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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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Different effects of actin-binding drugs on nuclear export of GST–Rev–NES and GST–PKI–NES. (A) Oocyte injection protocol. 1 h after nuclear injection of latrunculin B, swinholide A or PBS (control) and the export substrate GST–Rev–NES or GST–PKI–NES, together with BSA, were microinjected into oocyte nuclei. 2 h later, oocytes were manually dissected into nuclear (N) and cytoplasmic (C) fractions. (B and C) Proteins were separated by 18% SDS-PAGE, and blots were analyzed with antibodies to GST and BSA. A substantial fraction of the injected export substrate migrates into the cytoplasm in the presence of PBS (lane 2) or swinholide A (lane 6). In contrast, the export substrate remains exclusively in the nuclei in presence of latrunculin B (lane 3). Detection of BSA in the nuclei confirms the specificity of the nuclear injections.
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Figure 7: Different effects of actin-binding drugs on nuclear export of GST–Rev–NES and GST–PKI–NES. (A) Oocyte injection protocol. 1 h after nuclear injection of latrunculin B, swinholide A or PBS (control) and the export substrate GST–Rev–NES or GST–PKI–NES, together with BSA, were microinjected into oocyte nuclei. 2 h later, oocytes were manually dissected into nuclear (N) and cytoplasmic (C) fractions. (B and C) Proteins were separated by 18% SDS-PAGE, and blots were analyzed with antibodies to GST and BSA. A substantial fraction of the injected export substrate migrates into the cytoplasm in the presence of PBS (lane 2) or swinholide A (lane 6). In contrast, the export substrate remains exclusively in the nuclei in presence of latrunculin B (lane 3). Detection of BSA in the nuclei confirms the specificity of the nuclear injections.

Mentions: Since nuclei of somatic cells and amphibian oocytes are usually not stained by fluorescent phalloidin, a specific marker of F-actin in immunofluorescence microscopy, it is possible that nuclear actin may exist in an unpolymerized or oligomeric form that is not recognized by phalloidin or organized in higher order structures different from F-actin (Gonsior et al. 1999). To further test the role of actin polymerization in nuclear export, we studied the effect of two actin-binding drugs with different specificities. The toxin latrunculin binds to G-actin and prevents its polymerization (Coue et al. 1987). Microinjection of latrunculin B into oocyte nuclei inhibited not only the export of Rev–NES (Fig. 7 B, lanes 3 and 4) and RRE RNA (data not shown) but also the export of PKI–NES (Fig. 7 C, lanes 3 and 4) and of the MPMV CTE (data not shown). In contrast, nuclear injection of swinholide A, a drug that severs F-actin and stabilizes actin dimers (Bubb et al. 1995), did not inhibit nuclear export (Fig. 7B and Fig. C, lanes 5 and 6). These results support the idea that a soluble form of nuclear actin, rather than F-actin, participate in multiple nuclear export pathways in Xenopus oocytes.


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)

Different effects of actin-binding drugs on nuclear export of GST–Rev–NES and GST–PKI–NES. (A) Oocyte injection protocol. 1 h after nuclear injection of latrunculin B, swinholide A or PBS (control) and the export substrate GST–Rev–NES or GST–PKI–NES, together with BSA, were microinjected into oocyte nuclei. 2 h later, oocytes were manually dissected into nuclear (N) and cytoplasmic (C) fractions. (B and C) Proteins were separated by 18% SDS-PAGE, and blots were analyzed with antibodies to GST and BSA. A substantial fraction of the injected export substrate migrates into the cytoplasm in the presence of PBS (lane 2) or swinholide A (lane 6). In contrast, the export substrate remains exclusively in the nuclei in presence of latrunculin B (lane 3). Detection of BSA in the nuclei confirms the specificity of the nuclear injections.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198816&req=5

Figure 7: Different effects of actin-binding drugs on nuclear export of GST–Rev–NES and GST–PKI–NES. (A) Oocyte injection protocol. 1 h after nuclear injection of latrunculin B, swinholide A or PBS (control) and the export substrate GST–Rev–NES or GST–PKI–NES, together with BSA, were microinjected into oocyte nuclei. 2 h later, oocytes were manually dissected into nuclear (N) and cytoplasmic (C) fractions. (B and C) Proteins were separated by 18% SDS-PAGE, and blots were analyzed with antibodies to GST and BSA. A substantial fraction of the injected export substrate migrates into the cytoplasm in the presence of PBS (lane 2) or swinholide A (lane 6). In contrast, the export substrate remains exclusively in the nuclei in presence of latrunculin B (lane 3). Detection of BSA in the nuclei confirms the specificity of the nuclear injections.
Mentions: Since nuclei of somatic cells and amphibian oocytes are usually not stained by fluorescent phalloidin, a specific marker of F-actin in immunofluorescence microscopy, it is possible that nuclear actin may exist in an unpolymerized or oligomeric form that is not recognized by phalloidin or organized in higher order structures different from F-actin (Gonsior et al. 1999). To further test the role of actin polymerization in nuclear export, we studied the effect of two actin-binding drugs with different specificities. The toxin latrunculin binds to G-actin and prevents its polymerization (Coue et al. 1987). Microinjection of latrunculin B into oocyte nuclei inhibited not only the export of Rev–NES (Fig. 7 B, lanes 3 and 4) and RRE RNA (data not shown) but also the export of PKI–NES (Fig. 7 C, lanes 3 and 4) and of the MPMV CTE (data not shown). In contrast, nuclear injection of swinholide A, a drug that severs F-actin and stabilizes actin dimers (Bubb et al. 1995), did not inhibit nuclear export (Fig. 7B and Fig. C, lanes 5 and 6). These results support the idea that a soluble form of nuclear actin, rather than F-actin, participate in multiple nuclear export pathways in Xenopus oocytes.

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