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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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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.
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fig2: 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.

Mentions: Solution binding assays and pull-down experiments from whole cell extract have shown that transport receptor–cargo complexes have the highest binding affinity for Nups located nearest to their destination compartment. For example, importin β and its yeast orthologue Kap95 have a strong preference for Nups that are in the nuclear basket versus those localized to the cytoplasmic NPC surface or within the central channel (Allen et al., 2001; Ben-Efraim and Gerace, 2001; Pyhtila and Rexach, 2003). Conversely, Nup159 is the major NPC-binding partner of the export factor Xpo1/Crm1 within the NPC (Allen et al., 2002; unpublished data). Because biochemical and structural studies have established that the interaction between nuclear transport factors and Nups is directly mediated by the FG repeats, we predicted that swapping the FG domains between Nup1 and Nup159 would change their binding preference for Xpo1 and Kap95. To test this possibility, Xpo1 and Kap95 were fused to a carboxy-terminal ZZ tag and immunoprecipitated to assess their association with the Nup1 and Nup159 FG alleles (Fig. 2).


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)

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.
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Related In: Results  -  Collection

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fig2: 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.
Mentions: Solution binding assays and pull-down experiments from whole cell extract have shown that transport receptor–cargo complexes have the highest binding affinity for Nups located nearest to their destination compartment. For example, importin β and its yeast orthologue Kap95 have a strong preference for Nups that are in the nuclear basket versus those localized to the cytoplasmic NPC surface or within the central channel (Allen et al., 2001; Ben-Efraim and Gerace, 2001; Pyhtila and Rexach, 2003). Conversely, Nup159 is the major NPC-binding partner of the export factor Xpo1/Crm1 within the NPC (Allen et al., 2002; unpublished data). Because biochemical and structural studies have established that the interaction between nuclear transport factors and Nups is directly mediated by the FG repeats, we predicted that swapping the FG domains between Nup1 and Nup159 would change their binding preference for Xpo1 and Kap95. To test this possibility, Xpo1 and Kap95 were fused to a carboxy-terminal ZZ tag and immunoprecipitated to assess their association with the Nup1 and Nup159 FG alleles (Fig. 2).

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