Signal-dependent nuclear protein export was studied in perforated nuclei and isolated nuclear envelopes of Xenopus oocytes by optical single transporter recording. Manually isolated and purified oocyte nuclei were attached to isoporous filters and made permeable for macromolecules by perforation. Export of a recombinant protein (GG-NES) containing the nuclear export signal (NES) of the protein kinase A inhibitor through nuclear envelope patches spanning filter pores could be induced by the addition of GTP alone. Export continued against a concentration gradient, and was NES dependent and inhibited by leptomycin B and GTPγS, a nonhydrolyzable GTP analogue. Addition of recombinant RanBP3, a potential cofactor of CRM1-dependent export, did not promote GG-NES export at stoichiometric concentration but gradually inhibited export at higher concentrations. In isolated filter-attached nuclear envelopes, export of GG-NES was virtually abolished in the presence of GTP alone. However, a preformed export complex consisting of GG-NES, recombinant human CRM1, and RanGTP was rapidly exported. Unexpectedly, export was strongly reduced when the export complex contained RanGTPγS or RanG19V/Q69L-GTP, a GTPase-deficient Ran mutant. This paper shows that nuclear transport, previously studied in intact and permeabilized cells only, can be quantitatively analyzed in perforated nuclei and isolated nuclear envelopes.
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2 September 2002
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August 26 2002
Reconstitution of nuclear protein export in isolated nuclear envelopes
Jan Peter Siebrasse,
Jan Peter Siebrasse
1Institut für Medizinische Physik und Biophysik, Universität Münster, 48149 Münster, Germany
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Elias Coutavas,
Elias Coutavas
2Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, NY 10021
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Reiner Peters
Reiner Peters
1Institut für Medizinische Physik und Biophysik, Universität Münster, 48149 Münster, Germany
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Jan Peter Siebrasse
1Institut für Medizinische Physik und Biophysik, Universität Münster, 48149 Münster, Germany
Elias Coutavas
2Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, NY 10021
Reiner Peters
1Institut für Medizinische Physik und Biophysik, Universität Münster, 48149 Münster, Germany
Address correspondence to Dr. Reiner Peters, Institut für Medizinische Physik und Biophysik, Universität Münster, Robert-Koch-Strasse 31, 48149 Münster, Germany. Tel.: 49-251-835-6933. Fax: 49-251-835-5121. E-mail: [email protected]
*
Abbreviations used in this paper: NES, nuclear export signal; NLS, nuclear localization sequence; NPC, nuclear pore complex; OSTR, optical single transporter recording; TRD70, Texas red–labeled 70-kD dextran.
Received:
January 31 2002
Revision Received:
July 16 2002
Accepted:
July 17 2002
Online ISSN: 1540-8140
Print ISSN: 0021-9525
The Rockefeller University Press
2002
J Cell Biol (2002) 158 (5): 849–854.
Article history
Received:
January 31 2002
Revision Received:
July 16 2002
Accepted:
July 17 2002
Citation
Jan Peter Siebrasse, Elias Coutavas, Reiner Peters; Reconstitution of nuclear protein export in isolated nuclear envelopes . J Cell Biol 2 September 2002; 158 (5): 849–854. doi: https://doi.org/10.1083/jcb.200201130
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