The Drosophila nucleoporin DNup88 localizes DNup214 and CRM1 on the nuclear envelope and attenuates NES-mediated nuclear export

Abstract

Many cellular responses rely on the control of nucleocytoplasmic transport of transcriptional regulators. The Drosophila nucleoporin Nup88 is selectively required for nuclear accumulation of Rel proteins and full activation of the innate immune response. Here, we investigate the mechanisms underlying its role in nucleocytoplasmic transport. Nuclear import of an nuclear localization signal-enhanced green fluorescent protein (NLS-EGFP) reporter is not affected in DNup88 (members only; mbo) mutants, whereas the level of CRM1-dependent EGFP-nuclear export signal (EGFP-NES) export is increased. We show that the nuclear accumulation of the Drosophila Rel protein Dorsal requires CRM1. DNup88 binds to DNup214 and DCRM1 in vitro, and both proteins become mislocalized from the nuclear rim into the nucleus of mbo mutants. Overexpression of DNup88 is sufficient to relocalize DNup214 and CRM1 on the nuclear envelope and revert the mutant phenotypes. We propose that a major function of DNup88 is to anchor DNup214 and CRM1 on the nuclear envelope and thereby attenuate NES-mediated nuclear export.

Figure 1.

DNup88 attenuates EGFP-NES export. (A) EGFP, NLS-EGFP, and EGFP-NES in wild-type, mbo, and emb ³ larvae. All panels show gut cells stained for EGFP (red) or DAPI (blue). In each row, the left panel shows background levels in wild-type (wt) untreated animals. The middle panel shows EGFP reporters after heat shock in the indicated genotypes. Bar, 35 μm. (B) EGFP-NES localization in heat shock– induced salivary glands of wild-type (left) and mbo mutants (middle). Treatment with LMB (+LMB) can revert the EGFP-NES mislocalization in mbo mutants (right). Bar, 35 μm.

Figure 2.

Nuclear export of Dorsal is mediated by DCRM1. (A) LMB inhibits nuclear export of EGFP-Dorsal and EGFP-NES in S2 cells. The fusion proteins are visualized by GFP, and nuclei are visualized by DAPI. LMB treatment enhanced the nuclear localization of both proteins. Bar, 2.5 μm. (B) Fat bodies from wild-type (wt) and emb ² mutant larvae stained for Dorsal (red) before (control) and after (induced) bacterial infection. Nuclei are shown by DAPI. Bar, 35 μm.

Figure 3.

DNup214 is mislocalized in mbo mutants. (A) DNup214 localization in fat body cells of wild-type, mbo mutants, and mbo larvae overexpressing DNup88. The top row shows DNup214 staining (red), and the bottom row shows DAPI. Confocal sections are shown in the insets. (B) Confocal sections of fat body cells from wild-type and mbo larvae stained with anti-mAb414 and anti-Nup153. Bars (A and B), 2 μm. (C) Western blot of S2 cells before (−) and after (+) Nup214 RNAi probed with the anti-DNup214 antibody. β-Tubulin is a loading control.

Figure 4.

DCRM1 is mislocalized in mbo mutants. (A) Confocal analysis of DCRM1 localization in fat body cells of wild-type, mbo mutants, and mbo larvae overexpressing DNup88. In each row, the left panel shows DCRM1 (red) and the middle panel shows lamin (green). To the right is the overlay. (B) Confocal sections of fat body cells from wild-type, early and late mbo mutant 3rd instar larvae stained for DRanGAP, DImportin-β, and DNXF-1. Localization and levels of these proteins are not changed in early mbo 3rd instar larvae (4 d after egg laying). In 6-d-old mutants, the nuclear rim staining appears reduced and punctuated. Bars (A and B), 2 μm. (C) Western blot of larval extracts from wild-type, emb ³, and mbo mutants probed for DCRM1. β-Tubulin and HSP70 are loading controls.

Figure 5.

DNup88 interacts with DCRM1 in vitro. (A) GST-fusions of DCRM1 and His₆ fusions of DNup88 were used in a GST pull-down assay. The binding is analyzed by Western blot with α-His₆ antibodies (top right). Arrows point to the products of the DNup88 deletions. A quantitation of the same blot with GST antibody (asterisk) is shown at the bottom. (B) DCRM1 interacts with DNup88 in the yeast two-hybrid system. 10-fold serial dilutions of strain PJ69-4A expressing the indicated protein combinations were spotted on synthetic complete medium lacking either adenine, tryptophan, and leucine (−ADE, −TRP, −LEU) or tryptophan and leucine (−TRP, −LEU). Growth in the absence of ADE indicates protein–protein interaction.