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Ordered Regions of Channel Nucleoporins Nup62, Nup54, and Nup58 Form Dynamic Complexes in Solution*

  • Alok Sharma
    Footnotes
    Affiliations
    Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, New York 10065
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  • Sozanne R. Solmaz
    Footnotes
    Affiliations
    Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, New York 10065
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  • Günter Blobel
    Correspondence
    An Investigator of the Howard Hughes Medical Institute. To whom correspondence may be addressed: Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY 10065. Tel.: 212-327-8096; Fax: 212-327-7880;
    Affiliations
    Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, New York 10065
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  • Ivo Melčák
    Correspondence
    To whom correspondence may be addressed: Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, 1230 York Ave., New York, NY 10065. Tel.: 212-327-8181; Fax: 212-327-7880;
    Affiliations
    Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, New York 10065
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  • Author Footnotes
    * The authors declare that they have no conflicts of interest with the contents of this article.
    1 Both authors contributed equally to this work.
    2 Present address: Dept. of Chemistry, State University of New York at Binghamton, P. O. Box 6000, Binghamton, NY, 13902.
Open AccessPublished:May 29, 2015DOI:https://doi.org/10.1074/jbc.M115.663500
      Three out of ∼30 nucleoporins, Nup62, Nup54, and Nup58, line the nuclear pore channel. These “channel” nucleoporins each contain an ordered region of ∼150–200 residues, which is predicted to be segmented into 3–4 α-helical regions of ∼40–80 residues. Notably, these segmentations are evolutionarily conserved between uni- and multicellular eukaryotes. Strikingly, the boundaries of these segments match our previously reported mapping and crystal data, which collectively identified two “cognate” segments of Nup54, each interacting with cognate segments, one in Nup58 and the other one in Nup62. Because Nup54 and Nup58 cognate segments form crystallographic hetero- or homo-oligomers, we proposed that these oligomers associate into inter-convertible “mid-plane” rings: a single large ring (40–50 nm diameter, consisting of eight hetero-dodecamers) or three small rings (10–20 nm diameter, each comprising eight homo-tetramers). Each “ring cycle” would recapitulate “dilation” and “constriction” of the nuclear pore complex's central transport channel. As for the Nup54·Nup62 interactome, it forms a 1:2 triple helix (“finger”), multiples of which project alternately up and down from mid-plane ring(s). Collectively, our previous crystal data suggested a copy number of 128, 64, and 32 for Nup62, Nup54, and Nup58, respectively, that is, a 4:2:1 stoichiometry. Here, we carried out solution analysis utilizing the entire ordered regions of Nup62, Nup54, and Nup58, and demonstrate that they form a dynamic “triple complex” that is heterogeneously formed from our previously characterized Nup54·Nup58 and Nup54·Nup62 interactomes. These data are consistent both with our crystal structure-deduced copy numbers and stoichiometries and also with our ring cycle model for structure and dynamics of the nuclear pore channel.

      Introduction

      The ∼30 distinct nucleoporins (nups)
      The abbreviations used are: nup
      nucleoporin
      NPC
      nuclear pore complex
      MALS
      multiangle light scattering
      MBP
      maltose-binding protein.
      building the nuclear pore complex (NPC) are organized into a gigantic protein ensemble estimated to be 120 MDa in mass in vertebrates (for review, see Ref.
      • Hoelz A.
      • Debler E.W.
      • Blobel G.
      The structure of the nuclear pore complex.
      ). However, an accurate copy number per NPC for each of the ∼30 nups (
      • Cronshaw J.M.
      • Krutchinsky A.N.
      • Zhang W.
      • Chait B.T.
      • Matunis M.J.
      Proteomic analysis of the mammalian nuclear pore complex.
      ,
      • Rout M.P.
      • Aitchison J.D.
      • Suprapto A.
      • Hjertaas K.
      • Zhao Y.
      • Chait B.T.
      The yeast nuclear pore complex: composition, architecture, and transport mechanism.
      ) has been difficult to determine experimentally as most procedures employ cell fractionation, where differential losses of nups, especially weakly attached ones, are likely to occur at the numerous theoretical plates of cell fractionation. Nevertheless, symmetry considerations provide important clues. Exhibiting an 8-fold rotational symmetry, each NPC contains at least eight (or multiples thereof) copies of a given nucleoporin. Moreover, as the central core of the NPC displays an additional 2-fold axis of symmetry (coincident with the NPC's mid-plane), the copy number for nucleoporins of the NPC core is at least 16 (or multiples thereof).
      A case in point for discrepancies in reported copy numbers and stoichiometries are the three “channel” nucleoporins, Nup62, Nup54, and Nup58 (Refs.
      • Cronshaw J.M.
      • Krutchinsky A.N.
      • Zhang W.
      • Chait B.T.
      • Matunis M.J.
      Proteomic analysis of the mammalian nuclear pore complex.
      and
      • Finlay D.R.
      • Meier E.
      • Bradley P.
      • Horecka J.
      • Forbes D.J.
      A complex of nuclear pore proteins required for pore function.
      ,
      • Buss F.
      • Stewart M.
      Macromolecular interactions in the nucleoporin p62 complex of rat nuclear pores: binding of nucleoporin p54 to the rod domain of p62.
      ,
      • Kita K.
      • Omata S.
      • Horigome T.
      Purification and characterization of a nuclear pore glycoprotein complex containing p62.
      ,
      • Ulrich A.
      • Partridge J.R.
      • Schwartz T.U.
      The stoichiometry of the nucleoporin 62 subcomplex of the nuclear pore in solution.
      ,
      • Guan T.
      • Müller S.
      • Klier G.
      • Panté N.
      • Blevitt J.M.
      • Haner M.
      • Paschal B.
      • Aebi U.
      • Gerace L.
      Structural analysis of the p62 complex, an assembly of O-linked glycoproteins that localizes near the central gated channel of the nuclear pore complex.
      ; see also Ref.
      • Solmaz S.R.
      • Chauhan R.
      • Blobel G.
      • Melčák I.
      Molecular architecture of the transport channel of the nuclear pore complex.
      ). Each of these three nups contains an ordered region of ∼150–200 residues in length (
      • Guan T.
      • Müller S.
      • Klier G.
      • Panté N.
      • Blevitt J.M.
      • Haner M.
      • Paschal B.
      • Aebi U.
      • Gerace L.
      Structural analysis of the p62 complex, an assembly of O-linked glycoproteins that localizes near the central gated channel of the nuclear pore complex.
      ,
      • Hu T.
      • Guan T.
      • Gerace L.
      Molecular and functional characterization of the p62 complex, an assembly of nuclear pore complex glycoproteins.
      ). However, this region is not contiguous, but is subdivided into 3–4 segments of ∼40–80 residues in length, each predicted to exhibit α-helical secondary structure. Remarkably, the topology of segmentation is evolutionarily highly conserved between channel nups of uni- and multicellular eukaryotes indicating functional relevance for the segmentation of each ordered region. Notably, our mapping and crystal studies on the three channel nups matched the boundaries of these segments (
      • Solmaz S.R.
      • Chauhan R.
      • Blobel G.
      • Melčák I.
      Molecular architecture of the transport channel of the nuclear pore complex.
      ,
      • Solmaz S.R.
      • Blobel G.
      • Melčák I.
      Ring cycle for dilating and constricting the nuclear pore.
      ,
      • Melčák I.
      • Hoelz A.
      • Blobel G.
      Structure of Nup58/45 suggests flexible nuclear pore diameter by intermolecular sliding.
      ). Collectively, these studies led us to propose a model for the dynamics and structure of the central channel of the NPC. The salient features of this model are mid-plane rings (consisting of “dilated” and “constricted” conformers of Nup54 and Nup58) with attached triple helices of Nup62 and Nup54 (“fingers”), projecting alternately to either the nucleoplasmic or the cytoplasmic side of the mid-plane rings (
      • Solmaz S.R.
      • Chauhan R.
      • Blobel G.
      • Melčák I.
      Molecular architecture of the transport channel of the nuclear pore complex.
      ,
      • Solmaz S.R.
      • Blobel G.
      • Melčák I.
      Ring cycle for dilating and constricting the nuclear pore.
      ).
      Our structure-informed data suggested a copy number for Nup62, Nup54, and Nup58 of 128, 64, and 32 molecules, respectively, that is, a 4:2:1 stoichiometry. However, a recent study by Ulrich et al. (
      • Ulrich A.
      • Partridge J.R.
      • Schwartz T.U.
      The stoichiometry of the nucleoporin 62 subcomplex of the nuclear pore in solution.
      ) concluded that the three channel nups associate in a 1:1:1 stoichiometry. Ulrich et al. (
      • Ulrich A.
      • Partridge J.R.
      • Schwartz T.U.
      The stoichiometry of the nucleoporin 62 subcomplex of the nuclear pore in solution.
      ) arrived at this conclusion based on analytical ultracentrifugation studies of recombinant fragments comprising the entire ordered region of each of the three channel nucleoporins. However, as these authors utilized only a single loading concentration instead of the multiple loading concentrations that are required for global modeling of multi-component systems to accurately determine equilibrium constants (
      • Vistica J.
      • Dam J.
      • Balbo A.
      • Yikilmaz E.
      • Mariuzza R.A.
      • Rouault T.A.
      • Schuck P.
      Sedimentation equilibrium analysis of protein interactions with global implicit mass conservation constraints and systematic noise decomposition.
      ,
      • Roark D.E.
      Sedimentation equilibrium techniques: multiple speed analyses and an overspeed procedure.
      ,
      • Lebowitz J.
      • Lewis M.S.
      • Schuck P.
      Modern analytical ultracentrifugation in protein science: a tutorial review.
      ,
      • Cole J.L.
      • Lary J.W.
      • Moody T.P.
      • Laue T.M.
      Analytical ultracentrifugation: sedimentation velocity and sedimentation equilibrium.
      ), their conclusions are flawed a priori. Despite these drawbacks, Ulrich et al. (
      • Ulrich A.
      • Partridge J.R.
      • Schwartz T.U.
      The stoichiometry of the nucleoporin 62 subcomplex of the nuclear pore in solution.
      ) went on to suggest that the biochemically and crystal structure-defined interactomes of segments of these ordered regions (
      • Solmaz S.R.
      • Chauhan R.
      • Blobel G.
      • Melčák I.
      Molecular architecture of the transport channel of the nuclear pore complex.
      ,
      • Solmaz S.R.
      • Blobel G.
      • Melčák I.
      Ring cycle for dilating and constricting the nuclear pore.
      ,
      • Melčák I.
      • Hoelz A.
      • Blobel G.
      Structure of Nup58/45 suggests flexible nuclear pore diameter by intermolecular sliding.
      ) are “non-canonical” in nature and therefore of doubtful physiological relevance.
      In this study, we report data on assembly and disassembly of complexes formed by the ordered region of each of the three channel nups. By employing several biochemical and biophysical approaches, we found that these fragments form dynamic assemblies of heterogeneous stoichiometries rather than forming complex(es) of a uniform 1:1:1 stoichiometry as proposed by Ulrich et al. (
      • Ulrich A.
      • Partridge J.R.
      • Schwartz T.U.
      The stoichiometry of the nucleoporin 62 subcomplex of the nuclear pore in solution.
      ). Importantly, we detected the previously characterized interacting domains of ordered segments of channel nups (
      • Solmaz S.R.
      • Chauhan R.
      • Blobel G.
      • Melčák I.
      Molecular architecture of the transport channel of the nuclear pore complex.
      ,
      • Solmaz S.R.
      • Blobel G.
      • Melčák I.
      Ring cycle for dilating and constricting the nuclear pore.
      ,
      • Melčák I.
      • Hoelz A.
      • Blobel G.
      Structure of Nup58/45 suggests flexible nuclear pore diameter by intermolecular sliding.
      ), which we refer to as interactomes, to be the principal contributors, even in the context of the heterogeneous and dynamic assemblies formed in solution by the entire ordered regions of the three channel nups. Hence, the previously detected interactomes (
      • Solmaz S.R.
      • Chauhan R.
      • Blobel G.
      • Melčák I.
      Molecular architecture of the transport channel of the nuclear pore complex.
      ,
      • Solmaz S.R.
      • Blobel G.
      • Melčák I.
      Ring cycle for dilating and constricting the nuclear pore.
      ,
      • Melčák I.
      • Hoelz A.
      • Blobel G.
      Structure of Nup58/45 suggests flexible nuclear pore diameter by intermolecular sliding.
      ) between segments of the ordered regions of the three channel nucleoporins are indeed “canonical.” The data reported here also provide further support for our “ring cycle” model for the molecular structure and dynamics of the central channel of the nuclear pore complex (
      • Solmaz S.R.
      • Chauhan R.
      • Blobel G.
      • Melčák I.
      Molecular architecture of the transport channel of the nuclear pore complex.
      ,
      • Solmaz S.R.
      • Blobel G.
      • Melčák I.
      Ring cycle for dilating and constricting the nuclear pore.
      ).

      Discussion

      Here, we report solution studies on a triple complex formed by the ∼150–200-residue-long ordered region of each of the three channel nucleoporins, Nup62, Nup54, and Nup58. We show that recombinant proteins (each representing the entire ordered region of the three channel nups) form a dynamic mixture of heterogeneous complexes. We biophysically and biochemically characterized this triple complex and employed truncated forms or cognate segments of the ordered region for probing assembly and disassembly of the triple complex. We find that our previously identified hetero-interactomes (
      • Solmaz S.R.
      • Chauhan R.
      • Blobel G.
      • Melčák I.
      Molecular architecture of the transport channel of the nuclear pore complex.
      ) formed by cognate segments of the ordered region of the three channel nups are indeed also the principal drivers for triple complex assembly in solution.
      Cognate segments of the ordered region of each of the channel nups have previously been identified and characterized by mapping and atomic resolution crystal structures, which led us to propose a model for the structure and dynamics of the central channel of the nuclear pore (
      • Solmaz S.R.
      • Chauhan R.
      • Blobel G.
      • Melčák I.
      Molecular architecture of the transport channel of the nuclear pore complex.
      ,
      • Solmaz S.R.
      • Blobel G.
      • Melčák I.
      Ring cycle for dilating and constricting the nuclear pore.
      ). Two salient structural features of this model are mid-plane rings and attached triple helices (fingers) that form cytoplasmic and nucleoplasmic entries to the mid-plane rings (Fig. 10). With regard to dynamics, we have proposed a ring cycle model, whereby conformers of a single large (dilated) ring convert to conformers of three small (constricted) rings, reflecting opened and closed states of the central channel of the nuclear pore (Fig. 10), with transport factors suggested to stabilize the open form of the central channel (
      • Solmaz S.R.
      • Chauhan R.
      • Blobel G.
      • Melčák I.
      Molecular architecture of the transport channel of the nuclear pore complex.
      ,
      • Solmaz S.R.
      • Blobel G.
      • Melčák I.
      Ring cycle for dilating and constricting the nuclear pore.
      ). Indeed, a recent study yielded data in support of the proposed regulation of the central transport channel. Transport factor binding to the Nup58 segment (extended C-terminally to contain the requisite phenylalanine-glycine repeat binding site for the transport factor) was shown to allosterically stabilize dilated Nup54·Nup58 conformers, at the expense of their constricted counterparts (
      • Koh J.
      • Blobel G.
      Allosteric regulation in gating the central channel of the nuclear pore complex.
      ).
      Figure thumbnail gr10
      FIGURE 10Piecing together crystal structures of channel nucleoporin segments into a model for a nuclear pore channel. A and B, schematic representation of inter-convertible dilated (A) and constricted (B) channel states. The path of segments of ordered regions of channel nups, Nup62 (gray), Nup54 (blue), and Nup58 (red), through the channel's two principal structural elements, mid-plane ring and attached fingers, is shown. The latter are projecting to nucleoplasm and cytoplasm. C and D, a single module (out of eight) is represented each for the dilated (C) or constricted (D) states of the channel. The “linker region” of Nup54 is indicated either by curved lines (A and B) or by dotted lines (C and D).
      Collectively, our studies led to crystal structure-informed copy numbers of 128, 64, and 32 molecules of Nup62, Nup54, and Nup58, respectively, amounting to a mass of 12.3 MDa for the central channel, roughly 10% of the estimated mass of the entire NPC (
      • Solmaz S.R.
      • Chauhan R.
      • Blobel G.
      • Melčák I.
      Molecular architecture of the transport channel of the nuclear pore complex.
      ). Our model also suggests that the hitherto unassigned segments of the ordered regions of the three channel nups provide binding sites for neighboring nups. Specifically, a segment located in the N-terminal part of the ordered region of Nup58 was proposed to anchor mid-plane rings to neighboring nucleoporins, and a C-terminal segment of the ordered region of Nup62 was proposed to link the base of the fingers to a neighboring nucleoporin. Although structural information for interactions of channel nups with neighboring nups is still missing, there is biochemical evidence for this arrangement (
      • Grandi P.
      • Schlaich N.
      • Tekotte H.
      • Hurt E.C.
      Functional interaction of Nic96p with a core nucleoporin complex consisting of Nsp1p, Nup49p and a novel protein Nup57p.
      ,
      • Bailer S.M.
      • Balduf C.
      • Hurt E.
      The Nsp1p carboxy-terminal domain is organized into functionally distinct coiled-coil regions required for assembly of nucleoporin subcomplexes and nucleocytoplasmic transport.
      ). Hence, the path of segments of the ordered regions of each of the three channel nucleoporins has been tentatively traced (
      • Solmaz S.R.
      • Blobel G.
      • Melčák I.
      Ring cycle for dilating and constricting the nuclear pore.
      ) and assigned to both the principal elements of the central channel (mid-plane ring and fingers) and putative binding sites in neighboring nucleoporins. Of the three channel nups, Nup54 stands out as it provides two cognate segments, one for forming the mid-plane ring and the other for forming the fingers, respectively, whereas only one segment each of Nup62 and two adjacent segments of Nup58 (Fig. 1D) contribute to the finger and mid-plane elements of the central channel. Notably, the structure-informed stoichiometry of 4:2:1 for Nup62, Nup54, and Nup58 has been supported by in vivo quantitative fluorescence intensity data from the fission yeast where endogenous nups were genomically replaced with their GFP-tagged versions (
      • Asakawa H.
      • Yang H.J.
      • Yamamoto T.G.
      • Ohtsuki C.
      • Chikashige Y.
      • Sakata-Sogawa K.
      • Tokunaga M.
      • Iwamoto M.
      • Hiraoka Y.
      • Haraguchi T.
      Characterization of nuclear pore complex components in fission yeast Schizosaccharomyces pombe.
      ).
      Our present solution studies on the triple complex are in support of our proposals for tracing the segments of the ordered region (Fig. 10) (
      • Solmaz S.R.
      • Blobel G.
      • Melčák I.
      Ring cycle for dilating and constricting the nuclear pore.
      ). We show that the principal interactions of the triple complex are established by the Nup54·Nup58 interactome (responsible for mid-plane ring) and the Nup54·Nup62 interactome (building the fingers) (FIGURE 9, FIGURE 10). Accordingly, the hitherto “unsaturated” segments of Nup58 and Nup62 that potentially link the structural elements of the central channel to neighboring nucleoporins do not participate in assembly or in disassembly of the triple complex.
      Collectively, these studies on the triple complex are entirely consistent with our previous mapping and crystal studies (
      • Solmaz S.R.
      • Chauhan R.
      • Blobel G.
      • Melčák I.
      Molecular architecture of the transport channel of the nuclear pore complex.
      ,
      • Solmaz S.R.
      • Blobel G.
      • Melčák I.
      Ring cycle for dilating and constricting the nuclear pore.
      ,
      • Melčák I.
      • Hoelz A.
      • Blobel G.
      Structure of Nup58/45 suggests flexible nuclear pore diameter by intermolecular sliding.
      ) and validate our approach not only to gain insight into the dynamics and structure of the central channel of the NPC, but also to probe the structure and dynamics of surrounding nucleoporins, which are likely to undergo structural and dynamic changes accompanying and accommodating the huge diameter changes proposed for the central channel of the NPC (
      • Solmaz S.R.
      • Chauhan R.
      • Blobel G.
      • Melčák I.
      Molecular architecture of the transport channel of the nuclear pore complex.
      ,
      • Solmaz S.R.
      • Blobel G.
      • Melčák I.
      Ring cycle for dilating and constricting the nuclear pore.
      ).

      Author Contributions

      A. S, S. R. S., G. B., and I. M. designed experiments, analyzed data, and wrote the paper. A. S., S. R. S., and I. M. performed experiments.

      Acknowledgments

      Technical assistance from Amrita Venkateswaran, Ndeye Fatou Gueye, and Hyung Bum Kim is acknowledged. We thank Elias Coutavas, Richard Wing, Thalia Farazi, Erik Debler, Nimisha Singh, and Richa Jaiswal for critical reading of the manuscript and David King for mass spectrometry analysis.

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