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Reconstitution of nuclear protein export in isolated nuclear envelopes.
Siebrasse JP
,
Coutavas E
,
Peters R
.
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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.
Figure 1. The OSTR version used in this study. (A) Sequence of events during an OSTR experiment. (B) Microscopic appearance of an OSTR specimen incubated with the NPC-impermeable molecule TRD70. (B1) Largest perimeter of a filter-attached intact nucleus. (B2) Filter-attached area of same nucleus. The typical size and position of measuring and reference fields (white squares) are indicated. (B3) Nuclear envelope after dissection and washing. (B4) Filter pores visualized at higher magnification showing the boundary of the nuclear envelopeâsealed area.
Figure 2. Examples of export measurements. (A) In an OSTR chamber, a filter-attached perforated nucleus was incubated with a transport medium containing 2 μM GG-NES, 7.5 μM TRD70, and 500 μM GTP. The time after addition of the transport medium to the OSTR chamber is indicated. A region in the center of the area covered by the nucleus was selected and the filter pores imaged. The accumulation of GG-NES with time and the exclusion of TRD70 can be recognized. After completion of export, the nucleus-covered area was shifted out of the field of view and a reference field was imaged in a region of the filter where GG-NES and TRD70 had free access to the filter pores. (B) Analogue experiments with isolated filter-attached nuclear envelopes at conditions specified in Fig. 5 B.
Figure 3. Export kinetics in perforated nuclei. The mean fluorescence of filter pores was plotted in normalized form versus time after the addition of transport medium. Symbols are mean ± SD of five or more measurements, each comprising â¼40 membrane patches. Lines are fits of simple exponentials to the experimental data. GG-NES was strongly exported in the presence of GTP alone (squares). Export started after a lag time of â¼50â100 s, probably needed for diffusion of export substrate to the inner side of the nuclear envelope and formation of the export complex, and proceeded beyond equilibration. If GTP was omitted (circles), export was reduced. If GTP was replaced by the nonhydrolyzable analogue GTPγS (diamonds), export was strongly inhibited. In the presence of leptomycin B (LMB) and GTP (triangles), export of GG-NES was virtually abolished. No export was seen of GG, an inert protein, in the presence of GTP (inverted triangles).
Figure 4. Protein profiles of perforated nuclei and nuclear envelopes. Either two perforated nuclei or four nuclear envelopes were separated by SDS gel electrophoresis and transferred to a nitrocellulose membrane. (A) Amido black staining (the prominent band at â¼120 kD is probably due to contaminating yolk proteins). (B) Western blots probed with anti-CRM1, anti-p62, and anti-Ran.
Figure 5. Export kinetics in isolated nuclear envelopes. The mean fluorescence of filter pores was plotted in normalized form versus time after addition of transport medium. Symbols are mean ± SD of five or more measurements, each comprising the data of â¼40 filter pores. Lines are fits of a simple exponential with off-set to the experimental data. (A) In the presence of GTP alone (circles), GTP and CRM1 (triangles), or GTP and RanGTP (inverted triangles) little export of GG-NES was observed. However, when an export complex was preformed from GG-NES, CRM1, and RanGTP and applied together with GTP (squares), export was strong, without apparent lag time, and proceeded beyond equilibration. (B) Export of export complexes in the absence of free GTP. A complex of GG-NES, CRM1, and RanGTP (squares) was very rapidly exported and accumulated in filter pores. When hydrolysis of Ran-bound GTP was prevented by using for complex formation either RanGTPγS (circles) or a GTPase-deficient Ran mutant (triangles), export was strongly inhibited.
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