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. 1996 Jan 1;132(1):5–20. doi: 10.1083/jcb.132.1.5

Assembly of the nuclear pore: biochemically distinct steps revealed with NEM, GTP gamma S, and BAPTA

PMCID: PMC2120707  PMID: 8567730

Abstract

A key event in nuclear formation is the assembly of functional nuclear pores. We have used a nuclear reconstitution system derived from Xenopus eggs to examine the process of nuclear pore assembly in vitro. With this system, we have identified three reagents which interfere with nuclear pore assembly, NEM, GTP gamma S, and the Ca++ chelator, BAPTA. These reagents have allowed us to determine that the assembly of a nuclear pore requires the prior assembly of a double nuclear membrane. Inhibition of nuclear vesicle fusion by pretreatment of the membrane vesicle fraction with NEM blocks pore complex assembly. In contrast, NEM treatment of already fused double nuclear membranes does not block pore assembly. This indicates that NEM inhibits a single step in pore assembly--the initial fusion of vesicles required to form a double nuclear membrane. The presence of GTP gamma S blocks pore assembly at two distinct steps, first by preventing fusion between nuclear vesicles, and second by blocking a step in pore assembly that occurs on already fused double nuclear membranes. Interestingly, when the Ca2+ chelator BAPTA is added to a nuclear assembly reaction, it only transiently blocks nuclear vesicle fusion, but completely blocks nuclear pore assembly. This results in the formation of a nucleus surrounded by a double nuclear membrane, but devoid of nuclear pores. To order the positions at which GTP gamma S and BAPTA interfere with pore assembly, a novel anchored nuclear assembly assay was developed. This assay revealed that the BAPTA-sensitive step in pore assembly occurs after the second GTP gamma S-sensitive step. Thus, through use of an in vitro nuclear reconstitution system, it has been possible to biochemically define and order multiple steps in nuclear pore assembly.

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Selected References

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  1. 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]
  2. 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]
  3. Blow J. J., Laskey R. A. Initiation of DNA replication in nuclei and purified DNA by a cell-free extract of Xenopus eggs. Cell. 1986 Nov 21;47(4):577–587. doi: 10.1016/0092-8674(86)90622-7. [DOI] [PubMed] [Google Scholar]
  4. Boman A. L., Delannoy M. R., Wilson K. L. GTP hydrolysis is required for vesicle fusion during nuclear envelope assembly in vitro. J Cell Biol. 1992 Jan;116(2):281–294. doi: 10.1083/jcb.116.2.281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Boman A. L., Kahn R. A. Arf proteins: the membrane traffic police? Trends Biochem Sci. 1995 Apr;20(4):147–150. doi: 10.1016/s0968-0004(00)88991-4. [DOI] [PubMed] [Google Scholar]
  6. Boman A. L., Taylor T. C., Melançon P., Wilson K. L. A role for ADP-ribosylation factor in nuclear vesicle dynamics. Nature. 1992 Aug 6;358(6386):512–514. doi: 10.1038/358512a0. [DOI] [PubMed] [Google Scholar]
  7. Byrd D. A., Sweet D. J., Panté N., Konstantinov K. N., Guan T., Saphire A. C., Mitchell P. J., Cooper C. S., Aebi U., Gerace L. Tpr, a large coiled coil protein whose amino terminus is involved in activation of oncogenic kinases, is localized to the cytoplasmic surface of the nuclear pore complex. J Cell Biol. 1994 Dec;127(6 Pt 1):1515–1526. doi: 10.1083/jcb.127.6.1515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chaudhary N., Courvalin J. C. Stepwise reassembly of the nuclear envelope at the end of mitosis. J Cell Biol. 1993 Jul;122(2):295–306. doi: 10.1083/jcb.122.2.295. [DOI] [PMC free article] [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. Coverley D., Downes C. S., Romanowski P., Laskey R. A. Reversible effects of nuclear membrane permeabilization on DNA replication: evidence for a positive licensing factor. J Cell Biol. 1993 Sep;122(5):985–992. doi: 10.1083/jcb.122.5.985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Dabauvalle M. C., Scheer U. Assembly of nuclear pore complexes in Xenopus egg extract. Biol Cell. 1991;72(1-2):25–29. doi: 10.1016/0248-4900(91)90074-w. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Dingwall C., Dilworth S. M., Black S. J., Kearsey S. E., Cox L. S., Laskey R. A. Nucleoplasmin cDNA sequence reveals polyglutamic acid tracts and a cluster of sequences homologous to putative nuclear localization signals. EMBO J. 1987 Jan;6(1):69–74. doi: 10.1002/j.1460-2075.1987.tb04720.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Filson A. J., Lewis A., Blobel G., Fisher P. A. Monoclonal antibodies prepared against the major Drosophila nuclear Matrix-pore complex-lamina glycoprotein bind specifically to the nuclear envelope in situ. J Biol Chem. 1985 Mar 10;260(5):3164–3172. [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. Foisner R., Gerace L. Integral membrane proteins of the nuclear envelope interact with lamins and chromosomes, and binding is modulated by mitotic phosphorylation. Cell. 1993 Jul 2;73(7):1267–1279. doi: 10.1016/0092-8674(93)90355-t. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Gerace L., Foisner R. Integral membrane proteins and dynamic organization of the nuclear envelope. Trends Cell Biol. 1994 Apr;4(4):127–131. doi: 10.1016/0962-8924(94)90067-1. [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. Greber U. F., Gerace L. Depletion of calcium from the lumen of endoplasmic reticulum reversibly inhibits passive diffusion and signal-mediated transport into the nucleus. J Cell Biol. 1995 Jan;128(1-2):5–14. doi: 10.1083/jcb.128.1.5. [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. Hutchison C. J., Bridger J. M., Cox L. S., Kill I. R. Weaving a pattern from disparate threads: lamin function in nuclear assembly and DNA replication. J Cell Sci. 1994 Dec;107(Pt 12):3259–3269. doi: 10.1242/jcs.107.12.3259. [DOI] [PubMed] [Google Scholar]
  28. Kelly R. B. Neural transmission. Synaptotagmin is just a calcium sensor. Curr Biol. 1995 Mar 1;5(3):257–259. doi: 10.1016/s0960-9822(95)00054-6. [DOI] [PubMed] [Google Scholar]
  29. Kessel R. G. Annulate lamellae: a last frontier in cellular organelles. Int Rev Cytol. 1992;133:43–120. doi: 10.1016/s0074-7696(08)61858-6. [DOI] [PubMed] [Google Scholar]
  30. Lohka M. J., Maller J. L. Induction of nuclear envelope breakdown, chromosome condensation, and spindle formation in cell-free extracts. J Cell Biol. 1985 Aug;101(2):518–523. doi: 10.1083/jcb.101.2.518. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lohka M. J., Masui Y. Formation in vitro of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmic components. Science. 1983 May 13;220(4598):719–721. doi: 10.1126/science.6601299. [DOI] [PubMed] [Google Scholar]
  32. Lohka M. J., Masui Y. Roles of cytosol and cytoplasmic particles in nuclear envelope assembly and sperm pronuclear formation in cell-free preparations from amphibian eggs. J Cell Biol. 1984 Apr;98(4):1222–1230. doi: 10.1083/jcb.98.4.1222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Lourim D., Krohne G. Lamin-dependent nuclear envelope reassembly following mitosis: an argument. Trends Cell Biol. 1994 Sep;4(9):314–318. doi: 10.1016/0962-8924(94)90228-3. [DOI] [PubMed] [Google Scholar]
  34. Lourim D., Krohne G. Membrane-associated lamins in Xenopus egg extracts: identification of two vesicle populations. J Cell Biol. 1993 Nov;123(3):501–512. doi: 10.1083/jcb.123.3.501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Macaulay C., Meier E., Forbes D. J. Differential mitotic phosphorylation of proteins of the nuclear pore complex. J Biol Chem. 1995 Jan 6;270(1):254–262. doi: 10.1074/jbc.270.1.254. [DOI] [PubMed] [Google Scholar]
  36. Malviya A. N. The nuclear inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate receptors. Cell Calcium. 1994 Oct;16(4):301–313. doi: 10.1016/0143-4160(94)90094-9. [DOI] [PubMed] [Google Scholar]
  37. Maul G. G., Maul H. M., Scogna J. E., Lieberman M. W., Stein G. S., Hsu B. Y., Borun T. W. Time sequence of nuclear pore formation in phytohemagglutinin-stimulated lymphocytes and in HeLa cells during the cell cycle. J Cell Biol. 1972 Nov;55(2):433–447. doi: 10.1083/jcb.55.2.433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Moss J., Vaughan M. Structure and function of ARF proteins: activators of cholera toxin and critical components of intracellular vesicular transport processes. J Biol Chem. 1995 May 26;270(21):12327–12330. doi: 10.1074/jbc.270.21.12327. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. Newmeyer D. D., Wilson K. L. Egg extracts for nuclear import and nuclear assembly reactions. Methods Cell Biol. 1991;36:607–634. doi: 10.1016/s0091-679x(08)60299-x. [DOI] [PubMed] [Google Scholar]
  41. Newport J. W., Wilson K. L., Dunphy W. G. A lamin-independent pathway for nuclear envelope assembly. J Cell Biol. 1990 Dec;111(6 Pt 1):2247–2259. doi: 10.1083/jcb.111.6.2247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Newport J., Dunphy W. Characterization of the membrane binding and fusion events during nuclear envelope assembly using purified components. J Cell Biol. 1992 Jan;116(2):295–306. doi: 10.1083/jcb.116.2.295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Newport J. Nuclear reconstitution in vitro: stages of assembly around protein-free DNA. Cell. 1987 Jan 30;48(2):205–217. doi: 10.1016/0092-8674(87)90424-7. [DOI] [PubMed] [Google Scholar]
  44. Osborne M. A., Silver P. A. Nucleocytoplasmic transport in the yeast Saccharomyces cerevisiae. Annu Rev Biochem. 1993;62:219–254. doi: 10.1146/annurev.bi.62.070193.001251. [DOI] [PubMed] [Google Scholar]
  45. Park M. K., D'Onofrio M., Willingham M. C., Hanover J. A. A monoclonal antibody against a family of nuclear pore proteins (nucleoporins): O-linked N-acetylglucosamine is part of the immunodeterminant. Proc Natl Acad Sci U S A. 1987 Sep;84(18):6462–6466. doi: 10.1073/pnas.84.18.6462. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Peters J. M., Walsh M. J., Franke W. W. An abundant and ubiquitous homo-oligomeric ring-shaped ATPase particle related to the putative vesicle fusion proteins Sec18p and NSF. EMBO J. 1990 Jun;9(6):1757–1767. doi: 10.1002/j.1460-2075.1990.tb08300.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Pfaller R., Smythe C., Newport J. W. Assembly/disassembly of the nuclear envelope membrane: cell cycle-dependent binding of nuclear membrane vesicles to chromatin in vitro. Cell. 1991 Apr 19;65(2):209–217. doi: 10.1016/0092-8674(91)90155-r. [DOI] [PubMed] [Google Scholar]
  48. Philpott A., Leno G. H., Laskey R. A. Sperm decondensation in Xenopus egg cytoplasm is mediated by nucleoplasmin. Cell. 1991 May 17;65(4):569–578. doi: 10.1016/0092-8674(91)90089-h. [DOI] [PubMed] [Google Scholar]
  49. 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]
  50. 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]
  51. Rexach M. F., Schekman R. W. Distinct biochemical requirements for the budding, targeting, and fusion of ER-derived transport vesicles. J Cell Biol. 1991 Jul;114(2):219–229. doi: 10.1083/jcb.114.2.219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Richardson A., Taylor C. W. Effects of Ca2+ chelators on purified inositol 1,4,5-trisphosphate (InsP3) receptors and InsP3-stimulated Ca2+ mobilization. J Biol Chem. 1993 Jun 5;268(16):11528–11533. [PubMed] [Google Scholar]
  53. Rothman J. E. Mechanisms of intracellular protein transport. Nature. 1994 Nov 3;372(6501):55–63. doi: 10.1038/372055a0. [DOI] [PubMed] [Google Scholar]
  54. 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]
  55. Schwaninger R., Beckers C. J., Balch W. E. Sequential transport of protein between the endoplasmic reticulum and successive Golgi compartments in semi-intact cells. J Biol Chem. 1991 Jul 15;266(20):13055–13063. [PubMed] [Google Scholar]
  56. Sheehan M. A., Mills A. D., Sleeman A. M., Laskey R. A., Blow J. J. Steps in the assembly of replication-competent nuclei in a cell-free system from Xenopus eggs. J Cell Biol. 1988 Jan;106(1):1–12. doi: 10.1083/jcb.106.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Smythe C., Newport J. W. Systems for the study of nuclear assembly, DNA replication, and nuclear breakdown in Xenopus laevis egg extracts. Methods Cell Biol. 1991;35:449–468. doi: 10.1016/s0091-679x(08)60583-x. [DOI] [PubMed] [Google Scholar]
  58. Sullivan K. M., Busa W. B., Wilson K. L. Calcium mobilization is required for nuclear vesicle fusion in vitro: implications for membrane traffic and IP3 receptor function. Cell. 1993 Jul 2;73(7):1411–1422. doi: 10.1016/0092-8674(93)90366-x. [DOI] [PubMed] [Google Scholar]
  59. Vigers G. P., Lohka M. J. A distinct vesicle population targets membranes and pore complexes to the nuclear envelope in Xenopus eggs. J Cell Biol. 1991 Feb;112(4):545–556. doi: 10.1083/jcb.112.4.545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Vigers G. P., Lohka M. J. Regulation of nuclear envelope precursor functions during cell division. J Cell Sci. 1992 Jun;102(Pt 2):273–284. doi: 10.1242/jcs.102.2.273. [DOI] [PubMed] [Google Scholar]
  61. White J. M. Membrane fusion. Science. 1992 Nov 6;258(5084):917–924. doi: 10.1126/science.1439803. [DOI] [PubMed] [Google Scholar]
  62. Wilson K. L., Newport J. A trypsin-sensitive receptor on membrane vesicles is required for nuclear envelope formation in vitro. J Cell Biol. 1988 Jul;107(1):57–68. doi: 10.1083/jcb.107.1.57. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. 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]

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