Although nuclear envelope (NE) assembly is known to require the GTPase Ran, the membrane fusion machinery involved is uncharacterized. NE assembly involves formation of a reticular network on chromatin, fusion of this network into a closed NE and subsequent expansion. Here we show that p97, an AAA-ATPase previously implicated in fusion of Golgi and transitional endoplasmic reticulum (ER) membranes together with the adaptor p47, has two discrete functions in NE assembly. Formation of a closed NE requires the p97–Ufd1–Npl4 complex, not previously implicated in membrane fusion. Subsequent NE growth involves a p97–p47 complex. This study provides the first insights into the molecular mechanisms and specificity of fusion events involved in NE formation.
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Gerace, L. & Burke, B. Functional organization of the nuclear envelope. Annu. Rev. Cell Biol. 4, 335–374 (1988).
Gant, T. M. & Wilson K. L. Nuclear assembly. Annu. Rev. Cell Dev. Biol. 13, 669–695 (1997).
Franke, W. W., Scheer, U., Krohne, G. & Jarasch, E. D. The nuclear envelope and the architecture of the nuclear periphery. J. Cell Biol. 91, 39s–50s (1981).
Ellenberg, J. et al. Nuclear membrane dynamics and reassembly in living cells: targeting of an inner nuclear membrane protein in interphase and mitosis. J. Cell Biol. 138, 1193–1206 (1997).
Powell, L. & Burke, B. Internuclear exchange of an inner nuclear membrane protein (p55) in heterokaryons: in vivo evidence for the interaction of p55 with the nuclear lamina. J. Cell Biol. 111, 2225–2234 (1990).
Soullam, B. & Worman, H. J. Signals and structural features involved in integral membrane protein targeting to the inner nuclear membrane. J. Cell Biol. 130, 15–27 (1995).
Lohka, M. J. & Masui, Y. Formation in vitro of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmic components. Science 220, 719–721 (1983).
Burke, B. & Gerace, L. A cell free system to study reassembly of the nuclear envelope at the end of mitosis. Cell 28, 639–652 (1986).
Dreier, L. & Rapoport, T. A. In vitro formation of the endoplasmic reticulum occurs independently of microtubules by a controlled fusion reaction. J. Cell Biol. 148, 883–898 (2000).
Macaulay, C. & Forbes, D. J. Assembly of the nuclear pore: biochemically distinct steps revealed with NEM, GTPγS, and BAPTA. J. Cell Biol. 132, 5–20 (1996).
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. 112, 545–556 (1991).
Newport, J. & Dunphy, W. Characterization of the membrane binding and fusion events during nuclear envelope assembly using purified components. J. Cell Biol. 116, 295–306 (1992).
Marshall, I. C. B. & Wilson, K. L. Nuclear envelope assembly after mitosis. Trends Cell Biol. 7, 69–74 (1997).
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. 116, 281–294 (1992).
Hetzer, M., Bilbao-Cortes, D., Walther, T. C., Gruss, O. J. & Mattaj, I. W. GTP hydrolysis by Ran is required for nuclear envelope assembly. Mol. Cell 5, 1013–1024 (2000).
Zhang, C. & Clarke, P. R. Chromatin-independent nuclear envelope assembly induced by Ran GTPase in Xenopus egg extracts. Science 26, 1429–1432 (2000).
Zhang, C. & Clarke, P. R. Roles of Ran-GTP and Ran-GDP in precursor vesicle recruitment and fusion during nuclear envelope assembly in a human cell-free system. Curr. Biol. 6, 208–212 (2001).
Benavente, R., Dabauvalle, M. C., Scheer, U. & Chaly, N. Functional role of newly formed pore complexes in postmitotic nuclear reorganization. Chromosoma 98, 233–241 (1989).
Wilson, K. L. & Newport, J. A trypsin-sensitive receptor on membrane vesicles is required for nuclear envelope formation in vitro. J. Cell Biol. 107, 57–68 (1988).
Drummond, S. et al. Temporal differences in the appearance of NEP-B78 and a LBR-like protein during Xenopus nuclear envelope reassembly reflect the order recruitment of functionally discrete vesicle types. J. Cell Biol. 144, 225–240 (1999).
Sasagawa, S., Yamamoto, A., Ichimura, T., Omata, S. & Horigome, T. In vitro nuclear assembly with affinity-purified nuclear precursor vesicle fractions, PV1 and PV2. Euro. J. Cell Biol. 78, 593–600 (1999).
Patel, S. & Latterich, M. The AAA team: related ATPases with diverse functions. Trends Cell Biol. 8, 65–71 (1998).
Kondo, H et al. p47 is a cofactor for p97-mediated membrane fusion. Nature 388, 75–78 (1997).
Roy, L. et al. Role of p97 and syntaxin 5 in the assembly of transitional endoplasmic reticulum. Mol. Biol. Cell 11, 2529–2542 (2000).
Rabouille, C. et al. Syntaxin 5 is a common component of the NSF- and p97-mediated reassembly pathways of Golgi cisternae from mitotic Golgi fragments in vitro. Cell 92, 603–610 (1998).
Meyer, H. H., Shorter, J. G., Seemann, J., Pappin, D. & Warren, G. A complex of mammalian Ufd1 and Npl4 links the AAA-ATPase, p97, to ubiquitin and nuclear transport pathways. EMBO J. 19, 2181–2192 (2000).
Latterich, M., Frohlich, K. U. & Schekman, R. Membrane fusion and the cell cycle: Cdc48p participates in the fusion of ER membranes. Cell 82, 885–893 (1995).
Johnson, E. S., Ma, P. C., Ota, I. M. & Varshavsky, A. A proteolytic pathway that recognizes ubiquitin as a degradation signal. J. Biol. Chem. 270, 17442–17456 (1995).
Ghislain, M., Dohmen, R. J., Levy, F. & Varshavsky, A. Cdc48p interacts with Ufd3p, a WD repeat protein required for ubiquitin-mediated proteolysis in Saccharomyces cerevisiae. EMBO J. 15, 4884–4899 (1996).
Hoppe, T. et al. Activation of a membrane-bound transcription factor by regulated ubiquitin/proteasome-dependent processing. Cell 102, 577–586 (2000).
DeHoratius, C. & Silver, P. A. Nuclear transport defects and nuclear envelope alterations are associated with mutation of the Saccharomyces cerevisiae NPL4 gene. Mol. Biol. Cell 7, 1835–1855 (1996).
Whaley, W. G. The Golgi apparatus. Cell Biology Monographs, Continuation of Protoplasmatologia, 2 (Springer, Vienna, 1975).
Rossanese, O. W. et al. Golgi structure correlates with transitional endoplasmic reticulum organization in Pichia pastoris and Saccharomyces cerevisiae. J. Cell Biol. 145, 68–81 (1999).
Lavoie, C., Lanoix, J., Kan, F. W. & Paiement, J. Cell-free assembly of rough and smooth endoplasmic reticulum. J. Cell Sci. 109, 1415–1425 (1996).
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. USA 84, 7552–7556 (1987).
M.H. was supported by EMBO and H.H.M. by the Human Frontiers Science Programme Organisation. We thank Jan Ellenberg and the ELMF for help with microscopy. Thanks to W. Antonin, P. Askjaer, K. Czaplinski, L. Englmeier, J. Ellenberg, V. Hachet, B. Huelsmann, M. Ohno and C. Schatz for critical comments on the manuscript.
Figure S1 Nuclei formed in the presence of p97 are functional. (PDF 348 kb)
Figure S2 Characterization of NE proteins in p97-restored nuclei.
Figure S3 Nuclei formed in the absence of p47 have an intact NE.
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Hetzer, M., Meyer, H., Walther, T. et al. Distinct AAA-ATPase p97 complexes function in discrete steps of nuclear assembly. Nat Cell Biol 3, 1086–1091 (2001). https://doi.org/10.1038/ncb1201-1086
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