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. 1994 Aug 1;126(3):603–617. doi: 10.1083/jcb.126.3.603

Interactions and three-dimensional localization of a group of nuclear pore complex proteins

PMCID: PMC2120134  PMID: 8045926

Abstract

We have used antibodies directed against a number of nuclear pore complex (NPC) proteins to determine their mutual interactions and location within the three-dimensional structure of the NPC. A monoclonal antibody, termed QE5, recognized three NPC polypeptides, p250, NUP153, and p62 on Western blots, and labeled the nuclear envelope of several cultured cell lines by immunofluorescence microscopy. These three polypeptides contained O-linked N- acetylglucosamine residues and were released from the NPC by detergent/high-salt treatment as discrete high molecular weight complexes. p250 was found in association with a novel 75 kD protein, NUP153 was released as a homo-oligomer of about 1 megadalton, and p62 was associated with polypeptides of 58 and 54 kD (previously reported by Finlay, D. R., E. Meier, P. Bradley, J. Horecka, and D. J. Forbes. 1991. J. Cell Biol. 114:169-183). p75, p58, and p54 were not galactosylated in vitro. Xenopus oocyte NEs were labeled with gold- conjugated QE5 and prepared for electron microscopy by quick freezing/freeze drying/rotary metal shadowing. This EM preparation method enabled us to more precisely localize the epitopes of this antibody to the cytoplasmic filaments and the nuclear basket of the NPC. Since QE5 recognizes three O-linked NPC glycoproteins, its labeling was compared with that of the lectin wheat germ agglutinin which recognizes O-linked N-acetylglucosamine moieties. The two probes were found to yield similar, although not identical, distributions of label. To identify the individual proteins with particular NPC components, we have used an anti-peptide antibody against NUP153 and a monospecific anti-p250 polyclonal antibody. Labeling with these two antibodies has documented that NUP153 is a constituent of the nuclear basket with at least one of its epitopes residing in its terminal ring, whereas p250 is a constituent of the cytoplasmic filaments.

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Selected References

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  1. 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]
  2. Akey C. W., Goldfarb D. S. Protein import through the nuclear pore complex is a multistep process. J Cell Biol. 1989 Sep;109(3):971–982. doi: 10.1083/jcb.109.3.971. [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. 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]
  5. Ash J. F., Louvard D., Singer S. J. Antibody-induced linkages of plasma membrane proteins to intracellular actomyosin-containing filaments in cultured fibroblasts. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5584–5588. doi: 10.1073/pnas.74.12.5584. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Baschong W., Wrigley N. G. Small colloidal gold conjugated to Fab fragments or to immunoglobulin G as high-resolution labels for electron microscopy: a technical overview. J Electron Microsc Tech. 1990 Apr;14(4):313–323. doi: 10.1002/jemt.1060140405. [DOI] [PubMed] [Google Scholar]
  7. Blobel G., Potter V. R. Nuclei from rat liver: isolation method that combines purity with high yield. Science. 1966 Dec 30;154(3757):1662–1665. doi: 10.1126/science.154.3757.1662. [DOI] [PubMed] [Google Scholar]
  8. Burke B., Griffiths G., Reggio H., Louvard D., Warren G. A monoclonal antibody against a 135-K Golgi membrane protein. EMBO J. 1982;1(12):1621–1628. doi: 10.1002/j.1460-2075.1982.tb01364.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Burke B. On the cell-free association of lamins A and C with metaphase chromosomes. Exp Cell Res. 1990 Jan;186(1):169–176. doi: 10.1016/0014-4827(90)90223-w. [DOI] [PubMed] [Google Scholar]
  10. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Coffino P., Scharff M. D. Rate of somatic mutation in immunoglobulin production by mouse myeloma cells. Proc Natl Acad Sci U S A. 1971 Jan;68(1):219–223. doi: 10.1073/pnas.68.1.219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. 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]
  16. 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]
  17. Davis L. I., Blobel G. Nuclear pore complex contains a family of glycoproteins that includes p62: glycosylation through a previously unidentified cellular pathway. Proc Natl Acad Sci U S A. 1987 Nov;84(21):7552–7556. doi: 10.1073/pnas.84.21.7552. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Dingwall C. Nuclear pore structure: fingers in the pore. Curr Biol. 1993 May 1;3(5):297–299. doi: 10.1016/0960-9822(93)90184-p. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Gerace L., Comeau C., Benson M. Organization and modulation of nuclear lamina structure. J Cell Sci Suppl. 1984;1:137–160. doi: 10.1242/jcs.1984.supplement_1.10. [DOI] [PubMed] [Google Scholar]
  22. Gerace L. Molecular trafficking across the nuclear pore complex. Curr Opin Cell Biol. 1992 Aug;4(4):637–645. doi: 10.1016/0955-0674(92)90083-o. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  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. 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]
  27. 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]
  28. 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]
  29. 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]
  30. 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]
  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. Kraemer D., Wozniak R. W., Blobel G., Radu A. The human CAN protein, a putative oncogene product associated with myeloid leukemogenesis, is a nuclear pore complex protein that faces the cytoplasm. Proc Natl Acad Sci U S A. 1994 Feb 15;91(4):1519–1523. doi: 10.1073/pnas.91.4.1519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Kreis T. E. Microinjected antibodies against the cytoplasmic domain of vesicular stomatitis virus glycoprotein block its transport to the cell surface. EMBO J. 1986 May;5(5):931–941. doi: 10.1002/j.1460-2075.1986.tb04306.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. McMorrow I., Bastos R., Horton H., Burke B. Sequence analysis of a cDNA encoding a human nuclear pore complex protein, hnup153. Biochim Biophys Acta. 1994 Mar 1;1217(2):219–223. doi: 10.1016/0167-4781(94)90040-x. [DOI] [PubMed] [Google Scholar]
  36. Mitchell P. J., Cooper C. S. Nucleotide sequence analysis of human tpr cDNA clones. Oncogene. 1992 Feb;7(2):383–388. [PubMed] [Google Scholar]
  37. 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]
  38. Radu A., Blobel G., Wozniak R. W. Nup155 is a novel nuclear pore complex protein that contains neither repetitive sequence motifs nor reacts with WGA. J Cell Biol. 1993 Apr;121(1):1–9. doi: 10.1083/jcb.121.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. Ris H., Malecki M. High-resolution field emission scanning electron microscope imaging of internal cell structures after Epon extraction from sections: a new approach to correlative ultrastructural and immunocytochemical studies. J Struct Biol. 1993 Sep-Oct;111(2):148–157. doi: 10.1006/jsbi.1993.1045. [DOI] [PubMed] [Google Scholar]
  41. Savvidou G., Klein M., Grey A. A., Dorrington K. J., Carver J. P. Possible role for peptide-oligosaccharide interactions in differential oligosaccharide processing at asparagine-107 of the light chain and asparagine-297 of the heavy chain in a monoclonal IgG1 kappa. Biochemistry. 1984 Jul 31;23(16):3736–3740. doi: 10.1021/bi00311a026. [DOI] [PubMed] [Google Scholar]
  42. Shotton D. M., Burke B. E., Branton D. The molecular structure of human erythrocyte spectrin. Biophysical and electron microscopic studies. J Mol Biol. 1979 Jun 25;131(2):303–329. doi: 10.1016/0022-2836(79)90078-0. [DOI] [PubMed] [Google Scholar]
  43. Slot J. W., Geuze H. J. A new method of preparing gold probes for multiple-labeling cytochemistry. Eur J Cell Biol. 1985 Jul;38(1):87–93. [PubMed] [Google Scholar]
  44. 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]
  45. 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]
  46. 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]
  47. Taggart R. T., Samloff I. M. Stable antibody-producing murine hybridomas. Science. 1983 Mar 11;219(4589):1228–1230. doi: 10.1126/science.6402815. [DOI] [PubMed] [Google Scholar]
  48. 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]
  49. 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]
  50. 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]
  51. 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]
  52. Wrigley N. G. The lattice spacing of crystalline catalase as an internal standard of length in electron microscopy. J Ultrastruct Res. 1968 Sep;24(5):454–464. doi: 10.1016/s0022-5320(68)80048-6. [DOI] [PubMed] [Google Scholar]
  53. von Lindern M., Fornerod M., van Baal S., Jaegle M., de Wit T., Buijs A., Grosveld G. The translocation (6;9), associated with a specific subtype of acute myeloid leukemia, results in the fusion of two genes, dek and can, and the expression of a chimeric, leukemia-specific dek-can mRNA. Mol Cell Biol. 1992 Apr;12(4):1687–1697. doi: 10.1128/mcb.12.4.1687. [DOI] [PMC free article] [PubMed] [Google Scholar]

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