Reconstitution of nuclear protein export in isolated nuclear envelopes.
Journal Article (Journal Article)
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.
Full Text
Duke Authors
Cited Authors
- Siebrasse, JP; Coutavas, E; Peters, R
Published Date
- September 2, 2002
Published In
Volume / Issue
- 158 / 5
Start / End Page
- 849 - 854
PubMed ID
- 12196506
Pubmed Central ID
- PMC2173161
International Standard Serial Number (ISSN)
- 0021-9525
Digital Object Identifier (DOI)
- 10.1083/jcb.200201130
Language
- eng
Conference Location
- United States