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. 2004 Nov 22;167(4):583-90.
doi: 10.1083/jcb.200407156.

The FG-repeat asymmetry of the nuclear pore complex is dispensable for bulk nucleocytoplasmic transport in vivo

Bryan Zeitler  1 , Karsten Weis
Affiliations

The FG-repeat asymmetry of the nuclear pore complex is dispensable for bulk nucleocytoplasmic transport in vivo

Bryan Zeitler et al. J Cell Biol. .

Abstract

Nucleocytoplasmic transport occurs through gigantic proteinaceous channels called nuclear pore complexes (NPCs). Translocation through the NPC is exquisitely selective and is mediated by interactions between soluble transport carriers and insoluble NPC proteins that contain phenylalanine-glycine (FG) repeats. Although most FG nucleoporins (Nups) are organized symmetrically about the planar axis of the nuclear envelope, very few localize exclusively to one side of the NPC. We constructed Saccharomyces cerevisiae mutants with asymmetric FG repeats either deleted or swapped to generate NPCs with inverted FG asymmetry. The mutant Nups localize properly within the NPC and exhibit exchanged binding specificity for the export factor Xpo1. Surprisingly, we were unable to detect any defects in the Kap95, Kap121, Xpo1, or mRNA transport pathways in cells expressing the mutant FG Nups. These findings suggest that the biased distribution of FG repeats is not required for major nucleocytoplasmic trafficking events across the NPC.

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Figures

Figure 1.
Figure 1.
Mutant FG alleles correctly localize within the NPC. (A) The cartoon (left) depicts the overall architecture of the NPC embedded in the nuclear envelope. Shown are the cytoplasmic filaments (red), central channel (green), and nuclear basket (blue), as well as the corresponding FG Nups (right) from which these structures are partly derived (adapted from Rout et al., 2000). Note that PxFG/SxFG repeats are mainly found in cytoplasmic Nups, GxFG repeats are present in the Nups forming the central channel, and FxFG and FxF repeats are enriched in nuclear Nups. (B) The FxF/FxFG repeats of Nup1 (aa 401–967) were amplified by PCR and cloned into Nup159, replacing its FG repeat domain (aa 458–902, Nup159FG1). Similarly, the SxFG/PxFG repeat domain of Nup159 (aa 459–885) was used to replace the Nup1 FG repeats (aa 358–1,000, Nup1FG159). Additionally, alleles were created that completely lack the FG-repeat domains (deletion junctions are the same as in the FG-swap alleles). (C) Log-phase cultures were stained with DAPI and visualized by bright-field and fluorescence microscopy. Bar, 5 μm. (D) Log-phase cells expressing GFP-tagged FG alleles were prepared for immuno-EM by high pressure freezing (McDonald and Muller-Reichert, 2002). (Left) Representative images showing thin sections stained for the GFP epitope and labeled with a gold-conjugated secondary antibody. Nuclear pores were identified as breaks in the nuclear envelope (arrowheads). (Right) The perpendicular distance from the central plane of an NPC to a gold particle was measured and plotted in a histogram for each strain. The total number of gold particles and mean distance are given. Bar, 100 nm.
Figure 2.
Figure 2.
In vivo association of Kap95 and Xpo1 with the mutant FG alleles. (A) Whole cell extracts of strains expressing Kap95-ZZ were preincubated with 10 μM Gsp1ΔCQ71L-GTP or a mock treatment, immunoprecipitated, and detected by Western blot. (Left) 4 μg of input from each Kap95-ZZ strain was loaded and blotted with a highly sensitive rabbit anti-GFP antibody (rGFP; Seedorf et al., 1999). (Right) One fifth of the total bead volume was loaded from each pull down (± Gsp1ΔCQ71L-GTP) and blotted with highly specific mouse anti-GFP (mGFP; top) or anti-Kap95 antibodies (bottom). (B) Whole cell extracts of strains expressing Xpo1-ZZ were processed and detected as above for the Kap95-ZZ extracts. 10 μM Gsp1ΔCQ71L-GTP was included in all pull-down reactions. (Left) 4 μg of input from each Xpo1-ZZ strain was loaded, blotted, and detected with rGFP. (Right) One fifth of the total bead volume was loaded from each pull down and blotted with mGFP (top) or anti-Xpo1 antibodies (bottom). Note that all lanes are derived from the same blot and exposure, but have been repositioned for clarity.
Figure 3.
Figure 3.
The Kap95 import pathway is not affected in the FG mutants. (A) Log-phase cultures were induced to express the cNLS-RFP reporter with 2% galactose and visualized after 2–4 h by fluorescence microscopy. Bar, 5 μm. (B) Quantitation of cNLS localization shown in A. A minimum of 300 cells were counted four independent times. The mean and SEM are presented. (C) Each strain from A was induced to express the cNLS reporter, metabolically poisoned, washed, and rescued with 2% dextrose. Recovery of nuclear RFP enrichment was then scored for at least 60 cells every 90 s. Each time point represents the mean and SEM of four experiments.
Figure 4.
Figure 4.
Cytoplasmic FG repeats are not required for Xpo1-mediated export. (A) Wild-type or xpo1-1 cells were induced with 2% galactose for 3 h, stained with Hoechst dye, and visualized by fluorescence and bright field microscopy. xpo1-1 cells were shifted to 37°C for 30 min immediately before microscopy. Bar, 5 μm. (B) Early-log phase cultures were induced to express the cNLS/NES-RFP reporter as in A and visualized by fluorescence microscopy. Bar, 5 μm. (C) Quantitation of cNLS/NES localization shown in B. A minimum of 300 cells were counted four independent times. The mean and SEM are presented. (D) Overnight cultures were grown to stationary phase in rich media and titered onto YPD (left) or 5-fluoroorotic acid (5-FOA). An “x” in the pNUP159 column denotes strains that harbored NUP159-GFP on a URA3-marked plasmid. (E) nup42Δnup159 FG1 and nup42Δnup159 FGΔ cells containing the cNLS/NES-RFP reporter were examined for export defects as described for B. Note that for all strains tested, a large percentage of the cell population consistently failed to express the cNLS/NES reporter. Bar, 5 μm.
Figure 5.
Figure 5.
poly(A) RNA export is not inhibited in the FG mutants. Log-phase cells were fixed and stained for poly(A) RNA using an oligo-d(T) probe and detected by indirect immunofluorescence. DNA was labeled with Hoechst dye to visualize nuclei. mex67-5 cells were shifted to 37°C for 1 h before fixation. Bar, 5 μm.
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