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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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Transplantation of the eDID-FG repeat domain from Nsp1 and Nup1 onto Nup159 causes a loss of toxicity toward Dyn2 expression. (A) Schematic drawing of the involved FG repeat Nups, the transplantation of the indicated eDID-labeled FGNsp1 repeat domain onto Nup57, and the eDID-labeled FGNsp1 or FGNup1 repeat domain onto Nup159. C-domain, C-terminal domain. (B) To analyze the effect of Dyn2 expression, strains were transformed with an empty plasmid or pGAL-DYN2. The indicated cells were plated on SDC (glucose) and SGC (galactose) plates, and growth was analyzed after 2 and 3 d, respectively. (C) Poly(A)+ RNA export was analyzed after 3-h Dyn2 induction by in situ hybridization using a Cy3-labeled oligo d(T) probe. (D) Subcellular localization of pGAL-DYN2-GFP was analyzed in the NUP159ΔDID-eDID-FGNsp1 Δdyn2 and NUP159ΔDID-eDID-FGNup1 Δdyn2 strain after 30-min galactose induction. Bars, 5 µm.
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fig4: Transplantation of the eDID-FG repeat domain from Nsp1 and Nup1 onto Nup159 causes a loss of toxicity toward Dyn2 expression. (A) Schematic drawing of the involved FG repeat Nups, the transplantation of the indicated eDID-labeled FGNsp1 repeat domain onto Nup57, and the eDID-labeled FGNsp1 or FGNup1 repeat domain onto Nup159. C-domain, C-terminal domain. (B) To analyze the effect of Dyn2 expression, strains were transformed with an empty plasmid or pGAL-DYN2. The indicated cells were plated on SDC (glucose) and SGC (galactose) plates, and growth was analyzed after 2 and 3 d, respectively. (C) Poly(A)+ RNA export was analyzed after 3-h Dyn2 induction by in situ hybridization using a Cy3-labeled oligo d(T) probe. (D) Subcellular localization of pGAL-DYN2-GFP was analyzed in the NUP159ΔDID-eDID-FGNsp1 Δdyn2 and NUP159ΔDID-eDID-FGNup1 Δdyn2 strain after 30-min galactose induction. Bars, 5 µm.

Mentions: The data so far did not allow discrimination between whether the FG repeat domain modified with Dyn2 blocks nucleocytoplasmic transport because of the type of FG repeats or the topological location within the NPC scaffold. To distinguish between these possibilities, we sought to change the localization of eDID-FG repeat domains by genetic engineering. Hence, we replaced the FG repeat domain of Nup159 (putative “nontoxic” location) or Nup57 (putative “toxic” location) with the eDID-FG domain of Nsp1 (Fig. 4 A). Both chimera, Nup57-eDID-FGNsp1 and Nup159ΔDID-eDID-FGNsp1, were functional and complemented the respective mutants nup57Δ and nup159Δ (Fig. 4 B). Interestingly, the eDID-FGNsp1 attached to Nup57 still yielded a robust growth and mRNA export defect upon Dyn2 expression. However, when the eDID-FGNsp1 or the eDID-FGNup1 was transplanted onto Nup159, neither defective mRNA export nor growth inhibition was observed (Fig. 4, B and C; and Fig. S1 E). Affinity purification of Nup82-TAP from strain NUP159ΔDID-eDID-FGNsp1 showed that Dyn2 was recruited to this NPC module of the cytoplasmic pore filaments upon GAL::DYN2 expression (not depicted), which could also be confirmed by GFP-Dyn2 location experiments (Fig. 4 D). These data indicate separate roles of the unique Nsp1 FG repeat domain, which is part of two distinct NPC subcomplexes. Accordingly, the peripheral FG repeat domains of Nup159 and Nsp1 as part of the Nup82 complex may protrude into the cytoplasm to be used for recruitment of nuclear import receptors or termination of nuclear mRNA export (Stelter et al., 2007). Whereas the chemically identical other Nsp1 FG repeat protruding from the Nsp1–Nup49–Nup57 complex is crucially involved in lining the central transport channel to generate the permeability barrier.


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)

Transplantation of the eDID-FG repeat domain from Nsp1 and Nup1 onto Nup159 causes a loss of toxicity toward Dyn2 expression. (A) Schematic drawing of the involved FG repeat Nups, the transplantation of the indicated eDID-labeled FGNsp1 repeat domain onto Nup57, and the eDID-labeled FGNsp1 or FGNup1 repeat domain onto Nup159. C-domain, C-terminal domain. (B) To analyze the effect of Dyn2 expression, strains were transformed with an empty plasmid or pGAL-DYN2. The indicated cells were plated on SDC (glucose) and SGC (galactose) plates, and growth was analyzed after 2 and 3 d, respectively. (C) Poly(A)+ RNA export was analyzed after 3-h Dyn2 induction by in situ hybridization using a Cy3-labeled oligo d(T) probe. (D) Subcellular localization of pGAL-DYN2-GFP was analyzed in the NUP159ΔDID-eDID-FGNsp1 Δdyn2 and NUP159ΔDID-eDID-FGNup1 Δdyn2 strain after 30-min galactose induction. Bars, 5 µm.
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fig4: Transplantation of the eDID-FG repeat domain from Nsp1 and Nup1 onto Nup159 causes a loss of toxicity toward Dyn2 expression. (A) Schematic drawing of the involved FG repeat Nups, the transplantation of the indicated eDID-labeled FGNsp1 repeat domain onto Nup57, and the eDID-labeled FGNsp1 or FGNup1 repeat domain onto Nup159. C-domain, C-terminal domain. (B) To analyze the effect of Dyn2 expression, strains were transformed with an empty plasmid or pGAL-DYN2. The indicated cells were plated on SDC (glucose) and SGC (galactose) plates, and growth was analyzed after 2 and 3 d, respectively. (C) Poly(A)+ RNA export was analyzed after 3-h Dyn2 induction by in situ hybridization using a Cy3-labeled oligo d(T) probe. (D) Subcellular localization of pGAL-DYN2-GFP was analyzed in the NUP159ΔDID-eDID-FGNsp1 Δdyn2 and NUP159ΔDID-eDID-FGNup1 Δdyn2 strain after 30-min galactose induction. Bars, 5 µm.
Mentions: The data so far did not allow discrimination between whether the FG repeat domain modified with Dyn2 blocks nucleocytoplasmic transport because of the type of FG repeats or the topological location within the NPC scaffold. To distinguish between these possibilities, we sought to change the localization of eDID-FG repeat domains by genetic engineering. Hence, we replaced the FG repeat domain of Nup159 (putative “nontoxic” location) or Nup57 (putative “toxic” location) with the eDID-FG domain of Nsp1 (Fig. 4 A). Both chimera, Nup57-eDID-FGNsp1 and Nup159ΔDID-eDID-FGNsp1, were functional and complemented the respective mutants nup57Δ and nup159Δ (Fig. 4 B). Interestingly, the eDID-FGNsp1 attached to Nup57 still yielded a robust growth and mRNA export defect upon Dyn2 expression. However, when the eDID-FGNsp1 or the eDID-FGNup1 was transplanted onto Nup159, neither defective mRNA export nor growth inhibition was observed (Fig. 4, B and C; and Fig. S1 E). Affinity purification of Nup82-TAP from strain NUP159ΔDID-eDID-FGNsp1 showed that Dyn2 was recruited to this NPC module of the cytoplasmic pore filaments upon GAL::DYN2 expression (not depicted), which could also be confirmed by GFP-Dyn2 location experiments (Fig. 4 D). These data indicate separate roles of the unique Nsp1 FG repeat domain, which is part of two distinct NPC subcomplexes. Accordingly, the peripheral FG repeat domains of Nup159 and Nsp1 as part of the Nup82 complex may protrude into the cytoplasm to be used for recruitment of nuclear import receptors or termination of nuclear mRNA export (Stelter et al., 2007). Whereas the chemically identical other Nsp1 FG repeat protruding from the Nsp1–Nup49–Nup57 complex is crucially involved in lining the central transport channel to generate the permeability barrier.

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