Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Reconstitution of nuclear protein export in isolated nuclear envelopes.
Siebrasse JP, Coutavas E, Peters R.
???displayArticle.abstract???
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 envelope–sealed 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 ∼40 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 ∼50–100 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 ∼120 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 ∼40 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.
Arts,
Identification of a nuclear export receptor for tRNA.
1998, Pubmed,
Xenbase
Arts,
Identification of a nuclear export receptor for tRNA.
1998,
Pubmed
,
Xenbase Askjaer,
RanGTP-regulated interactions of CRM1 with nucleoporins and a shuttling DEAD-box helicase.
1999,
Pubmed
,
Xenbase Ben-Efraim,
Gradient of increasing affinity of importin beta for nucleoporins along the pathway of nuclear import.
2001,
Pubmed Bischoff,
Catalysis of guanine nucleotide exchange of Ran by RCC1 and stimulation of hydrolysis of Ran-bound GTP by Ran-GAP1.
1995,
Pubmed Black,
Identification of an NTF2-related factor that binds Ran-GTP and regulates nuclear protein export.
1999,
Pubmed Chook,
Karyopherins and nuclear import.
2001,
Pubmed Clouse,
A Ran-independent pathway for export of spliced mRNA.
2001,
Pubmed
,
Xenbase Cole,
mRNA export: the long and winding road.
2000,
Pubmed Coutavas,
Characterization of proteins that interact with the cell-cycle regulatory protein Ran/TC4.
1993,
Pubmed
,
Xenbase Englmeier,
RanBP3 influences interactions between CRM1 and its nuclear protein export substrates.
2001,
Pubmed Englmeier,
Receptor-mediated substrate translocation through the nuclear pore complex without nucleotide triphosphate hydrolysis.
1999,
Pubmed Görlich,
Transport between the cell nucleus and the cytoplasm.
1999,
Pubmed Görlich,
Identification of different roles for RanGDP and RanGTP in nuclear protein import.
1996,
Pubmed
,
Xenbase Grüter,
TAP, the human homolog of Mex67p, mediates CTE-dependent RNA export from the nucleus.
1998,
Pubmed
,
Xenbase Hellmuth,
Yeast Los1p has properties of an exportin-like nucleocytoplasmic transport factor for tRNA.
1998,
Pubmed Kehlenbach,
A role for RanBP1 in the release of CRM1 from the nuclear pore complex in a terminal step of nuclear export.
1999,
Pubmed Keminer,
Optical recording of signal-mediated protein transport through single nuclear pore complexes.
1999,
Pubmed
,
Xenbase Keminer,
Permeability of single nuclear pores.
1999,
Pubmed
,
Xenbase Kutay,
Identification of a tRNA-specific nuclear export receptor.
1998,
Pubmed
,
Xenbase Liker,
The structure of the mRNA export factor TAP reveals a cis arrangement of a non-canonical RNP domain and an LRR domain.
2000,
Pubmed Lindsay,
Ran-binding protein 3 is a cofactor for Crm1-mediated nuclear protein export.
2001,
Pubmed Lyman,
Nuclear pore complexes: dynamics in unexpected places.
2001,
Pubmed Mattaj,
Cell biology. Snail mail to the nucleus.
1999,
Pubmed Mueller,
Human RanBP3, a group of nuclear RanGTP binding proteins.
1998,
Pubmed Nakielny,
Import and export of the nuclear protein import receptor transportin by a mechanism independent of GTP hydrolysis.
1998,
Pubmed Radtke,
Kinetics of protein import into isolated Xenopus oocyte nuclei.
2001,
Pubmed
,
Xenbase Ribbeck,
Kinetic analysis of translocation through nuclear pore complexes.
2001,
Pubmed Ribbeck,
The translocation of transportin-cargo complexes through nuclear pores is independent of both Ran and energy.
1999,
Pubmed Richards,
Requirement of guanosine triphosphate-bound ran for signal-mediated nuclear protein export.
1997,
Pubmed Rout,
The yeast nuclear pore complex: composition, architecture, and transport mechanism.
2000,
Pubmed Segref,
Mex67p, a novel factor for nuclear mRNA export, binds to both poly(A)+ RNA and nuclear pores.
1997,
Pubmed Tschödrich-Rotter,
An optical method for recording the activity of single transporters in membrane patches.
1998,
Pubmed Wolff,
Leptomycin B is an inhibitor of nuclear export: inhibition of nucleo-cytoplasmic translocation of the human immunodeficiency virus type 1 (HIV-1) Rev protein and Rev-dependent mRNA.
1997,
Pubmed
