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Reconstitution of nuclear protein export in isolated nuclear envelopes.

Publication ,  Journal Article
Siebrasse, JP; Coutavas, E; Peters, R
Published in: J Cell Biol
September 2, 2002

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 GTPgammaS, 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 RanGTPgammaS 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.

Duke Scholars

Published In

J Cell Biol

DOI

ISSN

0021-9525

Publication Date

September 2, 2002

Volume

158

Issue

5

Start / End Page

849 / 854

Location

United States

Related Subject Headings

  • ran GTP-Binding Protein
  • Xenopus
  • Signal Transduction
  • Recombinant Proteins
  • Receptors, Cytoplasmic and Nuclear
  • Oocytes
  • Nuclear Proteins
  • Nuclear Envelope
  • Kinetics
  • Karyopherins
 

Citation

APA
Chicago
ICMJE
MLA
NLM
Siebrasse, J. P., Coutavas, E., & Peters, R. (2002). Reconstitution of nuclear protein export in isolated nuclear envelopes. J Cell Biol, 158(5), 849–854. https://doi.org/10.1083/jcb.200201130
Siebrasse, Jan Peter, Elias Coutavas, and Reiner Peters. “Reconstitution of nuclear protein export in isolated nuclear envelopes.J Cell Biol 158, no. 5 (September 2, 2002): 849–54. https://doi.org/10.1083/jcb.200201130.
Siebrasse JP, Coutavas E, Peters R. Reconstitution of nuclear protein export in isolated nuclear envelopes. J Cell Biol. 2002 Sep 2;158(5):849–54.
Siebrasse, Jan Peter, et al. “Reconstitution of nuclear protein export in isolated nuclear envelopes.J Cell Biol, vol. 158, no. 5, Sept. 2002, pp. 849–54. Pubmed, doi:10.1083/jcb.200201130.
Siebrasse JP, Coutavas E, Peters R. Reconstitution of nuclear protein export in isolated nuclear envelopes. J Cell Biol. 2002 Sep 2;158(5):849–854.

Published In

J Cell Biol

DOI

ISSN

0021-9525

Publication Date

September 2, 2002

Volume

158

Issue

5

Start / End Page

849 / 854

Location

United States

Related Subject Headings

  • ran GTP-Binding Protein
  • Xenopus
  • Signal Transduction
  • Recombinant Proteins
  • Receptors, Cytoplasmic and Nuclear
  • Oocytes
  • Nuclear Proteins
  • Nuclear Envelope
  • Kinetics
  • Karyopherins