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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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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.
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fig3: 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.

Mentions: To address the functional consequence of altering the asymmetric FG repeats, we first examined Kap95-mediated protein import using a monomeric red fluorescent protein (RFP; Campbell et al., 2002) reporter fused to the classical NLS (cNLS). When expressed in WT cells, the cNLS-RFP cargo localized exclusively to the nucleus, outlined by Nup1-GFP staining (Fig. 3 A). Identical results were obtained for each of the FG-swap and -deletion strains (Fig. 3, A and B).


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)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172579&req=5

fig3: 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.
Mentions: To address the functional consequence of altering the asymmetric FG repeats, we first examined Kap95-mediated protein import using a monomeric red fluorescent protein (RFP; Campbell et al., 2002) reporter fused to the classical NLS (cNLS). When expressed in WT cells, the cNLS-RFP cargo localized exclusively to the nucleus, outlined by Nup1-GFP staining (Fig. 3 A). Identical results were obtained for each of the FG-swap and -deletion strains (Fig. 3, A and B).

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