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Structural and functional analysis of Nup133 domains reveals modular building blocks of the nuclear pore complex.

Berke IC, Boehmer T, Blobel G, Schwartz TU - J. Cell Biol. (2004)

Bottom Line: We show that human Nup133 contains two domains: a COOH-terminal domain responsible for its interaction with its subcomplex through Nup107; and an NH2-terminal domain whose crystal structure reveals a seven-bladed beta-propeller.Other beta-propellers are predicted in a third of all nucleoporins.These and several other repeat-based motifs appear to be major elements of nucleoporins, indicating a level of structural repetition that may conceptually simplify the assembly and disassembly of this huge protein complex.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, NY 10021, USA.

ABSTRACT
Nucleocytoplasmic transport occurs through nuclear pore complexes (NPCs) whose complex architecture is generated from a set of only approximately 30 proteins, termed nucleoporins. Here, we explore the domain structure of Nup133, a nucleoporin in a conserved NPC subcomplex that is crucial for NPC biogenesis and is believed to form part of the NPC scaffold. We show that human Nup133 contains two domains: a COOH-terminal domain responsible for its interaction with its subcomplex through Nup107; and an NH2-terminal domain whose crystal structure reveals a seven-bladed beta-propeller. The surface properties and conservation of the Nup133 beta-propeller suggest it may mediate multiple interactions with other proteins. Other beta-propellers are predicted in a third of all nucleoporins. These and several other repeat-based motifs appear to be major elements of nucleoporins, indicating a level of structural repetition that may conceptually simplify the assembly and disassembly of this huge protein complex.

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Domain structure of Nup133. (a) Two domains were predicted, with the NTD having helical and sheet content, and the CTD being all helical. The construct used for crystallization is boxed. (b) In vitro binding of hNup133 domains to recombinant GST-hNup107. Top and bottom panels show the bound and unbound fractions of various [35S]methionine-labeled Nup133 translation products incubated with recombinant GST-Nup107 immobilized on affinity resin. (c) Localization of EGFP-tagged hNup133 domains in HeLa cells. The CTD shows punctate rim staining that overlaps with the mAb414 signal. The NTD shows diffuse EGFP-signal, with a weak concentration at the nuclear rim.
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fig1: Domain structure of Nup133. (a) Two domains were predicted, with the NTD having helical and sheet content, and the CTD being all helical. The construct used for crystallization is boxed. (b) In vitro binding of hNup133 domains to recombinant GST-hNup107. Top and bottom panels show the bound and unbound fractions of various [35S]methionine-labeled Nup133 translation products incubated with recombinant GST-Nup107 immobilized on affinity resin. (c) Localization of EGFP-tagged hNup133 domains in HeLa cells. The CTD shows punctate rim staining that overlaps with the mAb414 signal. The NTD shows diffuse EGFP-signal, with a weak concentration at the nuclear rim.

Mentions: Examination of the 1,156-residue human Nup133 by secondary structure prediction, disordered region prediction, and sequence conservation among homologues identified two domains: an NH2-terminal α/β domain of ∼400 residues and an all helical CTD of ∼640 residues (Fig. 1 a). Reconstitution of recombinant yeast Nup84 subcomplex revealed that Nup133 is anchored to the subcomplex via its direct interaction with Nup84, the yeast homologue of Nup107 (Lutzmann et al., 2002). Accordingly, Nup133 failed to assemble into NPCs of vertebrate cells depleted of Nup107 (Boehmer et al., 2003). In vitro binding experiments, performed with recombinant GST-Nup107 and in vitro transcribed and translated Nup133 proteins, show that the Nup133 CTD binds Nup107 (Fig. 1 b). GFP-tagged hNup133 (502–1156) shows punctate nuclear rim staining consistent with integration into the Nup107-160 subcomplex and the NPC (Fig. 1 c).


Structural and functional analysis of Nup133 domains reveals modular building blocks of the nuclear pore complex.

Berke IC, Boehmer T, Blobel G, Schwartz TU - J. Cell Biol. (2004)

Domain structure of Nup133. (a) Two domains were predicted, with the NTD having helical and sheet content, and the CTD being all helical. The construct used for crystallization is boxed. (b) In vitro binding of hNup133 domains to recombinant GST-hNup107. Top and bottom panels show the bound and unbound fractions of various [35S]methionine-labeled Nup133 translation products incubated with recombinant GST-Nup107 immobilized on affinity resin. (c) Localization of EGFP-tagged hNup133 domains in HeLa cells. The CTD shows punctate rim staining that overlaps with the mAb414 signal. The NTD shows diffuse EGFP-signal, with a weak concentration at the nuclear rim.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172596&req=5

fig1: Domain structure of Nup133. (a) Two domains were predicted, with the NTD having helical and sheet content, and the CTD being all helical. The construct used for crystallization is boxed. (b) In vitro binding of hNup133 domains to recombinant GST-hNup107. Top and bottom panels show the bound and unbound fractions of various [35S]methionine-labeled Nup133 translation products incubated with recombinant GST-Nup107 immobilized on affinity resin. (c) Localization of EGFP-tagged hNup133 domains in HeLa cells. The CTD shows punctate rim staining that overlaps with the mAb414 signal. The NTD shows diffuse EGFP-signal, with a weak concentration at the nuclear rim.
Mentions: Examination of the 1,156-residue human Nup133 by secondary structure prediction, disordered region prediction, and sequence conservation among homologues identified two domains: an NH2-terminal α/β domain of ∼400 residues and an all helical CTD of ∼640 residues (Fig. 1 a). Reconstitution of recombinant yeast Nup84 subcomplex revealed that Nup133 is anchored to the subcomplex via its direct interaction with Nup84, the yeast homologue of Nup107 (Lutzmann et al., 2002). Accordingly, Nup133 failed to assemble into NPCs of vertebrate cells depleted of Nup107 (Boehmer et al., 2003). In vitro binding experiments, performed with recombinant GST-Nup107 and in vitro transcribed and translated Nup133 proteins, show that the Nup133 CTD binds Nup107 (Fig. 1 b). GFP-tagged hNup133 (502–1156) shows punctate nuclear rim staining consistent with integration into the Nup107-160 subcomplex and the NPC (Fig. 1 c).

Bottom Line: We show that human Nup133 contains two domains: a COOH-terminal domain responsible for its interaction with its subcomplex through Nup107; and an NH2-terminal domain whose crystal structure reveals a seven-bladed beta-propeller.Other beta-propellers are predicted in a third of all nucleoporins.These and several other repeat-based motifs appear to be major elements of nucleoporins, indicating a level of structural repetition that may conceptually simplify the assembly and disassembly of this huge protein complex.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, NY 10021, USA.

ABSTRACT
Nucleocytoplasmic transport occurs through nuclear pore complexes (NPCs) whose complex architecture is generated from a set of only approximately 30 proteins, termed nucleoporins. Here, we explore the domain structure of Nup133, a nucleoporin in a conserved NPC subcomplex that is crucial for NPC biogenesis and is believed to form part of the NPC scaffold. We show that human Nup133 contains two domains: a COOH-terminal domain responsible for its interaction with its subcomplex through Nup107; and an NH2-terminal domain whose crystal structure reveals a seven-bladed beta-propeller. The surface properties and conservation of the Nup133 beta-propeller suggest it may mediate multiple interactions with other proteins. Other beta-propellers are predicted in a third of all nucleoporins. These and several other repeat-based motifs appear to be major elements of nucleoporins, indicating a level of structural repetition that may conceptually simplify the assembly and disassembly of this huge protein complex.

Show MeSH