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Probing the nucleoporin FG repeat network defines structural and functional features of the nuclear pore complex.

Stelter P, Kunze R, Fischer J, Hurt E - J. Cell Biol. (2011)

Bottom Line: Unraveling the organization of the FG repeat meshwork that forms the active transport channel of the nuclear pore complex (NPC) is key to understanding the mechanism of nucleocytoplasmic transport.In this paper, we develop a tool to probe the FG repeat network in living cells by modifying FG nucleoporins (Nups) with a binding motif (engineered dynein light chain-interacting domain) that can drag several copies of an interfering protein, Dyn2, into the FG network to plug the pore and stop nucleocytoplasmic transport.Our method allows us to specifically probe FG Nups in vivo, which provides insight into the organization and function of the NPC transport channel.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biochemie-Zentrum der Universität Heidelberg, D-69120 Heidelberg, Germany.

ABSTRACT
Unraveling the organization of the FG repeat meshwork that forms the active transport channel of the nuclear pore complex (NPC) is key to understanding the mechanism of nucleocytoplasmic transport. In this paper, we develop a tool to probe the FG repeat network in living cells by modifying FG nucleoporins (Nups) with a binding motif (engineered dynein light chain-interacting domain) that can drag several copies of an interfering protein, Dyn2, into the FG network to plug the pore and stop nucleocytoplasmic transport. Our method allows us to specifically probe FG Nups in vivo, which provides insight into the organization and function of the NPC transport channel.

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Recruitment of Dyn2 to eDID-modified FG repeat domains of FG Nups at the NPC can cause a toxic phenotype. (A) Schematic drawing of the FG repeat Nups tested in this study: Nup159 with its natural DID, Nup116, Nsp1, Nup49, Nup57, Nup2, and Nup1 and position of the various FG repeat motifs (FG, GLFG, and FxFG; according to Strawn et al., 2004). Also indicated is the eDID insertion site within the corresponding FG repeat domain after Cre-induced recombination (Nup159ΔDID-eDID[624; i.e., insertion at amino acid position 624], Nup159ΔDID-eDID(2)[904], Nsp1-eDID[275], Nsp1-eDID(2)[2], Nup49-eDID[81], Nup57-eDID[71], Nup2-eDID[407], Nup1-eDID[717], and Nup116-eDID[197]) and recruitment of six Dyn2 molecules (violet) to the eDID platform. The topological position of the various FG Nups within the NPC scaffold is also drawn. (B) Growth of dyn2Δ strains that carry the wild-type NUP in a dyn2- background (dyn2Δ) or the indicated eDID-labeled FG repeat Nup, transformed with either an empty pGAL plasmid (empty plasmid) or a plasmid containing pGAL-DYN2. pGAL-DYN2 was repressed in glucose medium and induced in galactose medium. Growth was analyzed on SDC-Leu (glucose) or SGC-Leu (galactose) plates after 2 and 3 d, respectively. (C) Subcellular localization of pGAL-DYN2-GFP after 30-min galactose induction in NUP159ΔDID dyn2Δ NUP-eDID strains. NUP159 dyn2Δ + pGAL-DYN2-GFP and NUP159ΔDID dyn2Δ + pGAL-DYN2-GFP strains served as positive and negative controls, respectively. Fluorescence microscopy and Nomarski micrographs of representative cells are shown. Bar, 5 µm. (D) TAP of Nup57-TAP from strain dyn2Δ NSP1-eDID (lane 3) or dyn2Δ NSP1-eDID + GAL::DYN2 (lane 2) after 3-h galactose induction. Nup57-TAP affinity purified from wild-type + GAL::DYN2 (lane 1) cells served as a control after 3 h galactose induction. Calmodulin beads and whole-cell lysates (homogenates [HOM], lanes 4–6) were boiled in sample buffer and analyzed by SDS-PAGE and Coomassie staining or Western blotting using anti-Dyn2 antibodies. CBP, calmodulin-binding peptide.
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fig1: Recruitment of Dyn2 to eDID-modified FG repeat domains of FG Nups at the NPC can cause a toxic phenotype. (A) Schematic drawing of the FG repeat Nups tested in this study: Nup159 with its natural DID, Nup116, Nsp1, Nup49, Nup57, Nup2, and Nup1 and position of the various FG repeat motifs (FG, GLFG, and FxFG; according to Strawn et al., 2004). Also indicated is the eDID insertion site within the corresponding FG repeat domain after Cre-induced recombination (Nup159ΔDID-eDID[624; i.e., insertion at amino acid position 624], Nup159ΔDID-eDID(2)[904], Nsp1-eDID[275], Nsp1-eDID(2)[2], Nup49-eDID[81], Nup57-eDID[71], Nup2-eDID[407], Nup1-eDID[717], and Nup116-eDID[197]) and recruitment of six Dyn2 molecules (violet) to the eDID platform. The topological position of the various FG Nups within the NPC scaffold is also drawn. (B) Growth of dyn2Δ strains that carry the wild-type NUP in a dyn2- background (dyn2Δ) or the indicated eDID-labeled FG repeat Nup, transformed with either an empty pGAL plasmid (empty plasmid) or a plasmid containing pGAL-DYN2. pGAL-DYN2 was repressed in glucose medium and induced in galactose medium. Growth was analyzed on SDC-Leu (glucose) or SGC-Leu (galactose) plates after 2 and 3 d, respectively. (C) Subcellular localization of pGAL-DYN2-GFP after 30-min galactose induction in NUP159ΔDID dyn2Δ NUP-eDID strains. NUP159 dyn2Δ + pGAL-DYN2-GFP and NUP159ΔDID dyn2Δ + pGAL-DYN2-GFP strains served as positive and negative controls, respectively. Fluorescence microscopy and Nomarski micrographs of representative cells are shown. Bar, 5 µm. (D) TAP of Nup57-TAP from strain dyn2Δ NSP1-eDID (lane 3) or dyn2Δ NSP1-eDID + GAL::DYN2 (lane 2) after 3-h galactose induction. Nup57-TAP affinity purified from wild-type + GAL::DYN2 (lane 1) cells served as a control after 3 h galactose induction. Calmodulin beads and whole-cell lysates (homogenates [HOM], lanes 4–6) were boiled in sample buffer and analyzed by SDS-PAGE and Coomassie staining or Western blotting using anti-Dyn2 antibodies. CBP, calmodulin-binding peptide.

Mentions: We sought to develop a tool for probing individual FG repeat nucleoporins in vivo to gain insight into the organization and function of the FG repeat network. Previously, we showed that under physiological conditions, Dyn2 is recruited to Nup159, a subunit of the Nup82–Nsp1–Nup159 complex located at the cytoplasmic pore filaments (Stelter et al., 2007). Specifically, Dyn2 is bound to a Dyn2 interaction motif (dynein light chain–interacting domain [DID]) present between the FG and coiled-coil domain of Nup159, generating a 20-nm elongated structure that could contribute to the formation of the cytoplasmic pore filaments (Fig. 1 A). Moreover, the Nup82 complex dimerizes via the Nup159DID in a Dyn2-dependent manner, which facilitates NPC assembly. Dyn2 forms only a small 20-kD dimer and, when depleted or overexpressed, in a wild-type background, does not significantly influence growth or nucleocytoplasmic transport. These characteristics, therefore, make Dyn2, which should overcome the permeability barrier of the NPC, a promising tool to probe the FG repeat network in vivo. Hence, we inserted an engineered DID (eDID) composed of six consecutive Dyn2 binding motifs (derived from Pac11; see also Flemming et al., 2010) into the FG repeat domain of different FG Nups to test for consequences in NPC function. The selected FG Nups were Nup159, Nsp1, Nup49, Nup57, Nup2, Nup1, and Nup116 (Fig. 1 A and Fig. S1 A), which are either representatives of a discrete topological location in the NPC or are part of distinct NPC modules. Subsequently, we tested whether these eDID-FG Nups can recruit dynein light chain (Dyn2) upon GAL-DYN2 induction.


Probing the nucleoporin FG repeat network defines structural and functional features of the nuclear pore complex.

Stelter P, Kunze R, Fischer J, Hurt E - J. Cell Biol. (2011)

Recruitment of Dyn2 to eDID-modified FG repeat domains of FG Nups at the NPC can cause a toxic phenotype. (A) Schematic drawing of the FG repeat Nups tested in this study: Nup159 with its natural DID, Nup116, Nsp1, Nup49, Nup57, Nup2, and Nup1 and position of the various FG repeat motifs (FG, GLFG, and FxFG; according to Strawn et al., 2004). Also indicated is the eDID insertion site within the corresponding FG repeat domain after Cre-induced recombination (Nup159ΔDID-eDID[624; i.e., insertion at amino acid position 624], Nup159ΔDID-eDID(2)[904], Nsp1-eDID[275], Nsp1-eDID(2)[2], Nup49-eDID[81], Nup57-eDID[71], Nup2-eDID[407], Nup1-eDID[717], and Nup116-eDID[197]) and recruitment of six Dyn2 molecules (violet) to the eDID platform. The topological position of the various FG Nups within the NPC scaffold is also drawn. (B) Growth of dyn2Δ strains that carry the wild-type NUP in a dyn2- background (dyn2Δ) or the indicated eDID-labeled FG repeat Nup, transformed with either an empty pGAL plasmid (empty plasmid) or a plasmid containing pGAL-DYN2. pGAL-DYN2 was repressed in glucose medium and induced in galactose medium. Growth was analyzed on SDC-Leu (glucose) or SGC-Leu (galactose) plates after 2 and 3 d, respectively. (C) Subcellular localization of pGAL-DYN2-GFP after 30-min galactose induction in NUP159ΔDID dyn2Δ NUP-eDID strains. NUP159 dyn2Δ + pGAL-DYN2-GFP and NUP159ΔDID dyn2Δ + pGAL-DYN2-GFP strains served as positive and negative controls, respectively. Fluorescence microscopy and Nomarski micrographs of representative cells are shown. Bar, 5 µm. (D) TAP of Nup57-TAP from strain dyn2Δ NSP1-eDID (lane 3) or dyn2Δ NSP1-eDID + GAL::DYN2 (lane 2) after 3-h galactose induction. Nup57-TAP affinity purified from wild-type + GAL::DYN2 (lane 1) cells served as a control after 3 h galactose induction. Calmodulin beads and whole-cell lysates (homogenates [HOM], lanes 4–6) were boiled in sample buffer and analyzed by SDS-PAGE and Coomassie staining or Western blotting using anti-Dyn2 antibodies. CBP, calmodulin-binding peptide.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3198172&req=5

fig1: Recruitment of Dyn2 to eDID-modified FG repeat domains of FG Nups at the NPC can cause a toxic phenotype. (A) Schematic drawing of the FG repeat Nups tested in this study: Nup159 with its natural DID, Nup116, Nsp1, Nup49, Nup57, Nup2, and Nup1 and position of the various FG repeat motifs (FG, GLFG, and FxFG; according to Strawn et al., 2004). Also indicated is the eDID insertion site within the corresponding FG repeat domain after Cre-induced recombination (Nup159ΔDID-eDID[624; i.e., insertion at amino acid position 624], Nup159ΔDID-eDID(2)[904], Nsp1-eDID[275], Nsp1-eDID(2)[2], Nup49-eDID[81], Nup57-eDID[71], Nup2-eDID[407], Nup1-eDID[717], and Nup116-eDID[197]) and recruitment of six Dyn2 molecules (violet) to the eDID platform. The topological position of the various FG Nups within the NPC scaffold is also drawn. (B) Growth of dyn2Δ strains that carry the wild-type NUP in a dyn2- background (dyn2Δ) or the indicated eDID-labeled FG repeat Nup, transformed with either an empty pGAL plasmid (empty plasmid) or a plasmid containing pGAL-DYN2. pGAL-DYN2 was repressed in glucose medium and induced in galactose medium. Growth was analyzed on SDC-Leu (glucose) or SGC-Leu (galactose) plates after 2 and 3 d, respectively. (C) Subcellular localization of pGAL-DYN2-GFP after 30-min galactose induction in NUP159ΔDID dyn2Δ NUP-eDID strains. NUP159 dyn2Δ + pGAL-DYN2-GFP and NUP159ΔDID dyn2Δ + pGAL-DYN2-GFP strains served as positive and negative controls, respectively. Fluorescence microscopy and Nomarski micrographs of representative cells are shown. Bar, 5 µm. (D) TAP of Nup57-TAP from strain dyn2Δ NSP1-eDID (lane 3) or dyn2Δ NSP1-eDID + GAL::DYN2 (lane 2) after 3-h galactose induction. Nup57-TAP affinity purified from wild-type + GAL::DYN2 (lane 1) cells served as a control after 3 h galactose induction. Calmodulin beads and whole-cell lysates (homogenates [HOM], lanes 4–6) were boiled in sample buffer and analyzed by SDS-PAGE and Coomassie staining or Western blotting using anti-Dyn2 antibodies. CBP, calmodulin-binding peptide.
Mentions: We sought to develop a tool for probing individual FG repeat nucleoporins in vivo to gain insight into the organization and function of the FG repeat network. Previously, we showed that under physiological conditions, Dyn2 is recruited to Nup159, a subunit of the Nup82–Nsp1–Nup159 complex located at the cytoplasmic pore filaments (Stelter et al., 2007). Specifically, Dyn2 is bound to a Dyn2 interaction motif (dynein light chain–interacting domain [DID]) present between the FG and coiled-coil domain of Nup159, generating a 20-nm elongated structure that could contribute to the formation of the cytoplasmic pore filaments (Fig. 1 A). Moreover, the Nup82 complex dimerizes via the Nup159DID in a Dyn2-dependent manner, which facilitates NPC assembly. Dyn2 forms only a small 20-kD dimer and, when depleted or overexpressed, in a wild-type background, does not significantly influence growth or nucleocytoplasmic transport. These characteristics, therefore, make Dyn2, which should overcome the permeability barrier of the NPC, a promising tool to probe the FG repeat network in vivo. Hence, we inserted an engineered DID (eDID) composed of six consecutive Dyn2 binding motifs (derived from Pac11; see also Flemming et al., 2010) into the FG repeat domain of different FG Nups to test for consequences in NPC function. The selected FG Nups were Nup159, Nsp1, Nup49, Nup57, Nup2, Nup1, and Nup116 (Fig. 1 A and Fig. S1 A), which are either representatives of a discrete topological location in the NPC or are part of distinct NPC modules. Subsequently, we tested whether these eDID-FG Nups can recruit dynein light chain (Dyn2) upon GAL-DYN2 induction.

Bottom Line: Unraveling the organization of the FG repeat meshwork that forms the active transport channel of the nuclear pore complex (NPC) is key to understanding the mechanism of nucleocytoplasmic transport.In this paper, we develop a tool to probe the FG repeat network in living cells by modifying FG nucleoporins (Nups) with a binding motif (engineered dynein light chain-interacting domain) that can drag several copies of an interfering protein, Dyn2, into the FG network to plug the pore and stop nucleocytoplasmic transport.Our method allows us to specifically probe FG Nups in vivo, which provides insight into the organization and function of the NPC transport channel.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biochemie-Zentrum der Universität Heidelberg, D-69120 Heidelberg, Germany.

ABSTRACT
Unraveling the organization of the FG repeat meshwork that forms the active transport channel of the nuclear pore complex (NPC) is key to understanding the mechanism of nucleocytoplasmic transport. In this paper, we develop a tool to probe the FG repeat network in living cells by modifying FG nucleoporins (Nups) with a binding motif (engineered dynein light chain-interacting domain) that can drag several copies of an interfering protein, Dyn2, into the FG network to plug the pore and stop nucleocytoplasmic transport. Our method allows us to specifically probe FG Nups in vivo, which provides insight into the organization and function of the NPC transport channel.

Show MeSH
Related in: MedlinePlus