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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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Inhibition of nuclear export of GFP–Rev–NES–GST fusion protein by antiactin antibodies in cultured Vero cells. A mixture of GFP–Rev–NES–GST fusion protein, together with either control IgM (A and B), or antiactin antibodies (C and D) was microinjected into nuclei of Vero cells. Phase–contrast images (left) and GFP fluorescence (right) were recorded at the indicated postinjection time points. The export substrate is efficiently translocated from the nucleus to the cytoplasm in the presence of control IgM (A′ and B′). In contrast, coinjected antiactin antibodies completely prevent nuclear export (C′ and D′). Microinjected cells are denoted by arrows. Bar, 20 μm.
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Figure 8: Inhibition of nuclear export of GFP–Rev–NES–GST fusion protein by antiactin antibodies in cultured Vero cells. A mixture of GFP–Rev–NES–GST fusion protein, together with either control IgM (A and B), or antiactin antibodies (C and D) was microinjected into nuclei of Vero cells. Phase–contrast images (left) and GFP fluorescence (right) were recorded at the indicated postinjection time points. The export substrate is efficiently translocated from the nucleus to the cytoplasm in the presence of control IgM (A′ and B′). In contrast, coinjected antiactin antibodies completely prevent nuclear export (C′ and D′). Microinjected cells are denoted by arrows. Bar, 20 μm.

Mentions: Since these data provided evidence for a novel export function of actin, we also wanted to test whether these results could be confirmed in somatic cells. Therefore, we microinjected nuclei of Vero cells with recombinant nuclear export substrate in which Rev–NES–GST was fused to the GFP. This GFP–Rev–NES–GST fusion protein allows the monitoring of Rev–NES-mediated nuclear export in live cells (Rosorius et al. 1999a). GFP-specific fluorescence and phase contrast images were taken immediately and 30 min after injection. Coinjection of nonspecific IgM did not interfere with Rev–NES-mediated nuclear export (Fig. 8A′ and B′). However, coinjection of antiactin antibodies completely abrogated the nucleocytoplasmic translocation of the export substrate (Fig. 8C′ and D′), confirming the data raised in Xenopus oocytes. As expected, antiactin antibodies were able to effectively block the nuclear export of GST–PKI–NES (data not shown), again confirming the data obtained 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)

Inhibition of nuclear export of GFP–Rev–NES–GST fusion protein by antiactin antibodies in cultured Vero cells. A mixture of GFP–Rev–NES–GST fusion protein, together with either control IgM (A and B), or antiactin antibodies (C and D) was microinjected into nuclei of Vero cells. Phase–contrast images (left) and GFP fluorescence (right) were recorded at the indicated postinjection time points. The export substrate is efficiently translocated from the nucleus to the cytoplasm in the presence of control IgM (A′ and B′). In contrast, coinjected antiactin antibodies completely prevent nuclear export (C′ and D′). Microinjected cells are denoted by arrows. Bar, 20 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 8: Inhibition of nuclear export of GFP–Rev–NES–GST fusion protein by antiactin antibodies in cultured Vero cells. A mixture of GFP–Rev–NES–GST fusion protein, together with either control IgM (A and B), or antiactin antibodies (C and D) was microinjected into nuclei of Vero cells. Phase–contrast images (left) and GFP fluorescence (right) were recorded at the indicated postinjection time points. The export substrate is efficiently translocated from the nucleus to the cytoplasm in the presence of control IgM (A′ and B′). In contrast, coinjected antiactin antibodies completely prevent nuclear export (C′ and D′). Microinjected cells are denoted by arrows. Bar, 20 μm.
Mentions: Since these data provided evidence for a novel export function of actin, we also wanted to test whether these results could be confirmed in somatic cells. Therefore, we microinjected nuclei of Vero cells with recombinant nuclear export substrate in which Rev–NES–GST was fused to the GFP. This GFP–Rev–NES–GST fusion protein allows the monitoring of Rev–NES-mediated nuclear export in live cells (Rosorius et al. 1999a). GFP-specific fluorescence and phase contrast images were taken immediately and 30 min after injection. Coinjection of nonspecific IgM did not interfere with Rev–NES-mediated nuclear export (Fig. 8A′ and B′). However, coinjection of antiactin antibodies completely abrogated the nucleocytoplasmic translocation of the export substrate (Fig. 8C′ and D′), confirming the data raised in Xenopus oocytes. As expected, antiactin antibodies were able to effectively block the nuclear export of GST–PKI–NES (data not shown), again confirming the data obtained 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