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Cofactor requirements for nuclear export of Rev response element (RRE)- and constitutive transport element (CTE)-containing retroviral RNAs. An unexpected role for actin.

Hofmann W, Reichart B, Ewald A, Müller E, Schmitt I, Stauber RH, Lottspeich F, Jockusch BM, Scheer U, Hauber J, Dabauvalle MC - J. Cell Biol. (2001)

Bottom Line: 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).

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, Biocenter of the University of Würzburg, D-97074 Würzburg, Germany.

ABSTRACT
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).

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The eIF-5A mutant protein M14 is not exported and is unable to bind the export factor CRM1–exportin 1. (A) Oocyte injection protocol. 2 h after nuclear injection of wt eIF-5A or mutant protein M14, oocytes were manually dissected into nuclear (N) and cytoplasmic (C) fractions. (B) Proteins were separated by 18% SDS-PAGE, and blots were analyzed with antibodies to GST and BSA. A substantial fraction of the injected wt GST–eIF-5A migrates into the cytoplasm within 2 h (lane 2). In contrast, most of the eIF-5A mutant protein M14 remains in the nuclei (compare lanes 3 and 4). The exclusive presence of BSA in the nuclei confirms the specificity of the nuclear injections. (C) Interaction of wt eIF-5A and the mutant M14 with CRM1 as determined by pull-down assay. Glutathione–Sepharose beads coupled to the GST proteins indicated were incubated with oocyte extract. Bound and unbound proteins were resolved by SDS-PAGE and analyzed by immunoblotting experiments with anti-CRM1 antiserum. Bound proteins are shown in lanes 1–3, unbound proteins are shown in lanes 1′–3′. CRM1 binds to the wt form of eIF-5A (lane 1) but not to the M14 mutant (lane 2) and GST alone (lane 3). (D) Role of eIF-5A for interaction between CRM1 and Rev–NES was analyzed by pull-down assay. GST–Rev–NES coupled to glutathione–Sepharose beads was incubated with wt eIF-5A/eIF-5A–M14 before incubation with oocyte extract. Bound (lanes 1–3) and unbound (1′–3′) proteins were analyzed as described above. CRM1 binds to GST–Rev–NES only in the presence of the wt form of eIF-5A (lane 1) but not of the M14 mutant (lane 2). (E) Role of eIF-5A for interaction between recombinant CRM1 fusion protein and Rev–NES was analyzed by pull-down assay. GST–Rev–NES or GST alone coupled to glutathione-Sepharose beads were incubated in various combinations with RanGTP (GST-RanQ69L), wt eIF-5A, or eIF-5A–M14 before addition of recombinant His-tagged CRM1. Bound (lanes 1–5) and unbound (lanes 1′–5′) CRM1 was visualized by using His-specific antibodies. CRM1 fusion protein binds to GST–Rev–NES only in the presence of the wt form of eIF-5A and RanGTP (lane 1 versus lanes 3 and 5) but not in presence of the M14 mutant (lane 2) and not to GST (lane 4). The 66- and 45-kD components apparently represent CRM1 degradation products. Molecular mass standards are indicated in kD.
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Figure 3: The eIF-5A mutant protein M14 is not exported and is unable to bind the export factor CRM1–exportin 1. (A) Oocyte injection protocol. 2 h after nuclear injection of wt eIF-5A or mutant protein M14, oocytes were manually dissected into nuclear (N) and cytoplasmic (C) fractions. (B) Proteins were separated by 18% SDS-PAGE, and blots were analyzed with antibodies to GST and BSA. A substantial fraction of the injected wt GST–eIF-5A migrates into the cytoplasm within 2 h (lane 2). In contrast, most of the eIF-5A mutant protein M14 remains in the nuclei (compare lanes 3 and 4). The exclusive presence of BSA in the nuclei confirms the specificity of the nuclear injections. (C) Interaction of wt eIF-5A and the mutant M14 with CRM1 as determined by pull-down assay. Glutathione–Sepharose beads coupled to the GST proteins indicated were incubated with oocyte extract. Bound and unbound proteins were resolved by SDS-PAGE and analyzed by immunoblotting experiments with anti-CRM1 antiserum. Bound proteins are shown in lanes 1–3, unbound proteins are shown in lanes 1′–3′. CRM1 binds to the wt form of eIF-5A (lane 1) but not to the M14 mutant (lane 2) and GST alone (lane 3). (D) Role of eIF-5A for interaction between CRM1 and Rev–NES was analyzed by pull-down assay. GST–Rev–NES coupled to glutathione–Sepharose beads was incubated with wt eIF-5A/eIF-5A–M14 before incubation with oocyte extract. Bound (lanes 1–3) and unbound (1′–3′) proteins were analyzed as described above. CRM1 binds to GST–Rev–NES only in the presence of the wt form of eIF-5A (lane 1) but not of the M14 mutant (lane 2). (E) Role of eIF-5A for interaction between recombinant CRM1 fusion protein and Rev–NES was analyzed by pull-down assay. GST–Rev–NES or GST alone coupled to glutathione-Sepharose beads were incubated in various combinations with RanGTP (GST-RanQ69L), wt eIF-5A, or eIF-5A–M14 before addition of recombinant His-tagged CRM1. Bound (lanes 1–5) and unbound (lanes 1′–5′) CRM1 was visualized by using His-specific antibodies. CRM1 fusion protein binds to GST–Rev–NES only in the presence of the wt form of eIF-5A and RanGTP (lane 1 versus lanes 3 and 5) but not in presence of the M14 mutant (lane 2) and not to GST (lane 4). The 66- and 45-kD components apparently represent CRM1 degradation products. Molecular mass standards are indicated in kD.

Mentions: Together, these data show that eIF-5A plays an essential role in Rev and Rev-dependent RRE RNA export in Xenopus oocytes. To date, however, it is not clear which precise function eIF-5A executes during the nuclear export of Rev–RRE-containing ribonucleoproteins. In particular, it is not known at which stage during nucleocytoplasmic translocation eIF-5A mutant proteins (e.g., M14) block Rev export. Therefore, we next investigated whether or not eIF-5A–M14 by itself is exported from the nuclear to the cytoplasmic compartment. As depicted in Fig. 3 A, wt GST–eIF-5A or GST–eIF-5A–M14 fusion proteins were microinjected into oocyte nuclei, and localization of the injected proteins was analyzed using specific antibodies, 2 h after injection. In agreement with previous data using somatic cells (Rosorius et al. 1999b), a significant amount of the nuclear-injected wt GST–eIF-5A protein was transported to the cytoplasm in these experiments (Fig. 3 B, lanes 1 and 2). In sharp contrast, however, the GST–eIF-5A–M14 protein remained predominantly in the injected oocyte nuclei (lanes 3 and 4). It has recently been shown by overlay blot assays and binding studies in solution that unmodified eIF-5A is able to interact with CRM1/exportin1 (Rosorius et al. 1999b). In our next set of experiments, therefore, we analyzed the binding of wt eIF-5A and eIF-5A–M14 to CRM1/exportin1 using GST–eIF-5A fusion proteins and total protein extracts from stage VI oocytes, which are a rich source for CRM1/exportin1. The use of oocyte extracts in these experiments has the additional advantage that these extracts also contain the cofactors that are possibly required for the formation of nuclear export complexes (e.g., RanGTP). wt GST–eIF-5A or GST–eIF-5A–M14 fusion proteins were immobilized on glutathione–Sepharose beads and incubated with oocyte protein extracts. The beads were then pelleted by centrifugation, and the bound and unbound material was analyzed by Western blots using antiserum raised against CRM1/exportin1 (Kudo et al. 1997). As shown in Fig. 3 C, CRM1/exportin1 bound wt GST–eIF-5A protein (lane 1) but not GST alone (lane 3). Interestingly, the Rev inhibitory mutant eIF-5A–M14 clearly failed to interact with CRM1/exportin1 (lane 2). Thus, the inability of the eIF-5A–M14 protein to interact with CRM1/exportin1 (Fig. 3 C) correlates with its diminished nuclear export activity. Please also note that the ∼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).


Cofactor requirements for nuclear export of Rev response element (RRE)- and constitutive transport element (CTE)-containing retroviral RNAs. An unexpected role for actin.

Hofmann W, Reichart B, Ewald A, Müller E, Schmitt I, Stauber RH, Lottspeich F, Jockusch BM, Scheer U, Hauber J, Dabauvalle MC - J. Cell Biol. (2001)

The eIF-5A mutant protein M14 is not exported and is unable to bind the export factor CRM1–exportin 1. (A) Oocyte injection protocol. 2 h after nuclear injection of wt eIF-5A or mutant protein M14, oocytes were manually dissected into nuclear (N) and cytoplasmic (C) fractions. (B) Proteins were separated by 18% SDS-PAGE, and blots were analyzed with antibodies to GST and BSA. A substantial fraction of the injected wt GST–eIF-5A migrates into the cytoplasm within 2 h (lane 2). In contrast, most of the eIF-5A mutant protein M14 remains in the nuclei (compare lanes 3 and 4). The exclusive presence of BSA in the nuclei confirms the specificity of the nuclear injections. (C) Interaction of wt eIF-5A and the mutant M14 with CRM1 as determined by pull-down assay. Glutathione–Sepharose beads coupled to the GST proteins indicated were incubated with oocyte extract. Bound and unbound proteins were resolved by SDS-PAGE and analyzed by immunoblotting experiments with anti-CRM1 antiserum. Bound proteins are shown in lanes 1–3, unbound proteins are shown in lanes 1′–3′. CRM1 binds to the wt form of eIF-5A (lane 1) but not to the M14 mutant (lane 2) and GST alone (lane 3). (D) Role of eIF-5A for interaction between CRM1 and Rev–NES was analyzed by pull-down assay. GST–Rev–NES coupled to glutathione–Sepharose beads was incubated with wt eIF-5A/eIF-5A–M14 before incubation with oocyte extract. Bound (lanes 1–3) and unbound (1′–3′) proteins were analyzed as described above. CRM1 binds to GST–Rev–NES only in the presence of the wt form of eIF-5A (lane 1) but not of the M14 mutant (lane 2). (E) Role of eIF-5A for interaction between recombinant CRM1 fusion protein and Rev–NES was analyzed by pull-down assay. GST–Rev–NES or GST alone coupled to glutathione-Sepharose beads were incubated in various combinations with RanGTP (GST-RanQ69L), wt eIF-5A, or eIF-5A–M14 before addition of recombinant His-tagged CRM1. Bound (lanes 1–5) and unbound (lanes 1′–5′) CRM1 was visualized by using His-specific antibodies. CRM1 fusion protein binds to GST–Rev–NES only in the presence of the wt form of eIF-5A and RanGTP (lane 1 versus lanes 3 and 5) but not in presence of the M14 mutant (lane 2) and not to GST (lane 4). The 66- and 45-kD components apparently represent CRM1 degradation products. Molecular mass standards are indicated in kD.
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Figure 3: The eIF-5A mutant protein M14 is not exported and is unable to bind the export factor CRM1–exportin 1. (A) Oocyte injection protocol. 2 h after nuclear injection of wt eIF-5A or mutant protein M14, oocytes were manually dissected into nuclear (N) and cytoplasmic (C) fractions. (B) Proteins were separated by 18% SDS-PAGE, and blots were analyzed with antibodies to GST and BSA. A substantial fraction of the injected wt GST–eIF-5A migrates into the cytoplasm within 2 h (lane 2). In contrast, most of the eIF-5A mutant protein M14 remains in the nuclei (compare lanes 3 and 4). The exclusive presence of BSA in the nuclei confirms the specificity of the nuclear injections. (C) Interaction of wt eIF-5A and the mutant M14 with CRM1 as determined by pull-down assay. Glutathione–Sepharose beads coupled to the GST proteins indicated were incubated with oocyte extract. Bound and unbound proteins were resolved by SDS-PAGE and analyzed by immunoblotting experiments with anti-CRM1 antiserum. Bound proteins are shown in lanes 1–3, unbound proteins are shown in lanes 1′–3′. CRM1 binds to the wt form of eIF-5A (lane 1) but not to the M14 mutant (lane 2) and GST alone (lane 3). (D) Role of eIF-5A for interaction between CRM1 and Rev–NES was analyzed by pull-down assay. GST–Rev–NES coupled to glutathione–Sepharose beads was incubated with wt eIF-5A/eIF-5A–M14 before incubation with oocyte extract. Bound (lanes 1–3) and unbound (1′–3′) proteins were analyzed as described above. CRM1 binds to GST–Rev–NES only in the presence of the wt form of eIF-5A (lane 1) but not of the M14 mutant (lane 2). (E) Role of eIF-5A for interaction between recombinant CRM1 fusion protein and Rev–NES was analyzed by pull-down assay. GST–Rev–NES or GST alone coupled to glutathione-Sepharose beads were incubated in various combinations with RanGTP (GST-RanQ69L), wt eIF-5A, or eIF-5A–M14 before addition of recombinant His-tagged CRM1. Bound (lanes 1–5) and unbound (lanes 1′–5′) CRM1 was visualized by using His-specific antibodies. CRM1 fusion protein binds to GST–Rev–NES only in the presence of the wt form of eIF-5A and RanGTP (lane 1 versus lanes 3 and 5) but not in presence of the M14 mutant (lane 2) and not to GST (lane 4). The 66- and 45-kD components apparently represent CRM1 degradation products. Molecular mass standards are indicated in kD.
Mentions: Together, these data show that eIF-5A plays an essential role in Rev and Rev-dependent RRE RNA export in Xenopus oocytes. To date, however, it is not clear which precise function eIF-5A executes during the nuclear export of Rev–RRE-containing ribonucleoproteins. In particular, it is not known at which stage during nucleocytoplasmic translocation eIF-5A mutant proteins (e.g., M14) block Rev export. Therefore, we next investigated whether or not eIF-5A–M14 by itself is exported from the nuclear to the cytoplasmic compartment. As depicted in Fig. 3 A, wt GST–eIF-5A or GST–eIF-5A–M14 fusion proteins were microinjected into oocyte nuclei, and localization of the injected proteins was analyzed using specific antibodies, 2 h after injection. In agreement with previous data using somatic cells (Rosorius et al. 1999b), a significant amount of the nuclear-injected wt GST–eIF-5A protein was transported to the cytoplasm in these experiments (Fig. 3 B, lanes 1 and 2). In sharp contrast, however, the GST–eIF-5A–M14 protein remained predominantly in the injected oocyte nuclei (lanes 3 and 4). It has recently been shown by overlay blot assays and binding studies in solution that unmodified eIF-5A is able to interact with CRM1/exportin1 (Rosorius et al. 1999b). In our next set of experiments, therefore, we analyzed the binding of wt eIF-5A and eIF-5A–M14 to CRM1/exportin1 using GST–eIF-5A fusion proteins and total protein extracts from stage VI oocytes, which are a rich source for CRM1/exportin1. The use of oocyte extracts in these experiments has the additional advantage that these extracts also contain the cofactors that are possibly required for the formation of nuclear export complexes (e.g., RanGTP). wt GST–eIF-5A or GST–eIF-5A–M14 fusion proteins were immobilized on glutathione–Sepharose beads and incubated with oocyte protein extracts. The beads were then pelleted by centrifugation, and the bound and unbound material was analyzed by Western blots using antiserum raised against CRM1/exportin1 (Kudo et al. 1997). As shown in Fig. 3 C, CRM1/exportin1 bound wt GST–eIF-5A protein (lane 1) but not GST alone (lane 3). Interestingly, the Rev inhibitory mutant eIF-5A–M14 clearly failed to interact with CRM1/exportin1 (lane 2). Thus, the inability of the eIF-5A–M14 protein to interact with CRM1/exportin1 (Fig. 3 C) correlates with its diminished nuclear export activity. Please also note that the ∼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).

Bottom Line: 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).

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, Biocenter of the University of Würzburg, D-97074 Würzburg, Germany.

ABSTRACT
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).

Show MeSH
Related in: MedlinePlus