Search tips
Search criteria 


Logo of jcellbiolHomeThe Rockefeller University PressEditorsContactInstructions for AuthorsThis issue
J Cell Biol. 1995 June 2; 129(6): 1459–1472.
PMCID: PMC2291182

Nuclear pore complex assembly studied with a biochemical assay for annulate lamellae formation


Formation of the nuclear pore is an intricate process involving membrane fusion and the ordered assembly of up to 1,000 pore proteins. As such, the study of pore assembly is not a simple one. Interestingly, annulate lamellae, a cytoplasmic organelle consisting of stacks of flattened membrane cisternae perforated by numerous pore complexes, have been found to form spontaneously in a reconstitution system derived from Xenopus egg extracts, as determined by electron microscopy (Dabauvalle et al., 1991). In this work, a biochemical assay for annulate lamellae (AL) formation was developed and used to study the mechanism of AL assembly in general and the assembly of individual nucleoporins into pore complexes in particular. Upon incubation of Xenopus egg cytosol and membrane vesicles, the nucleoporins nup58, nup60, nup97, nup153, and nup200 initially present in a disassembled form in the cytosol became associated with membranes and were pelletable. The association was time and temperature dependent and could be measured by immunoblotting. Thin-section electron microscopy as well as negative staining confirmed that annulate lamellae were forming coincident with the incorporation of pore proteins into membranes. Homogenization and subsequent flotation of the membrane fraction allowed us to separate a population of dense membranes, containing the integral membrane pore protein gp210 and all other nucleoporins tested, from the bulk of cellular membranes. Electron microscopy indicated that annulate lamellae were enriched in this dense, pore protein-containing fraction. GTP gamma S prevented incorporation of the soluble pore proteins into membranes. To address whether AL form in the absence of N-acetylglucosaminylated pore proteins, AL assembly was carried out in WGA-sepharose-depleted cytosol. Under these conditions, annulate lamellae formed but were altered in appearance. When the membrane fraction containing this altered AL was homogenized and subjected to flotation, the pore protein- containing membranes still sedimented in a distinct peak but were less dense than control annulate lamellae.

Full Text

The Full Text of this article is available as a PDF (2.8M).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Akey CW, Goldfarb DS. Protein import through the nuclear pore complex is a multistep process. J Cell Biol. 1989 Sep;109(3):971–982. [PMC free article] [PubMed]
  • Akey CW, 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. [PMC free article] [PubMed]
  • Allen JL, Douglas MG. Organization of the nuclear pore complex in Saccharomyces cerevisiae. J Ultrastruct Mol Struct Res. 1989 Aug;102(2):95–108. [PubMed]
  • Almouzni G, Wolffe AP. Nuclear assembly, structure, and function: the use of Xenopus in vitro systems. Exp Cell Res. 1993 Mar;205(1):1–15. [PubMed]
  • Balch WE. Biochemistry of interorganelle transport. A new frontier in enzymology emerges from versatile in vitro model systems. J Biol Chem. 1989 Oct 15;264(29):16965–16968. [PubMed]
  • Bement WM, Capco DG. Intracellular signals trigger ultrastructural events characteristic of meiotic maturation in oocytes of Xenopus laevis. Cell Tissue Res. 1989 Jan;255(1):183–191. [PubMed]
  • Benavente R, Dabauvalle MC, Scheer U, Chaly N. Functional role of newly formed pore complexes in postmitotic nuclear reorganization. Chromosoma. 1989 Oct;98(4):233–241. [PubMed]
  • Boman AL, Delannoy MR, Wilson KL. GTP hydrolysis is required for vesicle fusion during nuclear envelope assembly in vitro. J Cell Biol. 1992 Jan;116(2):281–294. [PMC free article] [PubMed]
  • Boman AL, Taylor TC, Melançon P, Wilson KL. A role for ADP-ribosylation factor in nuclear vesicle dynamics. Nature. 1992 Aug 6;358(6386):512–514. [PubMed]
  • Burke B, Gerace L. A cell free system to study reassembly of the nuclear envelope at the end of mitosis. Cell. 1986 Feb 28;44(4):639–652. [PubMed]
  • Byrd DA, Sweet DJ, Panté N, Konstantinov KN, Guan T, Saphire AC, Mitchell PJ, Cooper CS, 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. [PMC free article] [PubMed]
  • Chaudhary N, Courvalin JC. Stepwise reassembly of the nuclear envelope at the end of mitosis. J Cell Biol. 1993 Jul;122(2):295–306. [PMC free article] [PubMed]
  • Chen TY, Merisko EM. Annulate lamellae: comparison of antigenic epitopes of annulate lamellae membranes with the nuclear envelope. J Cell Biol. 1988 Oct;107(4):1299–1306. [PMC free article] [PubMed]
  • 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]
  • Cordes VC, Reidenbach S, Köhler A, Stuurman N, van Driel R, Franke WW. Intranuclear filaments containing a nuclear pore complex protein. J Cell Biol. 1993 Dec;123(6 Pt 1):1333–1344. [PMC free article] [PubMed]
  • Dabauvalle MC, 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. [PubMed]
  • Dabauvalle MC, Loos K, Scheer U. Identification of a soluble precursor complex essential for nuclear pore assembly in vitro. Chromosoma. 1990 Dec;100(1):56–66. [PubMed]
  • Dabauvalle MC, Loos K, Merkert H, Scheer U. Spontaneous assembly of pore complex-containing membranes ("annulate lamellae") in Xenopus egg extract in the absence of chromatin. J Cell Biol. 1991 Mar;112(6):1073–1082. [PMC free article] [PubMed]
  • Davis LI, Blobel G. Identification and characterization of a nuclear pore complex protein. Cell. 1986 Jun 6;45(5):699–709. [PubMed]
  • Davis LI, Fink GR. The NUP1 gene encodes an essential component of the yeast nuclear pore complex. Cell. 1990 Jun 15;61(6):965–978. [PubMed]
  • Fabre E, Hurt EC. Nuclear transport. Curr Opin Cell Biol. 1994 Jun;6(3):335–342. [PubMed]
  • Featherstone C, Darby MK, 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. [PMC free article] [PubMed]
  • Feldherr CM, Kallenbach E, Schultz N. Movement of a karyophilic protein through the nuclear pores of oocytes. J Cell Biol. 1984 Dec;99(6):2216–2222. [PMC free article] [PubMed]
  • Finlay DR, Forbes DJ. Reconstitution of biochemically altered nuclear pores: transport can be eliminated and restored. Cell. 1990 Jan 12;60(1):17–29. [PubMed]
  • Finlay DR, Newmeyer DD, Price TM, Forbes DJ. Inhibition of in vitro nuclear transport by a lectin that binds to nuclear pores. J Cell Biol. 1987 Feb;104(2):189–200. [PMC free article] [PubMed]
  • Finlay DR, Meier E, Bradley P, Horecka J, Forbes DJ. A complex of nuclear pore proteins required for pore function. J Cell Biol. 1991 Jul;114(1):169–183. [PMC free article] [PubMed]
  • 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. [PubMed]
  • Forbes DJ. Structure and function of the nuclear pore complex. Annu Rev Cell Biol. 1992;8:495–527. [PubMed]
  • Franke WW, Scheer U. The ultrastructure of the nuclear envelope of amphibian oocytes: a reinvestigation. I. The mature oocyte. J Ultrastruct Res. 1970 Feb;30(3):288–316. [PubMed]
  • Gerace L. Molecular trafficking across the nuclear pore complex. Curr Opin Cell Biol. 1992 Aug;4(4):637–645. [PubMed]
  • 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. [PMC free article] [PubMed]
  • Goldberg MW, Allen TD. 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. [PMC free article] [PubMed]
  • Goldfarb D, Michaud N. Pathways for the nuclear transport of proteins and RNAs. Trends Cell Biol. 1991 Jul;1(1):20–24. [PubMed]
  • Greber UF, 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. [PubMed]
  • Haino M, Kawahire S, Omata S, Horigome T. Purification of a 60 kDa nuclear localization signal binding protein in rat liver nuclear envelopes and characterization of its properties. J Biochem. 1993 Mar;113(3):308–313. [PubMed]
  • Hall A. The cellular functions of small GTP-binding proteins. Science. 1990 Aug 10;249(4969):635–640. [PubMed]
  • Hallberg E, Wozniak RW, 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. [PMC free article] [PubMed]
  • Hart GW, Haltiwanger RS, Holt GD, Kelly WG. Glycosylation in the nucleus and cytoplasm. Annu Rev Biochem. 1989;58:841–874. [PubMed]
  • Hartl P, Olson E, Dang T, Forbes DJ. Nuclear assembly with lambda DNA in fractionated Xenopus egg extracts: an unexpected role for glycogen in formation of a higher order chromatin intermediate. J Cell Biol. 1994 Feb;124(3):235–248. [PMC free article] [PubMed]
  • Hinshaw JE, Carragher BO, Milligan RA. Architecture and design of the nuclear pore complex. Cell. 1992 Jun 26;69(7):1133–1141. [PubMed]
  • Kessel RG. The structure and function of annulate lamellae: porous cytoplasmic and intranuclear membranes. Int Rev Cytol. 1983;82:181–303. [PubMed]
  • Kessel RG. Annulate lamellae: a last frontier in cellular organelles. Int Rev Cytol. 1992;133:43–120. [PubMed]
  • Kessel RG, Subtelny S. Alteration of annulate lamellae in the in vitro progesterone-treated, full-grown Rana pipiens oocyte. J Exp Zool. 1981 Jul;217(1):119–135. [PubMed]
  • Kraemer D, Wozniak RW, 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. [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Larabell CA, Chandler DE. Freeze-fracture analysis of structural reorganization during meiotic maturation in oocytes of Xenopus laevis. Cell Tissue Res. 1988 Jan;251(1):129–136. [PubMed]
  • Laskey RA, Leno GH. Assembly of the cell nucleus. Trends Genet. 1990 Dec;6(12):406–410. [PubMed]
  • Loeb JD, Davis LI, Fink GR. 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. [PMC free article] [PubMed]
  • Lohka MJ, Masui Y. Formation in vitro of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmic components. Science. 1983 May 13;220(4598):719–721. [PubMed]
  • Lohka MJ, 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. [PMC free article] [PubMed]
  • Macaulay C, Meier E, Forbes DJ. Differential mitotic phosphorylation of proteins of the nuclear pore complex. J Biol Chem. 1995 Jan 6;270(1):254–262. [PubMed]
  • Maul GG. Nuclear pore complexes. Elimination and reconstruction during mitosis. J Cell Biol. 1977 Aug;74(2):492–500. [PMC free article] [PubMed]
  • Maul GG, Maul HM, Scogna JE, Lieberman MW, Stein GS, Hsu BY, Borun TW. 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. [PMC free article] [PubMed]
  • Merisko EM. Annulate lamellae: an organelle in search of a function. Tissue Cell. 1989;21(3):343–354. [PubMed]
  • Miller MW, Hanover JA. Functional nuclear pores reconstituted with beta 1-4 galactose-modified O-linked N-acetylglucosamine glycoproteins. J Biol Chem. 1994 Mar 25;269(12):9289–9297. [PubMed]
  • Murray AW, Solomon MJ, Kirschner MW. The role of cyclin synthesis and degradation in the control of maturation promoting factor activity. Nature. 1989 May 25;339(6222):280–286. [PubMed]
  • Nehrbass U, Kern H, Mutvei A, Horstmann H, Marshallsay B, Hurt EC. 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. [PubMed]
  • Newmeyer DD, Forbes DJ. Nuclear import can be separated into distinct steps in vitro: nuclear pore binding and translocation. Cell. 1988 Mar 11;52(5):641–653. [PubMed]
  • Newmeyer DD, Wilson KL. Egg extracts for nuclear import and nuclear assembly reactions. Methods Cell Biol. 1991;36:607–634. [PubMed]
  • Newmeyer DD, Finlay DR, Forbes DJ. In vitro transport of a fluorescent nuclear protein and exclusion of non-nuclear proteins. J Cell Biol. 1986 Dec;103(6 Pt 1):2091–2102. [PMC free article] [PubMed]
  • Newmeyer DD, Lucocq JM, Bürglin TR, De Robertis EM. Assembly in vitro of nuclei active in nuclear protein transport: ATP is required for nucleoplasmin accumulation. EMBO J. 1986 Mar;5(3):501–510. [PubMed]
  • Newport J. Nuclear reconstitution in vitro: stages of assembly around protein-free DNA. Cell. 1987 Jan 30;48(2):205–217. [PubMed]
  • 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. [PMC free article] [PubMed]
  • Newport J, Spann T, Kanki J, Forbes D. The role of mitotic factors in regulating the timing of the midblastula transition in Xenopus. Cold Spring Harb Symp Quant Biol. 1985;50:651–656. [PubMed]
  • Newport JW, Wilson KL, Dunphy WG. A lamin-independent pathway for nuclear envelope assembly. J Cell Biol. 1990 Dec;111(6 Pt 1):2247–2259. [PMC free article] [PubMed]
  • Osborne MA, Silver PA. Nucleocytoplasmic transport in the yeast Saccharomyces cerevisiae. Annu Rev Biochem. 1993;62:219–254. [PubMed]
  • Panté N, Aebi U. The nuclear pore complex. J Cell Biol. 1993 Sep;122(5):977–984. [PMC free article] [PubMed]
  • 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. [PMC free article] [PubMed]
  • Pfaller R, Newport JW. Nuclear envelope assembly following mitosis. Methods Enzymol. 1992;219:60–72. [PubMed]
  • Pfaller R, Smythe C, Newport JW. 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. [PubMed]
  • Powers MA, Forbes DJ. Cytosolic factors in nuclear transport: what's importin? Cell. 1994 Dec 16;79(6):931–934. [PubMed]
  • Powers MA, Macaulay C, Masiarz FR, Forbes DJ. Reconstituted nuclei depleted of a vertebrate GLFG nuclear pore protein, p97, import but are defective in nuclear growth and replication. J Cell Biol. 1995 Mar;128(5):721–736. [PMC free article] [PubMed]
  • Radu A, Blobel G, Wozniak RW. 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. [PMC free article] [PubMed]
  • Reichelt R, Holzenburg A, Buhle EL, 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. [PMC free article] [PubMed]
  • Richardson WD, Mills AD, Dilworth SM, Laskey RA, 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. [PubMed]
  • Rout MP, Blobel G. Isolation of the yeast nuclear pore complex. J Cell Biol. 1993 Nov;123(4):771–783. [PMC free article] [PubMed]
  • Rout MP, Wente SR. Pores for thought: nuclear pore complex proteins. Trends Cell Biol. 1994 Oct;4(10):357–365. [PubMed]
  • Scheer U, Dabauvalle MC, Merkert H, Benevente R. The nuclear envelope and the organization of the pore complexes. Cell Biol Int Rep. 1988 Sep;12(9):669–689. [PubMed]
  • Sheehan MA, Mills AD, Sleeman AM, Laskey RA, Blow JJ. 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. [PMC free article] [PubMed]
  • Smith FE, Berlin JD. Cytoplasmic annulate lamellae in human spermatogenesis. Cell Tissue Res. 1977 Jan 12;176(2):235–242. [PubMed]
  • Smythe C, Newport JW. Systems for the study of nuclear assembly, DNA replication, and nuclear breakdown in Xenopus laevis egg extracts. Methods Cell Biol. 1991;35:449–468. [PubMed]
  • Snow CM, Senior A, Gerace L. Monoclonal antibodies identify a group of nuclear pore complex glycoproteins. J Cell Biol. 1987 May;104(5):1143–1156. [PMC free article] [PubMed]
  • Stafstrom JP, Staehelin LA. Are annulate lamellae in the Drosophila embryo the result of overproduction of nuclear pore components? J Cell Biol. 1984 Feb;98(2):699–708. [PMC free article] [PubMed]
  • Starr CM, D'Onofrio M, Park MK, Hanover JA. Primary sequence and heterologous expression of nuclear pore glycoprotein p62. J Cell Biol. 1990 Jun;110(6):1861–1871. [PMC free article] [PubMed]
  • Steinert G, Baltus E, Hanocq-Quertier J, Brachet J. Ultrastructure of Xenopus laevis oocytes after injection of an extract from progesterone-treated oocytes. J Ultrastruct Res. 1974 Nov;49(2):188–210. [PubMed]
  • 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. [PubMed]
  • Sullivan KM, Busa WB, Wilson KL. 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. [PubMed]
  • Vigers GP, Lohka MJ. 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. [PMC free article] [PubMed]
  • Vigers GP, Lohka MJ. Regulation of nuclear envelope precursor functions during cell division. J Cell Sci. 1992 Jun;102(Pt 2):273–284. [PubMed]
  • Wente SR, Rout MP, Blobel G. A new family of yeast nuclear pore complex proteins. J Cell Biol. 1992 Nov;119(4):705–723. [PMC free article] [PubMed]
  • Wessel D, Flügge UI. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem. 1984 Apr;138(1):141–143. [PubMed]
  • Wilken N, Kossner U, Senécal JL, Scheer U, Dabauvalle MC. 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. [PMC free article] [PubMed]
  • Wilson KL, 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. [PMC free article] [PubMed]
  • Wozniak RW, 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. [PMC free article] [PubMed]
  • Wunderlich F, Wallach DF, Speth V, Fischer H. Differential effects of temperature on the nuclear and plasma membranes of lymphoid cells. A study by freeze-etch electron microscopy. Biochim Biophys Acta. 1974 Nov 27;373(1):34–43. [PubMed]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press