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Published online 26 August 2002. doi:10.1083/jcb.200201130
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© The Rockefeller University Press, 0021-9525/2002/9/849 $5.00
The Journal of Cell Biology, Volume 158, Number 5, September 2, 2002 849-854

Report

Reconstitution of nuclear protein export in isolated nuclear envelopes



Jan Peter Siebrasse1, Elias Coutavas2 and Reiner Peters1

1 Institut für Medizinische Physik und Biophysik, Universität Münster, 48149 Münster, Germany
2 Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, NY 10021

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: petersr{at}uni-muenster.de

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 GTP{gamma}S, 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 RanGTP{gamma}S 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.

Key Words: nucleocytoplasmic transport; nuclear export; nuclear pore complex; reconstitution; oocyte


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