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

Zeitler B, Weis K - J. Cell Biol. (2004)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell and Developmental Biology, Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.

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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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) nup42Δnup159FG1 and nup42Δnup159FGΔ 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.
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fig4: 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) nup42Δnup159FG1 and nup42Δnup159FGΔ 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.

Mentions: Because the FG swap between Nup159 and Nup1 created an artificial binding site for Xpo1 in the nucleus (Fig. 2 B), we next sought to examine whether the FG mutants were deficient in Xpo1-mediated export. We took advantage of a highly sensitive in vivo export assay, which relies on the competition between a nuclear export signal (NES) and an NLS fused in cis to a reporter cargo (Stade et al., 1997). As expected, a cNLS/NES-RFP cargo was enriched in the cytoplasm of wild-type cells, but rapidly accumulated in the nucleus of xpo1-1 mutants at the nonpermissive temperature (Fig. 4 A). When the reporter was expressed in the nup1 and nup159 FG mutants, no defect in its export was detected (Fig. 4, B and C). Because cNLS-mediated protein import is unaffected in all FG mutants tested (Fig. 3), we conclude that moving the high affinity binding site for Xpo1 from one side of the pore to the other does not significantly affect the export of NES-containing cargos.


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

Zeitler B, Weis K - J. Cell Biol. (2004)

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) nup42Δnup159FG1 and nup42Δnup159FGΔ 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.
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fig4: 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) nup42Δnup159FG1 and nup42Δnup159FGΔ 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.
Mentions: Because the FG swap between Nup159 and Nup1 created an artificial binding site for Xpo1 in the nucleus (Fig. 2 B), we next sought to examine whether the FG mutants were deficient in Xpo1-mediated export. We took advantage of a highly sensitive in vivo export assay, which relies on the competition between a nuclear export signal (NES) and an NLS fused in cis to a reporter cargo (Stade et al., 1997). As expected, a cNLS/NES-RFP cargo was enriched in the cytoplasm of wild-type cells, but rapidly accumulated in the nucleus of xpo1-1 mutants at the nonpermissive temperature (Fig. 4 A). When the reporter was expressed in the nup1 and nup159 FG mutants, no defect in its export was detected (Fig. 4, B and C). Because cNLS-mediated protein import is unaffected in all FG mutants tested (Fig. 3), we conclude that moving the high affinity binding site for Xpo1 from one side of the pore to the other does not significantly affect the export of NES-containing cargos.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell and Developmental Biology, Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.

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.

Show MeSH