|
|
|
|
|
|
|
||
© The Rockefeller University Press,
0021-9525/2001//895 $5.00
The Journal of Cell Biology, Volume 152, Number 5,
, 2001 895-910
Original Article |
Cofactor Requirements for Nuclear Export of Rev Response Element (Rre)–And Constitutive Transport Element (Cte)–Containing Retroviral Rnas
: An Unexpected Role for Actin
b Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037
c Institute for Clinical and Molecular Virology, University Erlangen-Nürnberg, D-91054 Erlangen, Germany
d Max-Planck-Institute for Biochemistry, D-82152 Martinsried, Germany
e Cell Biology, Zoological Institute, Technical University of Braunschweig, D-38092 Braunschweig, Germany
Department of Cell and Developmental Biology, Biocenter of the University of Würzburg, Am Hubland, D-97074 Würzburg, Germany.49-931-888-425249-931-888-4273
mcd{at}biozentrum.uni-wuerzburg.de
Nuclear export of proteins containing leucine-rich nuclear export signals (NESs) is mediated by the export receptor CRM1/exportin1. However, additional protein factors interacting with leucine-rich NESs have been described. Here, we investigate human immunodeficiency virus type 1 (HIV-1) Rev-mediated nuclear export and Mason-Pfizer monkey virus (MPMV) constitutive transport element (CTE)–mediated nuclear export in microinjected Xenopus laevis oocytes. We show that eukaryotic initiation factor 5A (eIF-5A) is essential for Rev and Rev-mediated viral RNA export, but not for nuclear export of CTE RNA. In vitro binding studies demonstrate that eIF-5A is required for efficient interaction of Rev–NES with CRM1/exportin1 and that eIF-5A interacts with the nucleoporins CAN/nup214, nup153, nup98, and nup62. Quite unexpectedly, nuclear actin was also identified as an eIF-5A binding protein. We show that actin is associated with the nucleoplasmic filaments of nuclear pore complexes and is critically involved in export processes. Finally, actin- and energy-dependent nuclear export of HIV-1 Rev is reconstituted by using a novel in vitro egg extract system. In summary, our data provide evidence that actin plays an important functional role in nuclear export not only of retroviral RNAs but also of host proteins such as protein kinase inhibitor (PKI).
Key Words: eIF-5A • CRM1 • nuclear actin • nuclear export • HIV-1 Rev
© 2001 The Rockefeller University Press
 |
Introduction |
|---|
 TopAbstract Introduction Materials and MethodsResultsDiscussionReferences |
|---|
Competition experiments in Xenopus laevis oocytes suggested that different classes of RNA (e.g., mRNA, rRNA, U snRNA, tRNA) are exported from the nucleus by specific export factors (Jarmolowski et al. 1994). In general, RNA export seems to be a multistep mechanism that is mediated by RNA-binding proteins that contain nuclear export signals (NESs) (for reviews see Izaurralde and Adam 1998; Mattaj and Englmeier 1998; Stutz and Rosbash 1998; Görlich and Kutay 1999; Nakielny and Dreyfuss 1999). Typically, NESs are recognized by soluble export receptors that target the transport complexes to the NPCs. In addition, efficient export requires the action of several critical factors, of which the GTPase Ran/TC4 and associated components play a major functional role in determining the direction of nucleocytoplasmic transport (for reviews see Görlich and Kutay 1999; Melchior and Gerace 1998; Moore 1998).
To date, the most extensively investigated specific mRNA export factor is the Rev trans-activator protein of human immunodeficiency virus type 1 (HIV-1) (for review see Pollard and Malim 1998). Rev is a nucleocytoplasmic shuttle protein that directly binds to its Rev-response element (RRE) RNA target sequence, which is part of all unspliced and incompletely spliced viral mRNAs. Upon multimerization and subsequent interaction with multiple cellular cofactors, Rev promotes the translocation of these mRNAs across the nuclear envelope. The region of Rev that interacts with cellular cofactors that are required for nuclear export of Rev–RRE ribonucleoprotein particles has been defined as either the activation or effector domain. This domain contains a short stretch of hydrophobic, primarily leucine amino acids and constitutes a prototypic NES that, upon fusion to heterologous protein substrates, mediates the rapid and active nuclear export of these otherwise inert proteins (Fischer et al. 1995; Stauber et al. 1995; Wen et al. 1995; Meyer et al. 1996; Elfgang et al. 1999).
A series of studies has shown that the primary target of leucine-rich Rev-like NESs is the export receptor CRM1/exportin1 and, furthermore, that NES-CRM1/exportin1 interaction depends on the presence of RanGTP (Fornerod et al. 1997a; Fukuda et al. 1997; Ossareh-Nazari et al. 1997; Stade et al. 1997; Askjaer et al. 1998). Studies with leptomycin B, a specific inhibitor of CRM1/exportin1 (Kudo et al. 1998, Kudo et al. 1999) that prevents the formation of stable NES-CRM1/exportin1 complexes, demonstrated that CRM1/exportin1 indeed mediates the translocation of all Rev-like NES-containing export cargoes through the NPC (Fornerod et al. 1997a; Fukuda et al. 1997; Ossareh-Nazari et al. 1997; Wolff et al. 1997; Engel et al. 1998; Freedman and Levine 1998; Kudo et al. 1998; Toyoshima et al. 1998; Wada et al. 1998; Stommel et al. 1999). However, investigation of HIV-1 Rev function also led to the identification of another cellular protein, eukaryotic initiation factor 5A (eIF-5A), that binds to the Rev–NES (Ruhl et al. 1993). eIF-5A is unique because it is the only cellular protein known to date to contain the unusual amino acid hypusine (Park et al. 1993). The hypusine modification, which does occur in eukaryotes and archaebacteria but not in eubacteria, is a spermidine-dependent posttranslational modification that is essential for eIF-5A function, although its exact function is yet unknown. Distinct eIF-5A mutants have been described that block Rev activity and thereby HIV-1 replication in human T cells in trans (Bevec et al. 1996; Junker et al. 1996). Moreover, microinjection experiments in somatic cells have demonstrated that eIF-5A is an essential cofactor specifically required for the nuclear export of HIV-1 Rev and the functionally equivalent Rex protein of human T cell leukemia virus type I (HTLV-I) (Elfgang et al. 1999). Finally, studies using Xenopus oocytes have revealed that eIF-5A accumulates at NPC-associated intranuclear filaments, interacts with the export receptor CRM1/exportin1, and is by itself exported from the nucleus to the cytoplasm in a leptomycin B–sensitive manner (Rosorius et al. 1999b).
In this study, we investigated the cofactor requirements for nuclear export of the prototypic HIV-1 Rev RNA export factor in Xenopus oocytes. Microinjection experiments demonstrate that nuclear export of glutathione S-transferase (GST)–Rev–NES fusion proteins, as well as Rev-mediated RRE RNA export, depends in oocytes, as it does in mammalian cells, on the Rev cofactor eIF-5A. Furthermore, binding studies using recombinant GST–eIF-5A and nuclear envelope proteins from oocytes demonstrate that eIF-5A interacts specifically with nucleoporins that are known to participate in nuclear export. In the course of these experiments, we noted that eIF-5A interacts not only with bona fide nucleoporins but also with nuclear actin that was localized at the nucleoplasmic face of NPCs, particularly at the NPC-associated intranuclear filaments. We present evidence that nuclear actin is functionally involved in nuclear export processes. Finally, by using Xenopus egg extract, we establish an energy-dependent in vitro nuclear export system and demonstrate that actin also participates in Rev-NES–mediated nuclear export in this cell-free system.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Antibodies
Antibodies against the following antigens were used: eIF-5A and Rev (rabbit antibodies; Hammerschmid et al. 1994); CRM1 (rabbit serum; Kudo et al. 1997); actin (mAb 2G2; Gonsior et al. 1999); phenylalanine–glycine (FG) repeat nucleoporins (mAb 414; Hiss Diagnostics; Davis and Blobel 1986). mAbs specific for GST and BSA or His were obtained from Sigma-Aldrich or QIAGEN, respectively. The peroxidase or gold-conjugated secondary antibodies were from Dianova.
Recombinant Proteins
GST–Rev, GST–Rev–NES, GST–PKI–NES, wild-type (wt) GST–eIF-5A, and GST–eIF-5A–M14 fusion proteins expressed in Escherichia coli BL21 as well as wt eIF-5A and eIF-5A–M14 were previously described (Bevec et al. 1996; Elfgang et al. 1999). GST–Rev–NES–green fluorescent protein (GFP) and GST–RanQ69L was expressed in E. coli BL21 and purified as described (Rosorius et al. 1999a). His-tagged CRM1 was expressed in E. coli TG1 and affinity purified from crude lysates using Ni beads (QIAGEN) as essentially described (Guan et al. 2000), with the exception that a further purification step was added to this protocol by using size S200 exclusion chromatography (Amersham Pharmacia Biotech).
Molecular Clones and In Vitro Synthesis of Radiolabeled RNA
To generate the expression vector pGEX–RanQ69L, the Ran coding region was amplified by polymerase chain reaction from pET3dRanQ69L (Klebe et al. 1993), using appropriate oligonucleotides, and inserted into the BamHI and EcoRI sites of pGEX–3X (Amersham Pharmacia Biotech). A sequence encoding the NES of the HIV–Rev protein (LQLPPLERLTL) was cloned between the BamHI and NheI site of the bacterial expression vector pGEX–GFP (Rosorius et al. 1999a) by using double-stranded synthetic oligonucleotides, resulting in the plasmid pGEX–Rev–NES–GFP.
The plasmid template pGEM–RRE, which is a derivative of the vector pGEM–3Zf(+) (Promega), was used for the synthesis of a 252-nucleotide wt RRE probe and has been described in detail previously (Malim et al. 1990). The variant vector pGEM–RRE
SLII was used to synthesize a nuclear export–deficient RRE probe in which stem loop II (SLII, lacking nucleotides 41–105, as shown in Malim et al. 1990), the high affinity Rev binding site within the RRE, is deleted. The template and the method used for the synthesis of the wt constitutive transport element (CTE) and the double mutant M2/M11 CTE RNA has been described (Ernst et al. 1997). U6 RNA was synthesized using the vector SP6-U6 (Jarmolowski et al. 1994). wt and mutant HIV-1 RRE RNA and wt and mutant Mason-Pfizer monkey virus (MPMV) CTE RNA were radiolabeled by in vitro transcription using [
-32P]UTP and T7 RNA polymerase (Promega) as described previously (Daly et al. 1993a). Likewise, U6 RNA was synthesized using SP6 RNA polymerase (Promega).
Microinjection Experiments
Just before injection, the defolliculated Xenopus oocytes were placed with the animal hemisphere pointing up in a miniplate containing modified Barth's medium and were centrifuged at 1,200 g for 15 min at 22°C in order to visualize the site of the nucleus. For nuclear export analysis, 20 nl export substrates was injected into the nuclei of 20 full-grown Xenopus oocytes that had been injected 1 h before with either purified antibodies against eIF-5A (IgG; 0.5 mg/ml), purified antibodies against actin (IgM; 1 mg/ml), nonspecific rabbit IgG (0.5 mg/ml), nonspecific mouse IgM (1 mg/ml), the actin-binding drugs latrunculin B (3 mM; Calbiochem), or swinholide A (3 mM; Alexis). Injected export substrates were GST–Rev–NES or GST–PKI–NES (0.1 mg/ml), RRE RNA (0.6 µM wt or mutant RRE RNA, 0.8–1.0 x 106 cpm, incubated 30 min on ice with Rev recombinant protein as described by Fischer et al. 1995) and CTE RNA (wt or mutant 32P-labeled CTE RNA, 107 cpm). In some experiments, wt eIF-5A or eIF-5A–M14 proteins (using a 10-fold molar excess over the coinjected GST–Rev–NES or GST–Rev fusion proteins) were preincubated for 1 h at 4°C before microinjection into the oocyte nuclei. BSA (1 mg/ml; Sigma-Aldrich) or 32P-labeled U6 RNA were coinjected as internal markers for monitoring the site of injection. For the microinjection experiments in Vero cells, GFP–Rev–NES–GST fusion protein (1 mg/ml) was coinjected into the nuclei with actin antibodies (IgM, 1 mg/ml) or with nonspecific mouse IgM (1 mg/ml).
Preparation and Analysis of Injected Samples
After an incubation time of 2 h, nuclei and cytoplasms from Xenopus oocytes were manually isolated, and the samples prepared for protein gel electrophoreseis as described (Dabauvalle and Franke 1982). The proteins were separated by SDS-PAGE (Thomas and Kornberg 1975), using 18% acrylamide. For immunoblots, polypeptides were transferred electrophoretically to nitrocellulose filters. Nonspecific protein binding sites were blocked by overnight incubation with TBST (10% nonfat dry milk in 150 mM NaCl, 0.05% Tween-20, 10 mM Tris-HCl, pH 8.0) at 4°C. After several washes in TBST, the nitrocellulose filters were incubated for 2 h at room temperature with GST antibodies (diluted 1:1,000 in TBST). After washing again in TBST, the nitrocellulose filters were then incubated for 1 h at room temperature with the appropriate secondary antibodies coupled to peroxidase (diluted 1:10,000 in TBST containing 10% dry milk). After several further washes in TBST, the nitrocellulose filters were incubated for 2 h at room temperature with BSA antibodies (diluted 1:500 in TBST). The nitrocellulose filters were then washed several times in TBST and incubated for 1 h at room temperature with appropriate secondary antibodies coupled to peroxidase (diluted 1:10,000 in TBST containing 10% dry milk). Bound antibodies were visualized using the enhanced chemical luminescence detection system (ECL).
For RNA analysis, nuclei and cytoplasms were manually isolated as described (Dabauvalle and Franke 1982). RNA from each fraction was extracted separately by using peqGOLD TriFastTM according to the manufacturer's protocol (Peqlab). Labeled RNA molecules were separated by electrophoresis on denaturating polyacrylamide gels (6% acrylamide/bis-acrylamide 29:1; 8 M urea) and visualized by autoradiography.
Overlay Blot Analyses and Pull-Down Experiments
Proteins obtained from 100 manually isolated nuclear envelopes were separated by SDS-PAGE (Thomas and Kornberg 1975), using 12% acrylamide, and processed for overlay blot assays as described previously (Lounsbury et al. 1994), using GST–eIF-5A. Bound proteins were visualized by incubation with anti-GST antibodies (diluted 1:1,000). Then, the nitrocellulose filters were stripped of bound antibodies and reprobed as described in the manufacturer's protocol (Amersham Pharmacia Biotech) with mAb 414 (diluted 1:1,000), antiactin antibodies (diluted 1: 1,000), or anti-CRM1 antiserum (diluted 1:250).
Pull-down experiments were performed as described previously (Yang et al. 1998), by using GST, wt GST–eIF-5A, GST–eIF-5A–M14, or GST–Rev–NES fusion proteins (1 µg/µl) and 150 manually isolated oocyte nuclei or total oocyte extract (corresponding to 150 oocytes). In another set of experiments, GST or GST–Rev–NES fusion protein (1 µg/µl) coupled to glutathione–Sepharose beads was incubated with a 10-fold molar excess of wt eIF-5A or eIF-5A–M14 protein before incubation with oocyte extract or with His-tagged CRM1 fusion protein (0.5 µg/µl). For the pull-down experiments with His-tagged CRM1, the glutathione–Sepharose beads were saturated by using 5% BSA, and the binding was performed with or without addition of the GTPase-deficient RanQ69L mutant. The bound and unbound proteins were separated by 12% SDS-PAGE, followed by silver staining (Switzer et al. 1979) or by immunoblotting using anti-CRM1 antiserum (diluted 1:250), anti-His antibodies (diluted 1:1,000), anti–eIF-5A antibodies, mAB 414, or antiactin antibodies, diluted as described above.
Mass Spectrometry Analysis and Peptide Sequencing
Proteins from manually isolated oocyte nuclear envelopes were separated by electrophoresis through a 12% SDS-PAGE gel and stained with Coomassie brilliant blue, and the 45-kD band was excised. Tryptic digestion of the protein sample within the polyacrylamide matrix was performed as previously described (Eckerskorn and Lottspeich 1989). After extensive washing in water, the gel pieces were lyophylized and resoaked with buffer containing endoproteinase LysC (Boehringer). The resulting peptide fragments were eluted with formic acid/acetonitrile/water (5:50:45). Peptide mass fingerprinting on a Reflex III MALDI mass spectrometer (Bruker) identified the sample protein as actin. This was further confirmed by peptide sequencing after separation of the peptide mixture by reversed phase HPLC on a Microcart 150-1 Purospher C18 5-µm column (Merck). The eluents used in the HPLC were solvent A (0.1% trifluoroacetic acid in water) and solvent B (0.1% trifluoroacetic acid in acetonitrile). The gradient was developed in 0–60% solvent B for 60 min at a flow rate of 60 µl/min. Eluted peptides were detected at 206 nm, collected manually, and sequenced in an amino acid sequencer (PE Biosystems) according to the manufacturer's instructions. The sequence obtained ([D]SYVGDEAQSK) fitted the amino acid sequence of X. laevis actin isoforms, perfectly.
Immunogold Electron Microscopy
Manually isolated and unfixed nuclear envelopes were incubated with either the antiactin antibody 2G2 (diluted 1:200) alone or in combination with anti–eIF-5A antibodies (diluted 1:250), followed by incubation for 2 h with secondary antibodies coupled to 6- or 12-nm gold particles (diluted 1:10). Procedures used for preembedding immunoelectron microscopy of isolated nuclear envelopes from Xenopus and Pleurodeles oocytes have been described previously.
Nuclear Export In Vitro
Nuclei from HeLa cells constitutively expressing HIV-1 Rev protein (Wolff et al. 1995) were isolated as described (Krohne et al. 1981). Extract was prepared from activated Xenopus eggs using standard protocols, which include the use of cytochalasin B (Dabauvalle et al. 1991; Newmeyer and Wilson 1991) or, alternatively, without this actin-binding drug. The nuclei were incubated in egg extract for 1 h with or without the addition of an ATP regenerating system (2 mM ATP, 20 mM phosphocreatine, 50 µg/ml creatine kinase). After incubation, the probes were centrifuged onto coverslips and prepared for immunofluorescence microscopy as described (Dabauvalle et al. 1991).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The Rev–NES has been shown previously to interact with HeLa cell–derived and recombinant eIF-5A (Ruhl et al. 1993; Bevec et al. 1996). To verify the interaction of the Rev–NES with oocyte-derived eIF-5A, we first performed binding studies in solution. For this, purified GST–Rev–NES fusion protein was immobilized on glutathione–Sepharose beads and incubated with total protein extracts from Xenopus oocytes. The beads were then pelleted by centrifugation, and the bound and unbound material was analyzed by Western analysis using antibodies directed against eIF-5A (Elfgang et al. 1999). As shown, eIF-5A bound the GST–Rev–NES fusion protein (Fig. 1 A, compare lanes 1 and 1') but not GST alone (Fig. 1 A, compare lanes 2 and 2').
|
Next, we used the same experimental design to investigate Rev-mediated nuclear export of HIV-1–derived RNA (Fig. 1 B). For this, the RRE RNA target sequence was radiolabeled by in vitro transcription and microinjected together with recombinant Rev protein into oocyte nuclei. Coinjection of export-deficient radiolabeled U6 RNA served as the control for accurate nuclear injection in these experiments (Hamm and Mattaj 1989). 2 h after microinjection of the RNA export substrate, the oocyte nuclear components and cytoplasmic components were separated, and localization of the various RNAs was analyzed by gel electrophoresis and autoradiography. In agreement with the data discussed above, efficient nuclear export of RRE RNA occurred in the presence of nonspecific IgG (Fig. 1 D, lanes 1 and 2), whereas its export was inhibited by anti–eIF-5A antibodies (Fig. 1 D, lanes 3 and 4). Notably, translocation of RRE RNA across the nuclear envelope was only observed in the presence of Rev trans-activator protein (data not shown).
All retroviruses have to export unspliced intron-containing RNA from the nucleus to the cytoplasm. However, simple retroviruses such as the type D retrovirus MPMV do not encode Rev-like activities. In the RNA genome of these viruses is a cis-active structure, termed the CTE, which mediates efficient nuclear export of their unspliced RNAs (Bray et al. 1994; Ernst et al. 1997; Pasquinelli et al. 1997; Saavedra et al. 1997). CTE-mediated export depends exclusively on endogenous cellular factors. Indeed, the TAP protein, which is a human protein involved in mRNA transport, was recently found to bind to the CTE and stimulate nuclear export of CTE-containing viral RNAs (Grüter et al. 1998; Braun et al. 1999; Bachi et al. 2000). Importantly, the CTE-TAP–dependent nuclear RNA export pathway appears to be distinct from the specific RNA export pathway that is accessed by the HIV-1 Rev protein (Pasquinelli et al. 1997; Bogerd et al. 1998; Kang and Cullen 1999; Zolotukhin and Felber 1999). Thus, inhibition of Rev export should not negatively affect the translocation of CTE-containing RNA across the oocyte nuclear envelope.
To test this, we investigated the effect of anti–eIF-5A antibodies on nuclear export of the prototypic MPMV CTE (Ernst et al. 1997). As shown in Fig. 1 E, nuclear export of wt CTE RNA was easily detectable in the presence of nonspecific control IgG, whereas, in contrast, a coinjected export-deficient CTE deletion mutant (M2/M11; Pasquinelli et al. 1997) remained in the nuclear compartment of the injected oocytes (lanes 1 and 2). Coinjection of U6 RNA served as the injection control in these experiments. In the presence of anti–eIF-5A antibodies, nuclear export of CTE RNA continued (Fig. 1 E, lanes 3 and 4). Note that, under the same conditions, Rev-mediated RRE RNA export was completely blocked (Fig. 1 D, lanes 3 and 4). These results demonstrate that eIF-5A does not participate in the export of CTE RNA.
Previously, we have reported that, in somatic cells, eIF-5A is required for the nuclear export of the Rev–NES and Rex–NES, but not for the export of the PKI–NES (Elfgang et al. 1999). Therefore, we tested anti–eIF-5A antibodies for their ability to interfere with PKI-NES–mediated nuclear export in oocytes. To our initial surprise, the microinjected GST–PKI–NES fusion protein remained in the microinjected nuclei in absence of anti–eIF-5A antibodies, even after extended periods of incubation (data not shown). However, when we microinjected oocytes derived from hormone-induced animals, the GST–PKI–NES export cargo was rapidly exported from the nucleus to the cytoplasm (Fig. 1 F, lanes 1 and 2). These data already indicated that, in oocytes, significant differences exist with respect to the regulation of PKI-NES– versus Rev-NES–mediated nuclear export. Importantly, however, comicroinjection of anti–eIF-5A antibodies did not abrogate nuclear export of the GST–PKI–NES export substrate (Fig. 1 F, lanes 3 and 4), which is in full agreement with the data raised in somatic cells (Elfgang et al. 1999).
Constitutive expression of an eIF-5A mutant gene (eIF-5A–M14) in human T cells has been previously shown to efficiently inhibit HIV-1 replication in these cells (Bevec et al. 1996; Junker et al. 1996). Subsequently, microinjection studies in somatic cells revealed that eIF-5A–M14 interferes with the nuclear export activity of Rev (Bevec et al. 1996; Elfgang et al. 1999), explaining its pronounced inhibitory phenotype on virus replication. Therefore, we next included wt eIF-5A and eIF-5A–M14 mutant protein in our oocyte microinjection studies, as outlined in Fig. 2 A. The GST–Rev–NES export substrate was incubated either with PBS, wt eIF-5A, or eIF-5A–M14 protein for 1 h (using a 10-fold M excess over the GST–Rev–NES fusion protein), in order to allow binding of the Rev–NES to the recombinant eIF-5A proteins. Subsequent nuclear microinjection demonstrated that the eIF-5A–M14 mutant protein also inhibited GST–Rev–NES export in oocytes (Fig. 2 B, lanes 5 and 6). This inhibitory phenotype was not observed in the presence of PBS (Fig. 2 B, lanes 1 and 2, negative control) or wt eIF-5A protein (Fig. 2 B, lanes 3 and 4). Moreover, the presence of eIF-5A–M14 also blocked Rev-mediated RRE RNA export (Fig. 2 C, lanes 5 and 6). As expected, nuclear export of CTE RNA was not inhibited by eIF-5A–M14 (Fig. 2 D, lanes 5 and 6).
|
66-kD cross-reacting protein appears to be a specific CRM1–exportin 1 degradation product (Rosorius et al. 1999b). To further clarify the mode of action of the inhibitory phenotype of eIF-5A–M14 on Rev export (Bevec et al. 1996; Elfgang et al. 1999), we investigated whether eIF-5A acts as a Rev–CRM1/exportin1 bridging factor (Fig. 3 D). GST–Rev–NES fusion protein was immobilized on glutathione–Sepharose beads and then incubated with a 10-fold M excess of either wt eIF-5A or eIF-5A–M14 to allow saturation of the Rev–NES. Next, oocyte extract was added, and the relative amount of bound and unbound CRM1/exportin1 was determined as before. As shown in Fig. 3 D, CRM1/exportin1 only bound efficiently to the Rev–NES in the presence of wt eIF-5A (lane 1) and almost completely failed to interact with the NES in the presence of eIF-5A–M14 (lane 2). Next, we substituted the oocyte extract by recombinant CRM1/exportin1 and RanGTP (Fig. 3 E). As shown, His-tagged CRM1/exportin1 only bound to GST–Rev–NES fusion protein in the presence of wt eIF-5A and the GTP-bound form of Ran (GST-RanQ69L; compare lanes 1, 3, and 5). Again, no interaction of CRM1/exportin1 with the Rev–NES was visible in presence of eIF-5A–M14 (lane 2). Finally, formation of CRM1/exportin1-containing nuclear export complexes did not occur in absence of the Rev–NES (lane 4). Note that the observed CRM1 degradation products of 66 and 49 kD were also detectable in these experiments using CRM1-specific antibodies (not shown).
|
Interaction of the Rev Cofactor eIF-5A with Nuclear Envelope Proteins
It has already been reported that eIF-5A accumulates at the nucleoplasmic periphery of the NPC, particularly at the nucleoplasmic filaments that emanate from the NPC surface into the nucleoplasm (Rosorius et al. 1999b). These filaments might serve as docking sites at which export cargoes initially interact with the NPC before their exit through the pore channel (e.g., Franke and Scheer 1974; Cordes et al. 1993). After we had established that eIF-5A is required for Rev export in Xenopus oocytes, we used GST–eIF-5A fusion protein as a bait in order to identify components of the NPC that participate in this export pathway. Therefore, we manually isolated nuclear envelopes from oocytes. The nuclear envelope proteins were separated by SDS-PAGE, transferred onto nitrocellulose filters, renatured, and incubated with GST–eIF-5A fusion protein or GST alone as control. Bound proteins were detected with anti-GST antibodies. As shown in Fig. 4 A, GST–eIF-5A interacted with several distinct oocyte nuclear envelope proteins (lane 2, denoted by dots), whereas GST alone did not bind (lane 1). To identify these potential eIF-5A interaction partners, the nitrocellulose filters were stripped of the bound proteins and reprobed with specific antibodies. In agreement with the data presented above (Fig. 3 C), the signal detected at 110 kD was identified as the export receptor CRM1/exportin1 (lane 3), which in fact has been reported to accumulate at both the nucleoplasmic and cytoplasmic face of the NPC, particularly associating with the nucleoporin CAN/nup214 (Fornerod et al. 1996, Fornerod et al. 1997b). The use of the mAb 414, which recognizes several nucleoporins containing multiple copies of FG repeats revealed that the nucleoporins CAN/nup214, nup153, nup98, and nup62 are potential eIF-5A binding partners (Fig. 4 A, lane 4). These interactions were reproduced in blot assays, using two-dimensional gels (data not shown), allowing us to unequivocally confirm that the abundant band observed at 110 kD in the overlay blot (Fig. 4 A, lane 2) is in fact CRM1/exportin1 and not exportin4, which has recently been described as a novel importin-β family member with a similar molecular weight to CRM1/exportin1 (Lipowsky et al. 2000). Finally, these interactions were also confirmed in solution by pull-down experiments using wt GST–eIF-5A protein and extracts of manually isolated nuclei of Xenopus oocytes (Fig. 4 B, lanes 1–3). The corresponding silver-stained gel showing the pattern of bound (Fig. 4 B, lane 4) and unbound (Fig. 4 B, lane 5) proteins to GST–eIF-5A demonstrated the specificity of this binding assay. Only a very few specific binding proteins were visible (Fig. 4 B, compare lanes 4 and 5). Furthermore, an additional control experiment was performed using GST alone. The pattern of GST-bound (Fig. 4 B, lane 6) and unbound (Fig. 4 B, lane 7) proteins are shown. As expected, no GST-bound nuclear envelope–associated proteins were detected. Of note, the predominant signal that was observed in this experiment is BSA, an ingredient of the used blocking buffer (Fig. 4 B, compare lanes 6 and 7).
|
30 to 45 kD). These eIF-5A interacting proteins were analyzed by mass spectrometry and peptide sequencing. The data obtained identified the 45-kD protein as actin. This result was confirmed by reprobing the nitrocellulose filters with antiactin antibody 2G2 (Fig. 4 A, lane 5) as well as by pull-down experiments (Fig. 4 B, lane 3). Additional solution binding studies using purified actin monomers and nuclear extracts from Xenopus oocyte nuclei confirmed that actin indeed binds to eIF-5A. Under these conditions, actin also interacted with the nucleoporin nup62 (data not shown). Then, we characterized the nuclear envelope association of actin at the ultrastuctural level by preembedding immunogold electron microscopy. To this end, nuclear envelopes were manually isolated from Xenopus or Pleurodeles oocytes and incubated with antiactin antibody 2G2 (Gonsior et al. 1999), followed by gold-coupled secondary antibodies. Interestingly, actin was found specifically at the nucleoplasmic face of the NPC and along the nucleoplasmic NPC-attached fibrils that are particularly prominent in Pleurodeles oocytes (Fig. 5A and Fig. B). It should be stressed that the NPC-associated fibers are not actin filaments (F-actin). When isolated nuclear envelopes from Xenopus and Pleurodeles oocytes were incubated in the presence of Ca2+ with gelsolin, an actin severing protein, the NPC-associated filaments remained intact (data not shown). Thus, although actin is clearly associated with these filaments, their integrity is not based on polymerized actin but must involve other proteins.
|
|
|
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
It has previously been reported that eIF-5A is required in somatic cells specifically for the nucleocytoplasmic translocation of the Rev/Rex class of retroviral RNA nuclear export factors (Ruhl et al. 1993; Katahira et al. 1995; Bevec et al. 1996). Moreover, inhibition studies in somatic cells suggested that eIF-5A acts before or simultaneously with CRM1/exportin1 in the nuclear export of Rev and Rex (Elfgang et al. 1999). In agreement with the notion that eIF-5A is a protein that is part of this specific nuclear export pathway, we were able to demonstrate that eIF-5A accumulates in particular at the NPC-associated nucleoplasmic fibrils in Xenopus oocytes and somatic cells, interacts with the general export receptor CRM1/exportin1, and is by itself exported from the nucleus to the cytoplasm in a leptomycin B–sensitive manner (Rosorius et al. 1999b). In particular, the latter finding that eIF-5A is actively exported from the nucleus has been confirmed by a recent study (Lipowsky et al. 2000). In this work, it was demonstrated that eIF-5A also interacts with a novel export receptor, named exportin4. Importantly, binding of eIF-5A to exportin4 was shown to depend on the hypusine modification in eIF-5A, with the implication that hypusine is required for nuclear export of eIF-5A. On the other hand, however, unmodified eIF-5A has been shown to be efficiently exported from the nucleus to the cytoplasm in microinjected somatic cells (Rosorius et al. 1999b) and Xenopus oocytes (this study), as well as in permeabilized cells (Lipowsky et al. 2000). Moreover, human eIF-5A is fully functional in yeast strains that are disrupted for HYP2 encoding the yeast homologue of eIF-5A (Schwelberger et al. 1993; Magdolen et al. 1994), despite the fact that exportin4 is apparently not present in this organism (Lipowsky et al. 2000). In summary, these data suggest that the unique hypusine modification is not essential for nuclear export of eIF-5A and that it is possible that eIF-5A interacts with multiple export receptors (e.g., CRM1/exportin1 and exportin4). It remains to be investigated in future studies whether or not the interaction of eIF-5A with different export receptors reflects a distinct regulation of a specific nuclear export pathways in response to specific physiological conditions.
The results of this study clearly demonstrate that nuclear export of Rev and RRE RNA in Xenopus oocytes is also dependent on eIF-5A. In contrast, however, eIF-5A is not essential for CTE-mediated RNA export, which is in perfect agreement with previously published data that reported that the CTE RNA export is distinctly regulated when compared with Rev/Rex–mediated RNA export (Pasquinelli et al. 1997; Bogerd et al. 1998; Kang and Cullen 1999; Zolotukhin and Felber 1999).
The finding that the eIF-5A–M14 mutant protein is unable to bind to the general export receptor CRM1/exportin1, and that this correlates with an export-deficient phenotype of this protein, suggested a mechanism in which eIF-5A may act as an adapter that targets the Rev–NES to the nucleoplasmic face of the NPC and mediates efficient binding to CRM1/exportin1. CRM1/exportin1 and associated components (e.g., Ran) then translocate the Rev-NES/eIF-5A–containing ribonucleoprotein particle through the pore channel. Indeed, our subsequent in vitro binding studies demonstrated that efficient Rev-NES–CRM1 complex formation was only mediated by wt eIF-5A but not by the eIF-5A–M14 mutant protein.
The eIF-5A protein was originally detected as a potential Rev cofactor in HeLa nuclei using reversible cross-linker molecules in combination with structurally flexible Rev–NES peptide mimics (Ruhl et al. 1993). The binding experiments in this study were performed with GST–Rev–NES fusion proteins in which a structurally flexible glycine linker element separates the GST from the NES moiety (Elfgang et al. 1999). It should be noted that when full-length Rev protein was used in in vitro binding studies, an interaction with eIF-5A was only observed when Rev was bound to the RRE RNA at concentrations that allowed the formation of a dimeric Rev–specific complex (Bevec et al. 1996). In contrast, no in vitro interaction of Rev with eIF-5A was detected in a study in which Rev–RRE ratios were used that favored the formation of monomeric or multimeric Rev–specific complexes on the RRE RNA (Henderson and Percipalle 1997). More recent in vitro binding studies using full-length recombinant Rev protein and cytoplasmic HeLa cell extracts (as a source of eIF-5A) did also not detect binding of eIF-5A to Rev (Lipowsky et al. 2000). However, this experimental design is not suitable for the investigation of the interaction of full-length Rev protein with any nuclear export factor, since it neglects the well established fact that Rev homomultimer formation on the RRE RNA is essential for Rev function (Pollard and Malim 1998). As shown previously (Elfgang et al. 1999) and in this study, the requirement of RRE RNA binding and subsequent multimerization can apparently be overcome by using structurally flexible Rev–NES mimics. The combined data may therefore suggest that dimerization of Rev protein on its RRE RNA target is required for its interaction with nuclear export factors. In particular, a study in which Rev in vitro multimerization was investigated in combination with functional cell-based assays indeed concluded that two monomeric Rev proteins bound to the RRE RNA is sufficient for biological activity (Daly et al. 1993b). However, future studies are required to elucidate the exact protein composition of Rev-containing export complexes.
The concept that eIF-5A facilitates the interaction of the Rev–NES with CRM1/exportin1 is in full agreement with recent studies in which the binding affinities of Rev–NES to CRM1/exportin1 were investigated using recombinant proteins. When RRE RNA-bound Rev protein was incubated together with CRM1/exportin1 and RanGTP in RNA gel retardation assays, only an extremely poor level of complex formation was detected (Askjaer et al. 1998). It appears that, in these types of experiments, <1% of the preformed RRE–Rev ribonucleoprotein complexes interacted with recombinant CRM1/exportin1 and Ran- GTP. Moreover, in another study, efficient interaction of CRM1/exportin1 with RanGTP in the presence of Rev–NES peptides was only observed when CRM1/exportin1 derived from reticulocyte lysates was used (Fornerod et al. 1997a). Reticulocyte extracts contain high levels of eIF-5A (Schatz et al. 1998). Interestingly, when CRM1/exportin1 was produced by translation in an E. coli–derived lysate (which does not contain eIF-5A), binding was only observed using the PKI-derived NES (Fornerod et al. 1997a), again suggesting that factors in addition to RanGTP are required for efficient Rev-NES–CRM1 interaction. Finally, quantification of the Rev-NES–CRM1 affinity using an assay that measures the hydrolysis of Ran-bound GTP upon complex formation (RanGAP assay) clearly demonstrated that the Rev–NES displays an extremely low affinity for CRM1/exportin1, at a level that is almost indistinguishable from that of an export-deficient mutant NES (Askjaer et al. 1999). In sharp contrast, however, complex formation with CRM1/exportin1 was clearly observed when the PKI–NES was used in this assay (Askjaer et al. 1999). In this context it is also important to reemphasize that inhibition of eIF-5A has previously been shown to block Rev-NES–mediated but not PKI-NES–mediated nuclear export in somatic cells (Elfgang et al. 1999), a fact that was reproduced in Xenopus oocytes (this study). Clearly our data showing that eIF-5A mediates Rev-NES–CRM1 interaction provides an explanation for these findings. Moreover, our data reveal the mode of action of eIF-5A mutant proteins (e.g., M14) that have been shown to efficiently block HIV-1 replication in human T cells when constitutively expressed in trans (Bevec et al. 1996; Junker et al. 1996).
To analyze interactions of eIF-5A with components of the NPC, we performed in vitro binding studies using overlay blot assays and precipitation analyses. We were able to identify specific interactions between eIF-5A and the Xenopus oocyte nucleoporins nup153, nup98, nup62, and CAN/nup 214 (for review see Stoffler et al. 1999). Although the functional contribution of these interactions to eIF-5A export remains to be established, it is certainly intriguing that this assay only detected nucleoporins that have been implicated in nuclear export pathways (Bastos et al. 1995; Fornerod et al. 1997b; Powers et al. 1997; Askjaer et al. 1999; Kehlenbach et al. 1999; Nakielny et al. 1999; Pritchard et al. 1999; Ullman et al. 1999; Bachi et al. 2000), whereas other FG repeat–containing nucleoporins like RanBP2 are not detected. In this context it is also important to note that a series of recent studies indeed demonstrated the direct participation of CAN/nup214, nup153, and nup98 in the nuclear export of Rev and Rev-mediated viral mRNA export (Bogerd et al. 1998; Ullman et al. 1999; Zolotukhin and Felber 1999). Thus, the identification of NPC-associated proteins that interact with the Rev cofactor eIF-5A appears to be a promising experimental approach to detect novel factors that participate in nuclear export of Rev. In fact, in overlay assays and in solution binding studies using isolated nuclei from Xenopus oocytes, eIF-5A interacted not only with a specific set of nucleoporins but also with actin. By immunogold electron microscopy, we could exclude that the nuclear envelope–associated actin represented cytoplasmic contamination since it localized specifically to the nucleoplasmic face of the NPCs and the attached pore filaments. Amphibian oocyte nuclei contain high concentrations of nonmuscle β- and 
-actin, mostly in a soluble, i.e., unpolymerized or short polymer, form (Scheer et al. 1984; Ankenbauer et al. 1989; Gonsior et al. 1999). Our finding that eIF-5A colocalizes together with nuclear actin (this study) and nup153 (Rosorius et al. 1999b) at the filamentous arrays extending from the nucleoplasmic side of NPCs is in perfect agreement with the in vitro binding studies and, furthermore, suggests that the nucleoplasmic NPC filaments serve as docking sites for RRE RNA containing viral export complexes before nuclear exit.
Even with the inherent problems of cytoplasmic contamination, it is now generally accepted that actin is present not only in oocyte nuclei but also in the interphase nuclei of somatic cells (Scheer et al. 1984; Sahlas et al. 1993; Gonsior et al. 1999; Rando et al. 2000). Given its abundance and widespread occurrence, it is quite likely that nuclear actin is involved in specific nuclear processes that are, however, not necessarily dependent on filament formation. Among other functions (Gonsior et al. 1999; Rando et al. 2000), nuclear actin has been implicated in transcription of cellular and viral genes (Egly et al. 1984; Scheer et al. 1984; Burke et al. 1998) and chromatin remodelling events (Zhao et al. 1998). Based on nuclear injection experiments of antiactin antibodies and actin-binding drugs, this study now shows that nuclear actin also may be involved in the later stages of gene expression, namely the NPC-mediated transport of RNA out of the nucleus. This view is further corroborated by a recent study in which the authors also provide evidence for a functional involvement of nuclear actin in the nuclear export of Rev-dependent HIV-1 pre-mRNA (Kimura et al. 2000). However, although these authors emphasize the importance of a filamentous form of nuclear actin, our data suggest that it is the unpolymerized or short polymer form of nuclear actin that functions in nuclear export processes (see below).
At the moment, we do not know whether or not actin is a general cofactor that also participates in the nuclear export pathways of cellular RNA species. Nonetheless, our data demonstrate that actin plays a functional role in at least two distinct export pathways accessed by viral RNAs, the Rev-dependent RRE RNA, and the TAP-dependent CTE RNA export. Since TAP also contributes to the export of cellular mRNAs (Grüter et al. 1998; Braun et al. 1999; Görlich and Kutay 1999; Nakielny and Dreyfuss 1999), it is conceivable that actin could likewise function in this pathway. Furthermore, we show that nuclear actin participates in the nuclear export of the eIF-5A–independent, CRM1-dependent NES of PKI. Finally, it should be stressed that the inhibition of nuclear export by antibodies to actin was not restricted to oocytes but appears to be a general phenomenon as it was also observed in cultured mammalian cells.
Our findings that (a) actin is located at the inner pore fibrils, (b) actin interacts in vitro with the export factor eIF-5A and with the nucleoporin nup62, and (c) Rev and TAP-mediated nuclear export of viral RNAs as well as nuclear export of PKI can be inhibited by introducing either antiactin antibodies or the actin-binding drug latrunculin B into the cell nucleus, indicate a novel and unexpected role for nuclear actin. Since we observed the export-inhibitory effect with both agents, we conclude that it is caused by direct targeting and functional inactivation of nuclear actin rather than by more indirect effects such as precipitation of nuclear actin. Nuclear import was not inhibited under the same experimental conditions (not shown), indicating that the injected antibodies did not cause a nonspecific steric inhibition of transport complexes at the pore. Several lines of evidence suggest that it is the unpolymerized or short polymer form of nuclear actin that is involved in nuclear export rather than F-actin. First, most of the nuclear actin of Xenopus oocytes exists in a soluble, although polymerization-competent form (Scheer et al. 1984; Ankenbauer et al. 1989). Second, fluorescent phalloidin, which is selective for F-actin, does not stain F-actin in the nuclei of Xenopus oocytes and cultured vertebrate cells under normal conditions, i.e., without experimental manipulations (Sahlas et al. 1993; Gonsior et al. 1999). Third, the antiactin antibody we have used for the microinjection experiments (2G2) does not recognize native F-actin but is selective for a specific conformational state of actin that is predominantly found in nuclei (Gonsior et al. 1999). It is tempting to speculate that actin dimers, which have been reported to assume a conformation not present in polymerized F-actin (lower dimer or LD [Millonig et al. 1988]), are the major targets of this antibody. Fourth, Rev and PKI export are not affected by injection of swinholide A into nuclei. This actin-binding drug severs F-actin and stabilizes actin dimers (Bubb et al. 1995), demonstrating that neither the absence of F-actin nor a decrease in the G-actin level affects the nuclear export pathway. Finally, Rev and PKI export are inhibited by latrunculin B, which sequesters G-actin monomers and prevents its polymerization (Coue et al. 1987). The opposing effects of latrunculin B and swinholide A support the concept that actin dimers function in nuclear export.
The critical role of actin in the Rev export pathway was also demonstrated in a cell-free system using Xenopus egg extract. When we omitted cytochalasin B, which is normally added to the extract as a gelation inhibitor (Newmeyer and Wilson 1991), Rev was efficiently exported in an energy-dependent manner from incubated isolated nuclei. Since the interactions of cytochalasin with actin are complex and involve capping of the barbed ends of F-actin and hydrolysis of the monomeric ATP–actin pool (Sampath and Pollard 1991), the interpretation of the inhibitory mechanism of cytochalasin B in this experimental system is difficult. More importantly, the cell-free nuclear export assay described here should provide the means to further explore the export pathway of Rev–RRE and possibly also of cellular RNAs since the extract can be easily manipulated and nuclear export monitored by simple immunofluorescence microscopy. Furthermore, this model system should help to clarify whether or not the ability of soluble actin to shuttle between the nucleus and the cytoplasm (Wada et al. 1998) is linked to nuclear export processes. However, we consider this quite unlikely in view of the observation that the nuclear export of G-actin is inhibited by the drug leptomycin B (Wada et al. 1998), whereas the TAP–CTE export pathway is not affected (Otero et al. 1998). We have shown that nuclear actin participates in the export of CTE RNA. Obviously, the shuttling potential of actin is of no relevance in this context, since the CTE RNA export resists inhibition by leptomycin B.
In summary, our data demonstrate that actin accumulates at NPC-attached nucleoplasmic filaments and can contribute to the nuclear export of multiple export substrates. We propose that unpolymerized nuclear actin is critically involved in the assembly of multiprotein export complexes. Investigation of the exact functional role of actin as part of the NPC-associated nuclear export machinery is a major research goal for the future.
|
Acknowledgments |
|---|
This work received financial support from the Deutsche Forschungsgemeinschaft (SFB 466, SFB 176, and grant DA 243/1-1).
Submitted: 3 August 2000
Revised: 16 January 2001
Accepted: 16 January 2001
Abbreviations used in this paper: CTE, constitutive transport element; eIF-5A, eukaryotic initiation factor 5A; F-actin, actin filaments; FG, phenylalanine–glycine; GFP, green fluorescent protein; GST, glutathione S-transferase; HIV-1, human immunodeficiency virus type 1; MPMV, Mason-Pfizer monkey virus; NES, nuclear export signals; NPC, nuclear pore complex; PKI, protein kinase inhibitor; RRE, Rev response element; wt, wild-type.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Ankenbauer T., Kleinschmidt J.A., Walsh M.J., Weiner O.H. & Franke W.W.. Identification of a widespread nuclear actin binding protein, Nature., 342, 1989, 822–825.[Medline]
Askjaer P., Jensen T.H., Nilsson J., Englmeier L. & Kjems J.. The specificity of the CRM1-Rev nuclear export signal interaction is mediated by RanGTP, J. Biol. Chem., 273, 1998, 33414–33422.
Askjaer P., Bachi A., Wilm M., Bischoff F.R., Weeks D.L., Ogniewski V., Ohno M., Niehrs C., Kjems J., Mattaj I.W. & Fornerod M.. RanGTP-regulated interactions of CRM1 with nucleoporins and a shuttling DEAD-box helicase, Mol. Cell. Biol., 19, 1999, 6276–6285.
Bachi A., Braun I.C., Rodrigues J.P., Pante N., Ribbeck K., von Kobbe C., Kutay U., Wilm M., Görlich D., Carmo-Fonseca M. & Izaurralde E.. The C-terminal domain of TAP interacts with the nuclear pore complex and promotes export of specific CTE-bearing RNA substrates, RNA., 6, 2000, 136–158.[Abstract]
Bastos R., Pante N. & Burke B.. Nuclear pore complex proteins, Int. Rev. Cytol., 162B, 1995, 257–302.[Medline]
Bevec D., Jaksche H., Oft M., Wöhl T., Himmelspach M., Pacher A., Schebesta M., Koettnitz K., Dobrovnik M., Csonga R., Lottspeich F. & Hauber J.. Inhibition of HIV-1 replication in lymphocytes by mutants of the Rev cofactor eIF-5A, Science., 271, 1996, 1858–1860.[Abstract]
Bogerd H.P., Echarri A., Ross T.M. & Cullen B.R.. Inhibition of human immunodeficiency virus Rev and human T-cell leukemia virus Rex function, but not Mason-Pfizer monkey virus constitutive transport element activity, by a mutant human nucleoporin targeted to CRM1, J. Virol., 72, 1998, 8627–8635.
Braun I.C., Rohrbach E., Schmitt C. & Izaurralde E.. TAP binds to the constitutive transport element (CTE) through a novel RNA-binding motif that is sufficient to promote CTE-dependent RNA export from the nucleus, EMBO (Eur. Mol. Biol. Organ.) J., 18, 1999, 1953–1965.[Medline]
Bray M., Prasad S., Dubay J.W., Hunter E., Jeang K.-T., Rekosh D. & Hammarskjöld M.-L.. A small element from the Mason-Pfizer monkey virus genome makes human immunodeficiency virus type 1 expression and replication Rev-independent, Proc. Natl. Acad. Sci. USA., 91, 1994, 1256–1260.
Bubb M.R., Spector I., Bershadsky A.D. & Korn E.D.. Swinholide A is a microfilament disrupting marine toxin that stabilizes actin dimers and severs actin filaments, J. Biol. Chem., 270, 1995, 3463–3466.
Burke E., Dupuy L., Wall C. & Barik S.. Role of cellular actin in the gene expression and morphogenesis of human respiratory syncytial virus, Virology., 252, 1998, 137–148.[Medline]
Corbett A.H. & Silver P.A.. Nucleocytoplasmic transport of macromolecules, Microbiol. Mol. Biol. Rev., 61, 1997, 193–211.[Abstract]
Cordes V.C., Reidenbach S., Kohler A., Stuurman N., van Driel R. & Franke W.W.. Intranuclear filaments containing a nuclear pore complex protein, J. Cell Biol., 123, 1993, 1333–1344.
Coue M., Brenner S.L., Spector I. & Korn E.D.. Inhibition of actin polymerization by latrunculin A, FEBS Lett., 213, 1987, 316–318.[Medline]
Dabauvalle M.C. & Franke W.W.. Karyophilic proteinspolypeptides synthesized in vitro accumulate in the nucleus on microinjection into the cytoplasm of amphibian oocytes, Proc. Natl. Acad. Sci. USA., 79, 1982, 5302–5306.
Dabauvalle M.C., Loos K., Merkert H. & Scheer U.. Spontaneous assembly of pore complex-containing membranes ("annulate lamellae") in Xenopus egg extract in the absence of chromatin, J. Cell Biol., 112, 1991, 1073–1082.
Daly T.J., Doten R.C., Rennert P., Auer M., Jaksche H., Donner A., Fisk G. & Rusche J.R.. Biochemical characterization of binding of multiple HIV-1 Rev monomeric proteins to the Rev responsive element, Biochemistry., 32, 1993, 10497–10505a.[Medline]
Daly T.J., Rennert P., Lynch P., Barry J.K., Dundas M., Rusche J.R., Doten R.C., Auer M. & Farrington G.K.. Perturbation of the carboxy terminus of HIV-1 Rev affects multimerization on the Rev response element, Biochemistry, 32, 1993, 8945–8954b.[Medline]
Davis L.I. & Blobel G.. Identification and characterization of a nuclear pore complex protein, Cell., 45, 1986, 699–709.[Medline]
Eckerskorn C. & Lottspeich F.. Internal amino acid sequence analysis of proteins separated by gel electrophoresis after tryptic digestion in polyacrylamide matrix, Chromatographia., 28, 1989, 92–94.
Egly J.M., Miyamoto N.G., Moncollin V. & Chambon P.. Is actin a transcription initiation factor for RNA polymerase B?, EMBO (Eur. Mol. Biol. Organ.) J., 3, 1984, 2363–2371.[Medline]
Elfgang C., Rosorius O., Hofer L., Jaksche H., Hauber J. & Bevec D.. Evidence for specific nucleocytoplasmic transport pathways used by leucine-rich nuclear export signals, Proc. Natl. Acad. Sci. USA., 96, 1999, 6229–6234.
Engel K., Kotlyarov A. & Gaestel M.. Leptomycin B-sensitive nuclear export of MAPKAP kinase 2 is regulated by phosphorylation, EMBO (Eur. Mol. Biol. Organ.) J., 17, 1998, 3363–3371.[Medline]
Ernst R.K., Bray M., Rekosh D. & Hammarskjold M.L.. A structured retroviral RNA element that mediates nucleocytoplasmic export of intron-containing RNA, Mol. Cell. Biol., 17, 1997, 135–144.[Abstract]
Fischer U., Huber J., Boelens W.C., Mattaj I.W. & Lührmann R.. The HIV-1 Rev activation domain is a nuclear export signal that accesses an export pathway used by specific cellular RNAs, Cell., 82, 1995, 475–483.[Medline]
Fornerod M., Boer J., van Baal S., Morreau H. & Grosveld G.. Interaction of cellular proteins with the leukemia specific fusion proteins DEK-CAN and SET-CAN and their normal counterpart, the nucleoporin CAN, Oncogene, 13, 1996, 1801–1808.[Medline]
Fornerod M., Ohno M., Yoshida M. & Mattaj I.W.. CRM1 is an export receptor for leucine-rich nuclear export signals, Cell, 90, 1997, 1051–1060a.[Medline]
Fornerod M., van Deursen J., van Baal S., Reynolds A., Davis D., Murti K.G., Fransen J. & Grosveld G.. The human homologue of yeast CRM1 is in a dynamic subcomplex with CAN/Nup214 and a novel nuclear pore component Nup88, EMBO (Eur. Mol. Biol. Organ.) J., 16, 1997, 807–816b.[Medline]
Franke W.W. & Scheer U.. Structures and functions of the nuclear envelope, Busch H.. The Cell Nucleus, 1974, 219–347, Academic Press, New York.
Freedman D.A. & Levine A.J.. Nuclear export is required for degradation of endogenous p53 by MDM2 and human papillomavirus E6, Mol. Cell. Biol., 18, 1998, 7288–7293.
Fukuda M., Asano S., Nakamura T., Adachi M., Yoshida M., Yanagida M. & Nishida E.. CRM1 is responsible for intracellular transport mediated by the nuclear export signal, Nature, 390, 1997, 308–311.[Medline]
Gonsior S.M., Platz S., Buchmeier S., Scheer U., Jockusch B.M. & Hinssen H.. Conformational difference between nuclear and cytoplasmic actin as detected by a monoclonal antibody, J. Cell Sci., 112, 1999, 797–809.[Abstract]
Görlich D. & Kutay U.. Transport between the cell nucleus and the cytoplasm, Annu. Rev. Cell Dev. Biol., 15, 1999, 607–660.[Medline]
Grüter P., Tabernero C., von Kobbe C., Schmitt C., Saavedra C., Bachi A., Wilm M., Felber B.K. & Izaurralde E.. TAP, the human homolog of Mex67p, mediates CTE-dependent RNA export from the nucleus, Mol. Cell., 1, 1998, 649–659.[Medline]
Guan T., Kehlenbach R.H., Schirmer E.C., Kehlenbach A., Fan F., Clurman B.E., Arnheim N. & Gerace L.. Nup50, a nucleoplasmically oriented nucleoporin with a role in nuclear protein export, Mol. Cell. Biol., 20, 2000, 5619–5630.
Gurdon J.B.. Nuclear transplantation in Xenopus, Kay B.K. & Peng H.B., Methods in Cell Biology. Vol. 36, 1991, 608–634, Academic Press, San Diego, CA.
Hamm J. & Mattaj I.W.. An abundant U6 snRNP found in germ cells and embryos of Xenopus laevis, EMBO (Eur. Mol. Biol. Organ.) J., 8, 1989, 4179–4187.[Medline]
Hammerschmid M., Palmeri D., Ruhl M., Jaksche H., Weichselbraun I., Böhnlein E., Malim M.H. & Hauber J.. Scanning mutagenesis of the arginine-rich region of human immunodeficiency virus type 1 Rev trans activator, J. Virol., 68, 1994, 7329–7335.
Henderson B.R. & Percipalle P.. Interactions between HIV Rev and nuclear import and export factorsthe Rev nuclear localization signal mediates specific binding to human importin-beta, J. Mol. Biol., 274, 1997, 693–707.[Medline]
Izaurralde E. & Adam S.. Transport of macromolecules between the nucleus and the cytoplasm, RNA, 4, 1998, 351–364.[Abstract]
Jarmolowski A., Boelens W.C., Izaurralde E. & Mattaj I.W.. Nuclear export of different classes of RNA is mediated by specific factors, J. Cell Biol., 124, 1994, 627–635.
Jarnik M. & Aebi U.. Toward a more complete 3-D structure of the nuclear pore complex, J. Struct. Biol., 107, 1991, 291–308.[Medline]
Junker U., Bevec D., Barske C., Kalfoglou C., Escaich S., Dobrovnik M., Hauber J. & Böhnlein E.. Intracellular expression of cellular eIF-5A mutants inhibits HIV-1 replication in human T cellsa feasibility study, Hum. Gene Ther., 7, 1996, 1861–1869.[Medline]
Kang Y. & Cullen B.R.. The human Tap protein is a nuclear mRNA export factor that contains novel RNA-binding and nucleocytoplasmic transport sequences, Genes Dev., 13, 1999, 1126–1139.
Katahira J., Ishizaki T., Sakai H., Adachi A., Yamamoto K. & Shida H.. Effects of translation initiation factor eIF-5A on the functioning of human T-cell leukemia virus type I Rex and human immunodeficiency virus Rev inhibited trans dominantly by a Rex mutant deficient in RNA binding, J. Virol., 69, 1995, 3125–3133.[Abstract]
Kehlenbach R.H., Dickmanns A., Kehlenbach A., Guan T. & Gerace L.. A role for RanBP1 in the release of CRM1 from the nuclear pore complex in a terminal step of nuclear export, J. Cell Biol., 145, 1999, 645–657.
Kimura T., Hashimoto I., Yamamoto A., Nishikawa M. & Fujisawa J.. Rev-dependent association of the intron-containing HIV-1 gag mRNA with the nuclear actin bundles and the inhibition of its nucleocytoplasmic transport by latrunculin-B, Genes Cells., 5, 2000, 289–307.[Abstract]
Klebe C., Nishimoto T. & Wittinghofer F.. Functional expression in Escherichia coli of the mitotic regulator proteins p24ran and p45rcc1 and fluorescence measurements of their interaction, Biochemistry., 32, 1993, 11923–11928.[Medline]
Krohne G. & Franke W.W.. Proteins of pore complex-lamina structures from nuclei and nuclear membranes, Methods Enzymol., 96, 1983, 597–608.[Medline]
Krohne G., Dabauvalle M.C. & Franke W.W.. Cell type-specific differences in protein composition of nuclear pore complex-lamina structures in oocytes and erythrocytes of Xenopus laevis, J. Mol. Biol., 151, 1981, 121–141.[Medline]
Kudo N., Khochbin S., Nishi K., Kitano K., Yanagida M., Yoshida M. & Horinouchi S.. Molecular cloning and cell cycle-dependent expression of mammalian CRM1, a protein involved in nuclear export of proteins, J. Biol. Chem., 272, 1997, 29742–29751.
Kudo N., Wolff B., Sekimoto T., Schreiner E.P., Yoneda Y., Yanagida M., Horinouchi S. & Yoshida M.. Leptomycin B inhibition of signal-mediated nuclear export by direct binding to CRM1, Exp. Cell Res, 242, 1998, 540–547.[Medline]
Kudo N., Matsumori N., Taoka H., Fujiwara D., Schreiner E.P., Wolff B., Yoshida M. & Horinouchi S.. Leptomycin B inactivates CRM1/exportin1 by covalent modification at a cysteine residue in the central conserved region, Proc. Natl. Acad. Sci. USA., 96, 1999, 9112–9117.
Lipowsky G., Bischoff F.R., Schwarzmaier P., Kraft R., Kostka S., Hartmann E., Kutay U. & Görlich D.. Exportin 4a mediator of a novel nuclear export pathway in higher eukaryotes, EMBO (Eur. Mol. Biol. Organ.) J., 19, 2000, 4362–4371.[Medline]
Lounsbury K.M., Beddow A.L. & Macara I.G.. A family of proteins that stabilize the Ran/TC4 GTPase in its GTP-bound conformation, J. Biol. Chem., 269, 1994, 11285–11290.
Magdolen V., Klier H., Wöhl T., Klink F., Hirt H., Hauber J. & Lottspeich F.. The function of the hypusine-containing proteins in yeast and other eukaryotes is well conserved, Mol. Gen. Genet., 244, 1994, 646–652.[Medline]
Malim M.H., Tiley L.S., McCarn D.F., Rusche J.R., Hauber J. & Cullen B.R.. HIV-1 structural gene expression requires binding of the Rev trans-activator to its RNA target sequence, Cell, 60, 1990, 675–683.[Medline]
Mattaj I.W. & Englmeier L.. Nucleocytoplasmic transportthe soluble phase, Annu. Rev. Biochem., 67, 1998, 265–306.[Medline]
Melchior F. & Gerace L.. Two-way trafficking with Ran, Trends Cell Biol., 8, 1998, 175–179.[Medline]
Meyer B.E., Meinkoth J.L. & Malim M.H.. Nuclear transport of human immunodeficiency virus type 1, visna virus, and equine infectious anemia virus Rev proteinsidentification of a family of transferable nuclear export signals, J. Virol., 70, 1996, 2350–2359.[Abstract]
Millonig R., Salvo H. & Aebi U.. Probing actin polymerization by intermolecular cross-linking, J. Cell Biol., 106, 1988, 785–796.
Moore M.S.. Ran and nuclear transport, Genes Dev., 12, 1998, 3303–3319.
Nakielny S. & Dreyfuss G.. Transport of proteins and RNAs in and out of the nucleus, Cell., 99, 1999, 677–690.[Medline]
Nakielny S., Shaikh S., Burke B. & Dreyfuss G.. Nup153 is an M9-containing mobile nucleoporin with a novel Ran-binding domain, EMBO (Eur. Mol. Biol. Organ.) J., 18, 1999, 1982–1995.[Medline]
Newmeyer D.D. & Wilson K.L.. Egg extracts for nuclear import and nuclear assembly reactions, Kay B.K. & Peng H.B., Methods in Cell Biology. Vol. 36, 1991, 608–634, Academic Press, San Diego, CA.
Ossareh-Nazari B., Bachelerie F. & Dargemont C.. Evidence for a role of CRM1 in signal-mediated nuclear protein export, Science., 278, 1997, 141–144.
Otero G.C., Harris M.E., Donello J.E. & Hope T.J.. Leptomycin B inhibits equine infectious anemia virus Rev and feline immunodeficiency virus rev function but not the function of the hepatitis B virus posttranscriptional regulatory element, J. Virol., 72, 1998, 7593–7597.
Park M.H., Wolff E.C. & Folk J.E.. Hypusineits post-translational formation in eukaryotic initiation factor 5A and ist potential role in cellular regulation, Biofactors, 4, 1993, 95–104.[Medline]
Pasquinelli A.E., Ernst R.K., Lund E., Grimm C., Zapp M.L., Rekosh D., Hammarskjold M.L. & Dahlberg J.E.. The constitutive transport element (CTE) of Mason-Pfizer monkey virus (MPMV) accesses a cellular mRNA export pathway, EMBO (Eur. Mol. Biol. Organ.) J., 16, 1997, 7500–7510.[Medline]
Pollard V.W. & Malim M.H.. The HIV-1 Rev protein, Annu. Rev. Microbiol., 52, 1998, 491–532.[Medline]
Powers M.A., Forbes D.J., Dahlberg J.E. & Lund E.. The vertebrate GLFG nucleoporin, Nup98, is an essential component of multiple RNA export pathways, J. Cell Biol., 136, 1997, 241–250.
Pritchard C.E., Fornerod M., Kasper L.H. & van Deursen J.M.. RAE1 is a shuttling mRNA export factor that binds to a GLEBS-like NUP98 motif at the nuclear pore complex through multiple domains, J. Cell Biol., 145, 1999, 237–254.
Rando O.J., Zhao K. & Crabtree G.R.. Searching for a function for nuclear actin, Trends Cell Biol., 10, 2000, 92–97.[Medline]
Rosorius O., Heger P., Stelz G., Hirschmann N., Hauber J. & Stauber R.H.. Direct observation of nucleocytoplasmic transport by microinjection of GFP-tagged proteins in living cells, Biotechniques., 27, 1999, 350–355a.[Medline]
Rosorius O., Reichart B., Krätzer F., Heger P., Dabauvalle M.C. & Hauber J.. Nuclear pore localization and nucleocytoplasmic transport of eIF- 5Aevidence for direct interaction with the export receptor CRM1, J. Cell Sci., 112, 1999, 2369–2380b.[Abstract]
Ruhl M., Himmelspach M., Bahr G.M., Hammerschmid F., Jaksche H., Wolff B., Aschauer H., Farrington G.K., Probst H., Bevec D. & Hauber J.. Eukaryotic initiation factor 5A is a cellular target of the human immunodeficiency virus type 1 Rev activation domain mediating trans-activation, J. Cell Biol., 123, 1993, 1309–1320.
Saavedra C., Felber B. & Izaurralde E.. The simian retrovirus-1 constitutive transport element, unlike the HIV-1 RRE, uses factors required for cellular mRNA export, Curr. Biol., 7, 1997, 619–628.[Medline]
Sahlas D.J., Milankov K., Park P.C. & De Boni U.. Distribution of snRNPs, splicing factor SC-35 and actin in interphase nucleiimmunocytochemical evidence for differential distribution during changes in functional states, J. Cell Sci., 105, 1993, 347–357.[Abstract]
Sampath P. & Pollard T.D.. Effects of cytochalasin, phalloidin and pH on the elongation of actin filaments, Biochemistry., 30, 1991, 1973–1980.[Medline]
Schatz O., Oft M., Dascher C., Schebesta M., Rosorius O., Jaksche H., Dobrovnik M., Bevec D. & Hauber J.. Interaction of the HIV-1 Rev cofactor eukaryotic initiation factor 5A with ribosomal protein L5, Proc. Natl. Acad. Sci. USA.., 95, 1998, 1607–1612.
Scheer U., Hinssen H., Franke W.W. & Jockusch B.M.. Microinjection of actin-binding proteins and actin antibodies demonstrates involvement of nuclear actin in transcription of lampbrush chromosomes, Cell., 39, 1984, 111–122.[Medline]
Schwelberger H.G., Kang H.A. & Hershey J.W.B.. Translation initiation factor eIF-5A expressed from either of two yeast genes or from human cDNA, J. Biol. Chem., 268, 1993, 14018–14025.
Stade K., Ford C.S., Guthrie C. & Weis K.. Exportin 1 (Crm1p) is an essential nuclear export factor, Cell., 90, 1997, 1041–1050.[Medline]
Stauber R., Gaitanaris G.A. & Pavlakis G.N.. Analysis of trafficking of Rev and transdominant Rev proteins in living cells using green fluorescent protein fusionstransdominant Rev blocks the export of Rev from the nucleus to the cytoplasm, Virology., 213, 1995, 439–449.[Medline]
Stoffler D., Fahrenkrog B. & Aebi U.. The nuclear pore complexfrom molecular architecture to functional dynamics, Curr. Opin. Cell Biol., 11, 1999, 391–401.[Medline]
Stommel J.M., Marchenko N.D., Jimenez G.S., Moll U.M., Hope T.J. & Wahl G.M.. A leucine-rich nuclear export signal in the p53 tetramerization domainregulation of subcellular localization and p53 activity by NES masking, EMBO (Eur. Mol. Biol. Organ.) J., 18, 1999, 1660–1672.[Medline]
Stutz F. & Rosbash M.. Nuclear RNA export, Genes Dev., 12, 1998, 3303–3319.
Stutz F., Izzaurralde E., Mattaj I.W. & Rosbash M.. A role for nucleoporin FG repeat domains in export of human immunodeficiency virus type 1 Rev protein and RNA from the nucleus, Mol. Cell. Biol., 16, 1996, 7144–7150.[Abstract]
Switzer R.C., Merril C.R. & Shifrin S.. A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels, Anal. Biochem., 98, 1979, 231–237.[Medline]
Thomas J.O. & Kornberg R.D.. An octamer of histones in chromatin and free in solution, Proc. Natl. Acad. Sci. USA., 72, 1975, 2626–2630.
Toyoshima F., Moriguchi T., Wada A., Fukuda M. & Nishida E.. Nuclear export of cyclin B1 and its possible role in the DNA damage-induced G2 checkpoint, EMBO (Eur. Mol. Biol. Organ.) J., 17, 1998, 2728–2735.[Medline]
Ullman K.S., Shah S., Powers M.A. & Forbes D.J.. The nucleoporin nup153 plays a critical role in multiple types of nuclear export, Mol. Biol. Cell., 10, 1999, 649–664.
Wada A., Fukuda M., Mishima M. & Nishida E.. Nuclear export of actina novel mechanism regulating the subcellular localization of a major cytoskeletal protein, EMBO (Eur. Mol. Biol. Organ.) J., 17, 1998, 1635–1641.[Medline]
Wen W., Meinkoth J.L., Tsien R.Y. & Taylor S.S.. Identification of a signal for rapid export of proteins from the nucleus, Cell., 82, 1995, 463–473.[Medline]
Wolff B., Cohen G., Hauber J., Meshcheryakova D. & Rabeck C.. Nucleocytoplasmic transport of the Rev protein of human immunodeficiency virus type 1 is dependent on the activation domain of the protein, Exp. Cell Res., 217, 1995, 31–41.[Medline]
Wolff B., Sanglier J.J. & Wang Y.. Leptomycin B is an inhibitor of nuclear exportinhibition of nucleo-cytoplasmic translocation of the human immunodeficiency virus type 1 (HIV-1) Rev protein and Rev-dependent mRNA, Chem. Biol., 4, 1997, 139–147.[Medline]
Yang J., Bardes E.S.G., Moore J.D., Brennan J., Powers M.A. & Kornbluth S.. Control of cyclin B1 localization through regulated binding of the nuclear export factor CRM1, Genes Dev., 12, 1998, 2131–2143.
Zhao K., Wang W., Rando O.J., Xue Y., Swiderek K., Kuo A. & Crabtree G.R.. Rapid and phosphoinositol-dependent binding of the SWI/SNF-like BAF complex to chromatin after T lymphocyte receptor signaling, Cell., 95, 1998, 625–636.[Medline]
Zolotukhin A.S. & Felber B.K.. Nucleoporins Nup98 and Nup214 participate in nuclear export of human immunodeficiency virus type 1 Rev, J. Virol., 73, 1999, 120–127.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Facebook 
Reddit 
Technorati 
Twitter What's this?
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
|
