct 30;70(2):303–312. doi: 10.1016/0378-1119(88)90202-8. [DOI] [PubMed] [Google Scholar]
  17. 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. 1994 Jul 29;78(2):275–289. doi: 10.1016/0092-8674(94)90297-6. [DOI] [PubMed] [Google Scholar]
  18. Fabre E., Hurt E. C. Nuclear transport. Curr Opin Cell Biol. 1994 Jun;6(3):335–342. doi: 10.1016/0955-0674(94)90023-x. [DOI] [PubMed] [Google Scholar]
  19. Featherstone C., Darby M. K., Gerace L. A monoclonal antibody against the nuclear pore complex inhibits nucleocytoplasmic transport of protein and RNA in vivo. J Cell Biol. 1988 Oct;107(4):1289–1297. doi: 10.1083/jcb.107.4.1289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Feldherr C. M., Kallenbach E., Schultz N. Movement of a karyophilic protein through the nuclear pores of oocytes. J Cell Biol. 1984 Dec;99(6):2216–2222. doi: 10.1083/jcb.99.6.2216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Finlay D. R., Forbes D. J. Reconstitution of biochemically altered nuclear pores: transport can be eliminated and restored. Cell. 1990 Jan 12;60(1):17–29. doi: 10.1016/0092-8674(90)90712-n. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Forbes D. J. Structure and function of the nuclear pore complex. Annu Rev Cell Biol. 1992;8:495–527. doi: 10.1146/annurev.cb.08.110192.002431. [DOI] [PubMed] [Google Scholar]
  24. Gerace L., Ottaviano Y., Kondor-Koch C. Identification of a major polypeptide of the nuclear pore complex. J Cell Biol. 1982 Dec;95(3):826–837. doi: 10.1083/jcb.95.3.826. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Goldberg M. W., Allen T. D. High resolution scanning electron microscopy of the nuclear envelope: demonstration of a new, regular, fibrous lattice attached to the baskets of the nucleoplasmic face of the nuclear pores. J Cell Biol. 1992 Dec;119(6):1429–1440. doi: 10.1083/jcb.119.6.1429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Greber U. F., Senior A., Gerace L. A major glycoprotein of the nuclear pore complex is a membrane-spanning polypeptide with a large lumenal domain and a small cytoplasmic tail. EMBO J. 1990 May;9(5):1495–1502. doi: 10.1002/j.1460-2075.1990.tb08267.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Hallberg E., Wozniak R. W., Blobel G. An integral membrane protein of the pore membrane domain of the nuclear envelope contains a nucleoporin-like region. J Cell Biol. 1993 Aug;122(3):513–521. doi: 10.1083/jcb.122.3.513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Hinshaw J. E., Carragher B. O., Milligan R. A. Architecture and design of the nuclear pore complex. Cell. 1992 Jun 26;69(7):1133–1141. doi: 10.1016/0092-8674(92)90635-p. [DOI] [PubMed] [Google Scholar]
  30. Hurt E. C. A novel nucleoskeletal-like protein located at the nuclear periphery is required for the life cycle of Saccharomyces cerevisiae. EMBO J. 1988 Dec 20;7(13):4323–4334. doi: 10.1002/j.1460-2075.1988.tb03331.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Hurt E. C. Targeting of a cytosolic protein to the nuclear periphery. J Cell Biol. 1990 Dec;111(6 Pt 2):2829–2837. doi: 10.1083/jcb.111.6.2829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Jarnik M., Aebi U. Toward a more complete 3-D structure of the nuclear pore complex. J Struct Biol. 1991 Dec;107(3):291–308. doi: 10.1016/1047-8477(91)90054-z. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Kranz J. E., Holm C. Cloning by function: an alternative approach for identifying yeast homologs of genes from other organisms. Proc Natl Acad Sci U S A. 1990 Sep;87(17):6629–6633. doi: 10.1073/pnas.87.17.6629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Loeb J. D., Davis L. I., Fink G. R. NUP2, a novel yeast nucleoporin, has functional overlap with other proteins of the nuclear pore complex. Mol Biol Cell. 1993 Feb;4(2):209–222. doi: 10.1091/mbc.4.2.209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. McKeon F. D., Kirschner M. W., Caput D. Homologies in both primary and secondary structure between nuclear envelope and intermediate filament proteins. Nature. 1986 Feb 6;319(6053):463–468. doi: 10.1038/319463a0. [DOI] [PubMed] [Google Scholar]
  37. Morrison H. G., Desrosiers R. C. A PCR-based strategy for extensive mutagenesis of a target DNA sequence. Biotechniques. 1993 Mar;14(3):454–457. [PubMed] [Google Scholar]
  38. Mutvei A., Dihlmann S., Herth W., Hurt E. C. NSP1 depletion in yeast affects nuclear pore formation and nuclear accumulation. Eur J Cell Biol. 1992 Dec;59(2):280–295. [PubMed] [Google Scholar]
  39. 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]
  40. 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]
  41. Panté N., Aebi U. The nuclear pore complex. J Cell Biol. 1993 Sep;122(5):977–984. doi: 10.1083/jcb.122.5.977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Panté N., Bastos R., McMorrow I., Burke B., Aebi U. Interactions and three-dimensional localization of a group of nuclear pore complex proteins. J Cell Biol. 1994 Aug;126(3):603–617. doi: 10.1083/jcb.126.3.603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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]
  44. Richardson W. D., Mills A. D., Dilworth S. M., Laskey R. A., Dingwall C. Nuclear protein migration involves two steps: rapid binding at the nuclear envelope followed by slower translocation through nuclear pores. Cell. 1988 Mar 11;52(5):655–664. doi: 10.1016/0092-8674(88)90403-5. [DOI] [PubMed] [Google Scholar]
  45. 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]
  46. Rout M. P., Wente S. R. Pores for thought: nuclear pore complex proteins. Trends Cell Biol. 1994 Oct;4(10):357–365. doi: 10.1016/0962-8924(94)90085-x. [DOI] [PubMed] [Google Scholar]
  47. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. 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]
  49. Sherman F. Getting started with yeast. Methods Enzymol. 1991;194:3–21. doi: 10.1016/0076-6879(91)94004-v. [DOI] [PubMed] [Google Scholar]
  50. Sikorski R. S., Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19–27. doi: 10.1093/genetics/122.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Simonds W. F., Manji H. K., Garritsen A., Lupas A. N. G proteins and beta ARK: a new twist for the coiled coil. Trends Biochem Sci. 1993 Sep;18(9):315–317. doi: 10.1016/0968-0004(93)90062-r. [DOI] [PubMed] [Google Scholar]
  52. Snow C. M., Senior A., Gerace L. Monoclonal antibodies identify a group of nuclear pore complex glycoproteins. J Cell Biol. 1987 May;104(5):1143–1156. doi: 10.1083/jcb.104.5.1143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Steinert P. M., Roop D. R. Molecular and cellular biology of intermediate filaments. Annu Rev Biochem. 1988;57:593–625. doi: 10.1146/annurev.bi.57.070188.003113. [DOI] [PubMed] [Google Scholar]
  54. 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]
  55. Sukegawa J., Blobel G. A nuclear pore complex protein that contains zinc finger motifs, binds DNA, and faces the nucleoplasm. Cell. 1993 Jan 15;72(1):29–38. doi: 10.1016/0092-8674(93)90047-t. [DOI] [PubMed] [Google Scholar]
  56. Unwin P. N., Milligan R. A. A large particle associated with the perimeter of the nuclear pore complex. J Cell Biol. 1982 Apr;93(1):63–75. doi: 10.1083/jcb.93.1.63. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Wente S. R., Rout M. P., Blobel G. A new family of yeast nuclear pore complex proteins. J Cell Biol. 1992 Nov;119(4):705–723. doi: 10.1083/jcb.119.4.705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Wilken N., Kossner U., Senécal J. L., Scheer U., Dabauvalle M. C. Nup180, a novel nuclear pore complex protein localizing to the cytoplasmic ring and associated fibrils. J Cell Biol. 1993 Dec;123(6 Pt 1):1345–1354. doi: 10.1083/jcb.123.6.1345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. 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]
  60. Wozniak R. W., Bartnik E., Blobel G. Primary structure analysis of an integral membrane glycoprotein of the nuclear pore. J Cell Biol. 1989 Jun;108(6):2083–2092. doi: 10.1083/jcb.108.6.2083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Wozniak R. W., Blobel G., Rout M. P. POM152 is an integral protein of the pore membrane domain of the yeast nuclear envelope. J Cell Biol. 1994 Apr;125(1):31–42. doi: 10.1083/jcb.125.1.31. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Wozniak R. W., Blobel G. The single transmembrane segment of gp210 is sufficient for sorting to the pore membrane domain of the nuclear envelope. J Cell Biol. 1992 Dec;119(6):1441–1449. doi: 10.1083/jcb.119.6.1441. [DOI] [PMC free article] [PubMed] [Google Scholar]

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