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. 1992 Dec;11(13):5051–5061. doi: 10.1002/j.1460-2075.1992.tb05612.x

A new subclass of nucleoporins that functionally interact with nuclear pore protein NSP1.

C Wimmer 1, V Doye 1, P Grandi 1, U Nehrbass 1, E C Hurt 1
PMCID: PMC556983  PMID: 1464327

Abstract

NSP1 is a nuclear pore protein (nucleoporin) essential for cell growth. To identify the components that functionally interact with NSP1 in the living cell, we developed a genetic screen for mutants that are lethal in a genetic background of mutated, but not wild type NSP1. Fourteen synthetic lethal mutants were obtained, belonging to at least four different complementation groups. The genes of two complementation groups, NSP116 and NSP49, were cloned. Like the previously described nucleoporins, these genes encode proteins with many repeat sequences. NSP116 and NSP49, however, contain a new repetitive sequence motif 'GLFG', which classifies them as a subclass of nucleoporins. NSP116 and NSP49, tagged with the IgG binding domain of protein A and expressed in yeast, are located at the nuclear envelope. These data provide in vivo evidence that distinct subclasses of nucleoporins physically interact or share overlapping function in nuclear pore complexes.

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Selected References

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  1. Adam S. A., Gerace L. Cytosolic proteins that specifically bind nuclear location signals are receptors for nuclear import. Cell. 1991 Sep 6;66(5):837–847. doi: 10.1016/0092-8674(91)90431-w. [DOI] [PubMed] [Google Scholar]
  2. Adam S. A., Marr R. S., Gerace L. Nuclear protein import in permeabilized mammalian cells requires soluble cytoplasmic factors. J Cell Biol. 1990 Sep;111(3):807–816. doi: 10.1083/jcb.111.3.807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Aris J. P., Blobel G. Yeast nuclear envelope proteins cross react with an antibody against mammalian pore complex proteins. J Cell Biol. 1989 Jun;108(6):2059–2067. doi: 10.1083/jcb.108.6.2059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Becker D. M., Guarente L. High-efficiency transformation of yeast by electroporation. Methods Enzymol. 1991;194:182–187. doi: 10.1016/0076-6879(91)94015-5. [DOI] [PubMed] [Google Scholar]
  6. Bender A., Pringle J. R. Use of a screen for synthetic lethal and multicopy suppressee mutants to identify two new genes involved in morphogenesis in Saccharomyces cerevisiae. Mol Cell Biol. 1991 Mar;11(3):1295–1305. doi: 10.1128/mcb.11.3.1295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Boeke J. D., LaCroute F., Fink G. R. A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet. 1984;197(2):345–346. doi: 10.1007/BF00330984. [DOI] [PubMed] [Google Scholar]
  8. Carmo-Fonseca M., Hurt E. C. Across the nuclear pores with the help of nucleoporins. Chromosoma. 1991 Dec;101(4):199–205. doi: 10.1007/BF00365151. [DOI] [PubMed] [Google Scholar]
  9. 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]
  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. Costigan C., Gehrung S., Snyder M. A synthetic lethal screen identifies SLK1, a novel protein kinase homolog implicated in yeast cell morphogenesis and cell growth. Mol Cell Biol. 1992 Mar;12(3):1162–1178. doi: 10.1128/mcb.12.3.1162. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. D'Onofrio M., Starr C. M., Park M. K., Holt G. D., Haltiwanger R. S., Hart G. W., Hanover J. A. Partial cDNA sequence encoding a nuclear pore protein modified by O-linked N-acetylglucosamine. Proc Natl Acad Sci U S A. 1988 Dec;85(24):9595–9599. doi: 10.1073/pnas.85.24.9595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. 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]
  16. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Dingwall C., Laskey R. A. Protein import into the cell nucleus. Annu Rev Cell Biol. 1986;2:367–390. doi: 10.1146/annurev.cb.02.110186.002055. [DOI] [PubMed] [Google Scholar]
  18. Dykstra C. C., Kitada K., Clark A. B., Hamatake R. K., Sugino A. Cloning and characterization of DST2, the gene for DNA strand transfer protein beta from Saccharomyces cerevisiae. Mol Cell Biol. 1991 May;11(5):2583–2592. doi: 10.1128/mcb.11.5.2583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. 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]
  22. Finlay D. R., Newmeyer D. D., Price T. M., Forbes D. J. Inhibition of in vitro nuclear transport by a lectin that binds to nuclear pores. J Cell Biol. 1987 Feb;104(2):189–200. doi: 10.1083/jcb.104.2.189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Garcia-Bustos J., Heitman J., Hall M. N. Nuclear protein localization. Biochim Biophys Acta. 1991 Mar 7;1071(1):83–101. doi: 10.1016/0304-4157(91)90013-m. [DOI] [PubMed] [Google Scholar]
  24. Greber U. F., Gerace L. Nuclear protein import is inhibited by an antibody to a lumenal epitope of a nuclear pore complex glycoprotein. J Cell Biol. 1992 Jan;116(1):15–30. doi: 10.1083/jcb.116.1.15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Hanover J. A., Cohen C. K., Willingham M. C., Park M. K. O-linked N-acetylglucosamine is attached to proteins of the nuclear pore. Evidence for cytoplasmic and nucleoplasmic glycoproteins. J Biol Chem. 1987 Jul 15;262(20):9887–9894. [PubMed] [Google Scholar]
  27. Holt G. D., Hart G. W. The subcellular distribution of terminal N-acetylglucosamine moieties. Localization of a novel protein-saccharide linkage, O-linked GlcNAc. J Biol Chem. 1986 Jun 15;261(17):8049–8057. [PubMed] [Google Scholar]
  28. Holt G. D., Snow C. M., Senior A., Haltiwanger R. S., Gerace L., Hart G. W. Nuclear pore complex glycoproteins contain cytoplasmically disposed O-linked N-acetylglucosamine. J Cell Biol. 1987 May;104(5):1157–1164. doi: 10.1083/jcb.104.5.1157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Huffaker T. C., Hoyt M. A., Botstein D. Genetic analysis of the yeast cytoskeleton. Annu Rev Genet. 1987;21:259–284. doi: 10.1146/annurev.ge.21.120187.001355. [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., McDowall A., Schimmang T. Nucleolar and nuclear envelope proteins of the yeast Saccharomyces cerevisiae. Eur J Cell Biol. 1988 Aug;46(3):554–563. [PubMed] [Google Scholar]
  32. Hurt E. C., Mutvei A., Carmo-Fonseca M. The nuclear envelope of the yeast Saccharomyces cerevisiae. Int Rev Cytol. 1992;136:145–184. doi: 10.1016/s0074-7696(08)62052-5. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Jeoung D. I., Chen S., Windsor J., Pollack R. E. Human major HSP70 protein complements the localization and functional defects of cytoplasmic mutant SV40 T antigen in Swiss 3T3 mouse fibroblast cells. Genes Dev. 1991 Dec;5(12A):2235–2244. doi: 10.1101/gad.5.12a.2235. [DOI] [PubMed] [Google Scholar]
  36. Kalderon D., Roberts B. L., Richardson W. D., Smith A. E. A short amino acid sequence able to specify nuclear location. Cell. 1984 Dec;39(3 Pt 2):499–509. doi: 10.1016/0092-8674(84)90457-4. [DOI] [PubMed] [Google Scholar]
  37. Kim J., Ljungdahl P. O., Fink G. R. kem mutations affect nuclear fusion in Saccharomyces cerevisiae. Genetics. 1990 Dec;126(4):799–812. doi: 10.1093/genetics/126.4.799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Koshland D., Kent J. C., Hartwell L. H. Genetic analysis of the mitotic transmission of minichromosomes. Cell. 1985 Feb;40(2):393–403. doi: 10.1016/0092-8674(85)90153-9. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. Lee W. C., Mélèse T. Identification and characterization of a nuclear localization sequence-binding protein in yeast. Proc Natl Acad Sci U S A. 1989 Nov;86(22):8808–8812. doi: 10.1073/pnas.86.22.8808. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Meier U. T., Blobel G. A nuclear localization signal binding protein in the nucleolus. J Cell Biol. 1990 Dec;111(6 Pt 1):2235–2245. doi: 10.1083/jcb.111.6.2235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Moore M. S., Blobel G. The two steps of nuclear import, targeting to the nuclear envelope and translocation through the nuclear pore, require different cytosolic factors. Cell. 1992 Jun 12;69(6):939–950. doi: 10.1016/0092-8674(92)90613-h. [DOI] [PubMed] [Google Scholar]
  43. 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]
  44. Newmeyer D. D., Forbes D. J. An N-ethylmaleimide-sensitive cytosolic factor necessary for nuclear protein import: requirement in signal-mediated binding to the nuclear pore. J Cell Biol. 1990 Mar;110(3):547–557. doi: 10.1083/jcb.110.3.547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Newmeyer D. D., Forbes D. J. Nuclear import can be separated into distinct steps in vitro: nuclear pore binding and translocation. Cell. 1988 Mar 11;52(5):641–653. doi: 10.1016/0092-8674(88)90402-3. [DOI] [PubMed] [Google Scholar]
  46. 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]
  47. 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]
  48. Rothstein R. J. One-step gene disruption in yeast. Methods Enzymol. 1983;101:202–211. doi: 10.1016/0076-6879(83)01015-0. [DOI] [PubMed] [Google Scholar]
  49. 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]
  50. Schimmang T., Tollervey D., Kern H., Frank R., Hurt E. C. A yeast nucleolar protein related to mammalian fibrillarin is associated with small nucleolar RNA and is essential for viability. EMBO J. 1989 Dec 20;8(13):4015–4024. doi: 10.1002/j.1460-2075.1989.tb08584.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Shi Y., Thomas J. O. The transport of proteins into the nucleus requires the 70-kilodalton heat shock protein or its cytosolic cognate. Mol Cell Biol. 1992 May;12(5):2186–2192. doi: 10.1128/mcb.12.5.2186. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Silver P. A. How proteins enter the nucleus. Cell. 1991 Feb 8;64(3):489–497. doi: 10.1016/0092-8674(91)90233-o. [DOI] [PubMed] [Google Scholar]
  53. Silver P., Sadler I., Osborne M. A. Yeast proteins that recognize nuclear localization sequences. J Cell Biol. 1989 Sep;109(3):983–989. doi: 10.1083/jcb.109.3.983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. 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]
  55. Starr C. M., Hanover J. A. A common structural motif in nuclear pore proteins (nucleoporins) Bioessays. 1991 Mar;13(3):145–146. doi: 10.1002/bies.950130309. [DOI] [PubMed] [Google Scholar]
  56. Sterne-Marr R., Blevitt J. M., Gerace L. O-linked glycoproteins of the nuclear pore complex interact with a cytosolic factor required for nuclear protein import. J Cell Biol. 1992 Jan;116(2):271–280. doi: 10.1083/jcb.116.2.271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Stochaj U., Silver P. A. A conserved phosphoprotein that specifically binds nuclear localization sequences is involved in nuclear import. J Cell Biol. 1992 May;117(3):473–482. doi: 10.1083/jcb.117.3.473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Strathern J. N., Higgins D. R. Recovery of plasmids from yeast into Escherichia coli: shuttle vectors. Methods Enzymol. 1991;194:319–329. doi: 10.1016/0076-6879(91)94024-7. [DOI] [PubMed] [Google Scholar]
  59. 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]
  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. Yamasaki L., Kanda P., Lanford R. E. Identification of four nuclear transport signal-binding proteins that interact with diverse transport signals. Mol Cell Biol. 1989 Jul;9(7):3028–3036. doi: 10.1128/mcb.9.7.3028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Yoneda Y., Imamoto-Sonobe N., Yamaizumi M., Uchida T. Reversible inhibition of protein import into the nucleus by wheat germ agglutinin injected into cultured cells. Exp Cell Res. 1987 Dec;173(2):586–595. doi: 10.1016/0014-4827(87)90297-7. [DOI] [PubMed] [Google Scholar]

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