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Reconstitution of nuclear protein export in isolated nuclear envelopes

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. Ex...

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Detalles Bibliográficos
Autores principales: Siebrasse, Jan Peter, Coutavas, Elias, Peters, Reiner
Formato: Texto
Lenguaje:English
Publicado: The Rockefeller University Press 2002
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2173161/
https://www.ncbi.nlm.nih.gov/pubmed/12196506
http://dx.doi.org/10.1083/jcb.200201130
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author Siebrasse, Jan Peter
Coutavas, Elias
Peters, Reiner
author_facet Siebrasse, Jan Peter
Coutavas, Elias
Peters, Reiner
author_sort Siebrasse, Jan Peter
collection PubMed
description 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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spelling pubmed-21731612008-05-01 Reconstitution of nuclear protein export in isolated nuclear envelopes Siebrasse, Jan Peter Coutavas, Elias Peters, Reiner J Cell Biol Report 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. The Rockefeller University Press 2002-09-02 /pmc/articles/PMC2173161/ /pubmed/12196506 http://dx.doi.org/10.1083/jcb.200201130 Text en Copyright © 2002, The Rockefeller University Press This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).
spellingShingle Report
Siebrasse, Jan Peter
Coutavas, Elias
Peters, Reiner
Reconstitution of nuclear protein export in isolated nuclear envelopes
title Reconstitution of nuclear protein export in isolated nuclear envelopes
title_full Reconstitution of nuclear protein export in isolated nuclear envelopes
title_fullStr Reconstitution of nuclear protein export in isolated nuclear envelopes
title_full_unstemmed Reconstitution of nuclear protein export in isolated nuclear envelopes
title_short Reconstitution of nuclear protein export in isolated nuclear envelopes
title_sort reconstitution of nuclear protein export in isolated nuclear envelopes
topic Report
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2173161/
https://www.ncbi.nlm.nih.gov/pubmed/12196506
http://dx.doi.org/10.1083/jcb.200201130
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