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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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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.
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fig1: 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.

Mentions: As illustrated in Fig. 1 A, the composition of FG repeats varies and exhibits a biased distribution across the pore. To understand the functional importance of the asymmetric distribution of FG repeats, we generated yeast strains with the FG repeats of the cytoplasmic Nup159 and nuclear Nup1 deleted or swapped (Fig. 1 B). Nup159 and Nup1 preferentially bind to the transport factors Xpo1 (Allen et al., 2002; unpublished data) and Kap95 (Allen et al., 2001; Pyhtila and Rexach, 2003), respectively. We predicted that swapping FG repeats to the opposite side of the pore would create dominant NPC alleles with exchanged specificity for transport factors and thus would reverse the FG gradient.


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)

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.
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Related In: Results  -  Collection

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fig1: 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.
Mentions: As illustrated in Fig. 1 A, the composition of FG repeats varies and exhibits a biased distribution across the pore. To understand the functional importance of the asymmetric distribution of FG repeats, we generated yeast strains with the FG repeats of the cytoplasmic Nup159 and nuclear Nup1 deleted or swapped (Fig. 1 B). Nup159 and Nup1 preferentially bind to the transport factors Xpo1 (Allen et al., 2002; unpublished data) and Kap95 (Allen et al., 2001; Pyhtila and Rexach, 2003), respectively. We predicted that swapping FG repeats to the opposite side of the pore would create dominant NPC alleles with exchanged specificity for transport factors and thus would reverse the FG gradient.

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
Related in: MedlinePlus