The nucleoporin Nup214 sequesters CRM1 at the nuclear rim and modulates NFB activation in Drosophila

JCS03201 Supplementary Material

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• Supplemental Figure 1 -

  Fig. S1. Drosophila CRM1 binds to Nup214 in the yeast two-hybrid assay and in vitro. (A) Schematic drawings of the Nup214 and CRM1 fragments used in the yeast two-hybrid and pull-down assay. Interactions are indicated by +, no interactions by −. (B) Nup214 interacts with CRM1 in the yeast two-hybrid system. Yeast strain PJ69-4A containing pAS1 (Gal4-BD, bait-plasmid) fused to CAN1, CAN2 or CAN3 and pACT2 (Gal4-AD, prey plasmid) fused to either CRM1ΔC or CRM1ΔN, was tested for interaction by measuring β-Galactosidase (β-Gal) activity. High units of β-Gal indicate protein-protein interaction. CAN3, encoding the C-terminal, FG-rich part of the protein interacted strongly with the N-terminal (CRM1ΔC) part of CRM1. Additional weaker interactions were also detected between an N-terminal fragment of the protein (CAN1) and the N-terminus of CRM1 (CRM1ΔC) and between the central portion of Nup214 (CAN2) and the C-terminal part of CRM1 (CRM1ΔN) (C) Nup214 interacts with CRM1 in vitro. GST-fusions of wild-type and CRM1ΔC and His₆-fusions of the Nup214 fragments CAN1, CAN2 and CAN3 were used in the GST- pull down assay. The binding was analyzed by western blot with an anti-His₆ antibody (top). The same blot was probed with an anti-GST antibody for quantification. In this direct assay only the C-terminal part of Nup214 bound to full-length CRM1, indicating that the interaction between Nup214 with CRM1 occurs via the FG-repeats of the nucleoporin.

• Supplemental Figure 2 -

  Fig. S2. The localization of the Nup214-Nup88 complex depends on the relative amounts of its constituents. Nup214 and Nup88 were expressed under the hsp70 promoter in nup214 mutant larvae. The panels show a confocal section of larval fat body cells stained with Nup88 and Nup214 antibodies. The complex is found at the nuclear rim when the amount of Nup88 is higher than that of Nup214. When the levels of Nup88 are lower than that of Nup214, the complex is detected in the nucleus.

• Supplemental Figure 3 -

  Fig. S3. Nup214 is required for a subset of nuclear import events. (A) Ubx, Grh, NLS-GFP and GFP were expressed under the control of the hsp70 promoter in wild-type and nup214 mutant larvae and detected with the relevant antibodies. β-Gal-NLS expression was activated by heat-shock-induced GAL4 protein. The panels illustrate the localization of Ubx, Grh and β-Gal-NLS in fat bodies, or for NLS-GFP and GFP, in gut cells of treated animals. Nuclei are visualized by DAPI in the adjacent panels. In wild-type larvae Ubx, Grh, β-Gal-NLS and NLS-GFP accumulate in the nucleus after heat-shock. In fat bodies of nup214 mutants Ubx and Grh become nuclear whereas β-Gal-NLS remains predominantly cytoplasmic. NLS-GFP appears predominantly cytoplasmic in most of the mutant gut cells (arrowheads). Asterisks indicate cells with nuclear NLS-GFP. Bar, 35 μm. (B) Nuclear import rates of the NLS-GFP reporter are reduced in nup214 mutants. Comparison of the nuclear:cytoplasmic ratios of NLS-GFP intensities in early third (eL3) instar wild-type and nup214 mutant larval gut cells. The ratios are shown in a log₂ graph. For each genotype 30 cells were analyzed. nup214 mutants show a significant 44% decrease in nuclear accumulation of the NLS-GFP reporter compared to wild-type larvae (P<0.0001 by pair-wise t-test). Bars represent s.e.m.

• Supplemental Figure 4 -

  Fig. S4. Overexpression of Nup88 and Nup214 does not affect NLS-GFP import. (A) Nup214 (HS214), Nup88 (HS88) or Nup214+Nup88 (HS214+HS88) were expressed with the hsp70 promoter in wild-type third instar larvae. Heat-treated wild-type (wt) served as a control. All panels show confocal sections of malpighian tube nuclei stained either with Nup88, Nup214, CRM1, importin β, RanGAP or Lamin antiserum. Excess of Nup88 alone does not affect the localization of CRM1, importin β, Lamin and RanGAP. Overexpression of Nup214 causes a slight increase in the accumulation of CRM1 on the nuclear rim and a minor mislocalization of importin β. In animals expressing both Nups, importin β is slightly mislocalized. Bar, 5 μm. (B) NLS-GFP was expressed together with Nup88, Nup214 or Nup214+Nup88 with the hsp70 promoter in third instar wild-type larvae. The ratios of nuclear:cytoplasmic NLS-GFP intensities are shown in a log₂ graph. The localization of the reporter is not affected upon Nup88 and Nup214 overexpression (P<0.0001 by pair-wise t-test). Bars represent s.e.m.

• Supplemental Figure 5 -

  Fig. S5. Dorsal is a substrate for CRM1. (A) His₆-fusions of full-length Dorsal as well as Dorsal deletion (ΔNES, ΔC, ΔN) and point mutation (NES3* and NES4*) constructs were used for transfection of Drosophila S2 cells. Depicted as NES1-4 are the predicted Nuclear Export Signals. A functional nuclear localization signal (NLS) is marked in red. (B) Dorsal NES3 and NES4 function in CRM1-mediated protein export. Drosophila S2 cells transiently expressing GFP protein fused to Dorsal NES1, NES2, NES3 or NES4. GFP was used as a control. Cells were treated with dsRNA against crm1. Untreated cells served as a control. The images show the localization of the GFP fusion proteins in living cells. Hoechst staining visualizes the DNA. CRM1 dsRNA treatment enhances the nuclear localization of GFP-NES3 and GFP-NES4. Bar, 2.5 μm. (C) Dorsal NES4 is necessary for the cytoplasmic retention of Dorsal. Drosophila S2 cells transiently expressing either full length Dorsal, the deletion constructs ΔC, ΔN and ΔNES or Dorsal with mutated NES4 (NES4*) and NES3 (NES3*) before (−LMB) and after (+LMB) Leptomycin B treatment. Fusion proteins were visualized with the anti-His₆ antibody (red) whereas nuclei are shown by DAPI staining (blue). Full-length Dorsal, its C-terminus (ΔN; amino acid 325-679) and the Dorsal NES3 mutation are predominantly cytoplasmic and accumulate in the nucleus upon LMB treatment. By contrast, the N-terminal fragment of Dorsal (ΔC; amino acid 1-341) is restricted to the nucleus in untreated and LMB-treated cells. The Dorsal NES4 mutation converted Dorsal into a predominantly nuclear protein irrespective of LMB addition. Bar, 2.5 μm.