Structural and functional analysis of an essential nucleoporin heterotrimer on the cytoplasmic face of the nuclear pore complex

Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 09/2011; 108(40):16571-6. DOI: 10.1073/pnas.1112846108
Source: PubMed


So far, only a few of the interactions between the ≈ 30 nucleoporins comprising the modular structure of the nuclear pore complex have been defined at atomic resolution. Here we report the crystal structure, at 2.6 Å resolution, of a heterotrimeric complex, composed of fragments of three cytoplasmically oriented nucleoporins of yeast: Nup82, Nup116, and Nup159. Our data show that the Nup82 fragment, representing more than the N-terminal half of the molecule, folds into an extensively decorated, seven-bladed β-propeller that forms the centerpiece of this heterotrimeric complex and anchors both a C-terminal fragment of Nup116 and the C-terminal tail of Nup159. Binding between Nup116 and Nup82 is mutually reinforced via two loops, one emanating from the Nup82 β-propeller and the other one from the β-sandwich fold of Nup116, each contacting binding pockets in their counterparts. The Nup82-Nup159 interaction occurs through an amphipathic α-helix of Nup159, which is cradled in a large hydrophobic groove that is generated from several large surface decorations of the Nup82 β-propeller. Although Nup159 and Nup116 fragments bind to the Nup82 β-propeller in close vicinity, there are no direct contacts between them, consistent with the noncooperative binding that was detected biochemically. Extensive mutagenesis delineated hot-spot residues for these interactions. We also showed that the Nup82 β-propeller binds to other yeast Nup116 family members, Nup145N, Nup100 and to the mammalian homolog, Nup98. Notably, each of the three nucleoporins contains additional nuclear pore complex binding sites, distinct from those that were defined here in the heterotrimeric Nup82•Nup159•Nup116 complex.

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Available from: Erik W Debler, Mar 20, 2015
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    • "Therefore, after structural alignment (C  root-mean-square deviation < 3.5 Å), this model was used to build a heterodimer with the C-terminal Nup82  propeller. To generate a structural model of the Nup159–Nup82 heterodimer, the model of the C-terminal, -helical part of Nup159 and the x-ray structure of the C-terminal Nup82 -propeller (PDB ID: 3PBP; Yoshida et al., 2011) were superimposed with their overlapping counterpart of the Nup82–Nup159–Nup98 heterotrimeric x-ray structure (PDB ID: 3TKN; Stuwe et al., 2012). In the crystal structure, only 28 out of 39 residues of the construct are visible in the crystal structure, pointing to disordered termini. "
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    ABSTRACT: Nuclear pore complexes (NPCs) are huge assemblies formed from ∼30 different nucleoporins, typically organized in subcomplexes. One module, the conserved Nup82 complex at the cytoplasmic face of NPCs, is crucial to terminate mRNA export. To gain insight into the structure, assembly, and function of the cytoplasmic pore filaments, we reconstituted in yeast the Nup82-Nup159-Nsp1-Dyn2 complex, which was suitable for biochemical, biophysical, and electron microscopy analyses. Our integrative approach revealed that the yeast Nup82 complex forms an unusual asymmetric structure with a dimeric array of subunits. Based on all these data, we developed a three-dimensional structural model of the Nup82 complex that depicts how this module might be anchored to the NPC scaffold and concomitantly can interact with the soluble nucleocytoplasmic transport machinery. © 2015 Gaik et al.
    The Journal of Cell Biology 02/2015; 208(3):283-97. DOI:10.1083/jcb.201411003 · 9.83 Impact Factor
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    • "Nup98 proteins generated by these two mechanisms are identical in sequence and are thus expected to exhibit the same interactions with the NPC. In addition to Nup96, the autoproteolytic domain of Nup98 also binds the N-terminal domain of Nup88 (Griffis et al., 2003; Yoshida et al., 2011). Nup88 has recently been shown to reside on both sides of the NPC (Lussi et al., 2011). "
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    ABSTRACT: Nuclear pore complexes (NPCs) facilitate selective transport of macromolecules across the nuclear envelope in interphase eukaryotic cells. NPCs are composed of roughly 30 different proteins (nucleoporins) of which about one third are characterized by the presence of phenylalanine-glycine (FG) repeat domains that allow the association of soluble nuclear transport receptors with the NPC. Two types of FG (FG/FxFG and FG/GLFG) domains are found in nucleoporins and Nup98 is the sole vertebrate nucleoporin harboring the GLFG-type repeats. By immuno-electron microscopy using isolated nuclei from Xenopus oocytes we show here the localization of distinct domains of Nup98. We examined the localization of the C- and N-terminal domain of Nup98 by immunogold-labeling using domain-specific antibodies against Nup98 and by expressing epitope tagged versions of Nup98. Our studies revealed that anchorage of Nup98 to NPCs through its C-terminal autoproteolytic domain occurs in the center of the NPC, whereas its N-terminal GLFG domain is more flexible and is detected at multiple locations within the NPC. Additionally, we have confirmed the central localization of Nup98 within the NPC using super resolution structured illumination fluorescence microscopy (SIM) to position Nup98 domains relative to markers of cytoplasmic filaments and the nuclear basket. Our data support the notion that Nup98 is a major determinant of the permeability barrier of NPCs.
    Journal of Structural Biology 11/2011; 177(1):81-9. DOI:10.1016/j.jsb.2011.11.004 · 3.23 Impact Factor
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    ABSTRACT: The nuclear pore complex (NPC), embedded in the nuclear envelope, is a large, dynamic molecular assembly that facilitates exchange of macromolecules between the nucleus and the cytoplasm. The yeast NPC is an eightfold symmetric annular structure composed of ~456 polypeptide chains contributed by ~30 distinct proteins termed nucleoporins. Nup116, identified only in fungi, plays a central role in both protein import and mRNA export through the NPC. Nup116 is a modular protein with N-terminal "FG" repeats containing a Gle2p-binding sequence motif and a NPC targeting domain at its C-terminus. We report the crystal structure of the NPC targeting domain of Candida glabrata Nup116, consisting of residues 882-1034 [CgNup116(882-1034)], at 1.94 Å resolution. The X-ray structure of CgNup116(882-1034) is consistent with the molecular envelope determined in solution by small-angle X-ray scattering. Structural similarities of CgNup116(882-1034) with homologous domains from Saccharomyces cerevisiae Nup116, S. cerevisiae Nup145N, and human Nup98 are discussed.
    Proteins Structure Function and Bioinformatics 08/2012; 80(8):2110-6. DOI:10.1002/prot.24102 · 2.63 Impact Factor
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