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Murray Stewart - One of the best experts on this subject based on the ideXlab platform.
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Structural requirements for the UBA domain of the mRNA export receptor Mex67 to bind its specific targets, the transcription elongation THO complex component Hpr1 and Nucleoporin FXFG repeats.
Journal of Biological Chemistry, 2009Co-Authors: Maria Hobeika, Murray Stewart, David Neuhaus, Christoph Brockmann, Florian Gruessing, Gilles Divita, Catherine DargemontAbstract:The Ubiquitin-Associated domain (UBA) domain of the principal Saccharomyces cerevisiae mRNA nuclear export factor, Mex67, can bind both nuclear pore protein (Nucleoporin) FG repeats and Hpr1, a component of the TREX:THO complex that functions to link transcription and export. Using Fluorescence Resonance Energy Transfer-based assays, we show here that Hpr1 and the FG repeats interact with overlapping binding sites on the Mex67 UBA domain. We present the solution structure of the Mex67 UBA domain (UBA-Mex67) complexed with a FxFG Nucleoporin peptide and define residues engaged in the interaction and those involved in the FxFG-induced conformational change. We show by NMR titration that the binding of Hpr1 produces analogous changes in chemical shifts in similar regions of the UBA domain. Together the data presented here indicate that both Hpr1 and FxFG Nucleoporins may bind in a similar way to the UBA-Mex67 domain. However, whereas binding of Hpr1 allows UBA-Mex67 to interact with tetra-ubiquitin, the complex between UBA-Mex67 and FxFG is unable to bind mono- or tetra-ubiquitin, suggesting that both substrate binding and also the nature of the substrate may influence the affinity of the UBA-Mex67 domain for ubiquitin.
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structural basis for the high affinity binding of Nucleoporin nup1p to the saccharomyces cerevisiae importin β homologue kap95p
Journal of Molecular Biology, 2005Co-Authors: Sai Man Liu, Murray StewartAbstract:Macromolecules are transported across the nuclear envelope most frequently by karyopherin/importin-beta superfamily members that are constructed from HEAT repeats. Transport of Kap95p (yeast importin-beta), the principal carrier for protein import, through nuclear pore complexes is facilitated by interactions with Nucleoporins containing FG repeats. However, Nup1p interacts more strongly with Kap95p than other FG-Nucleoporins. To establish the basis of this increased affinity, we determined the structure of Kap95p complexed with Nup1p residues 963-1076 that contain the high-affinity Kap95p binding site. Nup1p binds Kap95p at three sites between the outer A-helices of HEAT repeats 5, 6, 7 and 8. At each site, phenylalanine residues from Nup1p are buried in hydrophobic depressions between adjacent HEAT repeats. Although the Nup1p and generic FG-Nucleoporin binding sites on Kap95p overlap, Nup1p binding differs markedly and has contributions from additional hydrophobic residues, together with interactions generated by the intimate contact of the linker between Nup1 residues 977-987 with Kap95p. The length and composition of this linker is crucial and suggests how differences in affinity for Kap95p both between and within FG-Nucleoporins arise.
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Structural basis for the interaction between the Tap/NXF1 UBA domain and FG Nucleoporins at 1A resolution.
Journal of molecular biology, 2003Co-Authors: Richard P. Grant, David Neuhaus, Murray StewartAbstract:The mRNA nuclear export function of Tap/NXF1 requires interactions with nuclear pore proteins (Nucleoporins) that contain characteristic Phe-Gly repeats based on FG, GLFG or FxFG cores separated by hydrophilic linkers. FG-Nucleoporins bind the two most C-terminal domains of Tap, which have NTF2 and UBA folds, respectively. We used a combination of NMR and X-ray crystallography to define the interaction interface between Tap UBA and FxFG Nucleoporins and show that it involves primarily the two aromatic rings of the FxFG core that bind in a hydrophobic surface depression centred on Tap Cys588. NMR evidence indicates that the same depression mediates the binding of GLFG Nucleoporins, which we confirmed by demonstrating competition between the two classes of repeat for binding to Tap UBA. Moreover, modification of Cys588 reduced the binding of Tap UBA to both GLFG and FxFG Nucleoporins as well as to nuclear envelopes. These data underscore the central role of the conserved FG-Nucleoporin repeat cores in binding to Tap UBA and indicate that functional differences between different classes of Nucleoporins depend more on their spatial distribution in nuclear pores than on their binding to different sites on Tap UBA.
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Structural basis for the interaction between NTF2 and Nucleoporin FxFG repeats.
The EMBO journal, 2002Co-Authors: Richard Bayliss, Anita H. Corbett, Sara W. Leung, Rosanna P. Baker, B. Booth Quimby, Murray StewartAbstract:Interactions with Nucleoporins containing FxFG‐repeat cores are crucial for the nuclear import of RanGDP mediated by nuclear transport factor 2 (NTF2). We describe here the 1.9 A resolution crystal structure of yeast NTF2‐N77Y bound to a FxFG‐Nucleoporin core, which provides a basis for understanding this interaction and its role in nuclear trafficking. The two identical FxFG binding sites on the dimeric molecule are formed by residues from each chain of NTF2. Engineered mutants at the interaction interface reduce the binding of NTF2 to nuclear pores and cause reduced growth rates and Ran mislocalization when substituted for the wild‐type protein in yeast. Comparison with the crystal structure of FG‐Nucleoporin cores bound to importin‐β and TAP/p15 identified a number of common features of their binding sites. The structure of the binding interfaces on these transport factors provides a rationale for the specificity of their interactions with Nucleoporins that, combined with their weak binding constants, facilitates rapid translocation through NPCs during nuclear trafficking.
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Structural basis for the interaction between FxFG Nucleoporin repeats and importin-beta in nuclear trafficking.
Cell, 2000Co-Authors: Richard Bayliss, Trevor Littlewood, Murray StewartAbstract:We describe the crystal structure of a complex between importin-beta residues 1-442 (Ib442) and five FxFG Nucleoporin repeats from Nsp1p. Nucleoporin FxFG cores bind on the convex face of Ib442 to a primary site between the A helices of HEAT repeats 5 and 6, and to a secondary site between HEAT repeats 6 and 7. Mutations at importin-beta Ile178 in the primary FxFG binding site reduce both binding and nuclear protein import, providing direct evidence for the functional significance of the importin-beta-FxFG interaction. The FxFG binding sites on importin-beta do not overlap with the RanGTP binding site. Instead, RanGTP may release importin-beta from FxFG Nucleoporins by generating a conformational change that alters the structure of the FxFG binding site.
Thomas U. Schwartz - One of the best experts on this subject based on the ideXlab platform.
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Structural and functional studies of Nup107/Nup133 interaction and its implications for the architecture of the nuclear pore complex.
Molecular cell, 2008Co-Authors: Thomas Boehmer, Sandra Jeudy, Ian C. Berke, Thomas U. SchwartzAbstract:Summary Nuclear pore complexes (NPCs) are 40–60 MDa protein assemblies embedded in the nuclear envelope of eukaryotic cells. NPCs exclusively mediate all transport between cytoplasm and nucleus. The Nucleoporins that build the NPC are arranged in a stable core of module-like subcomplexes with eight-fold rotational symmetry. To gain insight into the intricate assembly of the NPC, we have solved the crystal structure of a protein complex between two Nucleoporins, human Nup107 and Nup133. Both proteins form elongated structures that interact tightly via a compact interface in tail-to-tail fashion. Additional experiments using structure-guided mutants show that Nup107 is the critical anchor for Nup133 to the NPC, positioning Nup133 at the periphery of the NPC. The significant topological differences between Nup107 and Nup133 suggest that α-helical Nucleoporin domains of the NPC scaffold fall in different classes and fulfill largely nonredundant functions.
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structural and functional studies of nup107 nup133 interaction and its implications for the architecture of the nuclear pore complex
Molecular Cell, 2008Co-Authors: Thomas Boehmer, Sandra Jeudy, Ian C. Berke, Thomas U. SchwartzAbstract:Summary Nuclear pore complexes (NPCs) are 40–60 MDa protein assemblies embedded in the nuclear envelope of eukaryotic cells. NPCs exclusively mediate all transport between cytoplasm and nucleus. The Nucleoporins that build the NPC are arranged in a stable core of module-like subcomplexes with eight-fold rotational symmetry. To gain insight into the intricate assembly of the NPC, we have solved the crystal structure of a protein complex between two Nucleoporins, human Nup107 and Nup133. Both proteins form elongated structures that interact tightly via a compact interface in tail-to-tail fashion. Additional experiments using structure-guided mutants show that Nup107 is the critical anchor for Nup133 to the NPC, positioning Nup133 at the periphery of the NPC. The significant topological differences between Nup107 and Nup133 suggest that α-helical Nucleoporin domains of the NPC scaffold fall in different classes and fulfill largely nonredundant functions.
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crystal structure of Nucleoporin nic96 reveals a novel intricate helical domain architecture
Journal of Biological Chemistry, 2007Co-Authors: Sandra Jeudy, Thomas U. SchwartzAbstract:Abstract The nuclear pore complex (NPC) is an elaborate protein machine that mediates macromolecular transport across the nuclear envelope in all eukaryotes. The NPC is formed by Nucleoporins that assemble in multiple copies around an 8-fold symmetry axis. Homology modeling suggests that most architectural Nucleoporins are composed of simple β-propeller and α-helical repeat domains. Here we present the crystal structure of Nic96, the Nup93 homolog in Saccharomyces cerevisiae, one of the major components of the NPC. This is the first structure of an α-helical Nucleoporin domain. The protein folds into an elongated, mostly α-helical structure. Characteristically, non-canonical architectural features define the Nic96 structure. Sequence conservation among Nup93 homologs across all eukaryotes strongly suggests that the distinct topology is evolutionarily well maintained. We propose that the unique Nic96/Nup93 fold has a conserved function in all eukaryotes.
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Crystal structure of Nucleoporin Nic96 reveals a novel, intricate helical domain architecture.
The Journal of biological chemistry, 2007Co-Authors: Sandra Jeudy, Thomas U. SchwartzAbstract:The nuclear pore complex (NPC) is an elaborate protein machine that mediates macromolecular transport across the nuclear envelope in all eukaryotes. The NPC is formed by Nucleoporins that assemble in multiple copies around an 8-fold symmetry axis. Homology modeling suggests that most architectural Nucleoporins are composed of simple beta-propeller and alpha-helical repeat domains. Here we present the crystal structure of Nic96, the Nup93 homolog in Saccharomyces cerevisiae, one of the major components of the NPC. This is the first structure of an alpha-helical Nucleoporin domain. The protein folds into an elongated, mostly alpha-helical structure. Characteristically, non-canonical architectural features define the Nic96 structure. Sequence conservation among Nup93 homologs across all eukaryotes strongly suggests that the distinct topology is evolutionarily well maintained. We propose that the unique Nic96/Nup93 fold has a conserved function in all eukaryotes.
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Structural and functional analysis of Nup133 domains reveals modular building blocks of the nuclear pore complex
The Journal of cell biology, 2004Co-Authors: Ian C. Berke, Gunter Blobel, Thomas Boehmer, Thomas U. SchwartzAbstract:Nucleocytoplasmic transport occurs through nuclear pore complexes (NPCs) whose complex architecture is generated from a set of only ∼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 β-propeller. The surface properties and conservation of the Nup133 β-propeller suggest it may mediate multiple interactions with other proteins. Other β-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.
Gunter Blobel - One of the best experts on this subject based on the ideXlab platform.
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structure and nucleic acid binding activity of the Nucleoporin nup157
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Bartlomiej J Blus, Nina Z Jankovic, Gunter BlobelAbstract:The nuclear pore complex (NPC) is a multiprotein gating complex that allows for bidirectional transport across the nuclear membrane. A key feature of the NPC is a central transport channel that can undergo regulated diameter changes, thus enabling the trafficking of cargo of various sizes. Surrounding this channel is a group of proteins, named “adaptor” Nucleoporins, which are envisioned to accommodate and orchestrate these structural changes. Here we show the crystal structure of a fragment of an adaptor Nucleoporin, Nup157, which forms a compact C-shaped architecture. Notably, Nup157 contains a positively charged surface consistent with its nucleic acid binding capacity. Furthermore, the predicted hinge regions in Nup157 suggest its flexibility in agreement with the plastic nature of the NPC.
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Structural and functional analysis of Nup133 domains reveals modular building blocks of the nuclear pore complex
The Journal of cell biology, 2004Co-Authors: Ian C. Berke, Gunter Blobel, Thomas Boehmer, Thomas U. SchwartzAbstract:Nucleocytoplasmic transport occurs through nuclear pore complexes (NPCs) whose complex architecture is generated from a set of only ∼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 β-propeller. The surface properties and conservation of the Nup133 β-propeller suggest it may mediate multiple interactions with other proteins. Other β-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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a conserved biogenesis pathway for Nucleoporins proteolytic processing of a 186 kilodalton precursor generates nup98 and the novel Nucleoporin nup96
Journal of Cell Biology, 1999Co-Authors: Beatriz M A Fontoura, Gunter Blobel, Michael J MatunisAbstract:The mammalian nuclear pore complex (NPC) is comprised of ∼50 unique proteins, collectively known as Nucleoporins. Through fractionation of rat liver nuclei, we have isolated >30 potentially novel Nucleoporins and have begun a systematic characterization of these proteins. Here, we present the characterization of Nup96, a novel Nucleoporin with a predicted molecular mass of 96 kD. Nup96 is generated through an unusual biogenesis pathway that involves synthesis of a 186-kD precursor protein. Proteolytic cleavage of the precursor yields two Nucleoporins: Nup98, a previously characterized GLFG-repeat containing Nucleoporin, and Nup96. Mutational and functional analyses demonstrate that both the Nup98-Nup96 precursor and the previously characterized Nup98 (synthesized independently from an alternatively spliced mRNA) are proteolytically cleaved in vivo. This biogenesis pathway for Nup98 and Nup96 is evolutionarily conserved, as the putative Saccharomyces cerevisiae homologues, N-Nup145p and C-Nup145p, are also produced through proteolytic cleavage of a precursor protein. Using immunoelectron microscopy, Nup96 was localized to the nucleoplasmic side of the NPC, at or near the nucleoplasmic basket. The correct targeting of both Nup96 and Nup98 to the nucleoplasmic side of the NPC was found to be dependent on proteolytic cleavage, suggesting that the cleavage process may regulate NPC assembly. Finally, by biochemical fractionation, a complex containing Nup96, Nup107, and at least two Sec13- related proteins was identified, revealing that a major sub-complex of the NPC is conserved between yeast and mammals.
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A Conserved Biogenesis Pathway for Nucleoporins: Proteolytic Processing of a 186-Kilodalton Precursor Generates Nup98 and the Novel Nucleoporin, Nup96
The Journal of cell biology, 1999Co-Authors: Beatriz M A Fontoura, Gunter Blobel, Michael J MatunisAbstract:The mammalian nuclear pore complex (NPC) is comprised of approximately 50 unique proteins, collectively known as Nucleoporins. Through fractionation of rat liver nuclei, we have isolated >30 potentially novel Nucleoporins and have begun a systematic characterization of these proteins. Here, we present the characterization of Nup96, a novel Nucleoporin with a predicted molecular mass of 96 kD. Nup96 is generated through an unusual biogenesis pathway that involves synthesis of a 186-kD precursor protein. Proteolytic cleavage of the precursor yields two Nucleoporins: Nup98, a previously characterized GLFG-repeat containing Nucleoporin, and Nup96. Mutational and functional analyses demonstrate that both the Nup98-Nup96 precursor and the previously characterized Nup98 (synthesized independently from an alternatively spliced mRNA) are proteolytically cleaved in vivo. This biogenesis pathway for Nup98 and Nup96 is evolutionarily conserved, as the putative Saccharomyces cerevisiae homologues, N-Nup145p and C-Nup145p, are also produced through proteolytic cleavage of a precursor protein. Using immunoelectron microscopy, Nup96 was localized to the nucleoplasmic side of the NPC, at or near the nucleoplasmic basket. The correct targeting of both Nup96 and Nup98 to the nucleoplasmic side of the NPC was found to be dependent on proteolytic cleavage, suggesting that the cleavage process may regulate NPC assembly. Finally, by biochemical fractionation, a complex containing Nup96, Nup107, and at least two Sec13- related proteins was identified, revealing that a major sub-complex of the NPC is conserved between yeast and mammals.
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role of the nuclear transport factor p10 in nuclear import
Science, 1996Co-Authors: Ulf Nehrbass, Gunter BlobelAbstract:The nuclear import factor p10 was cloned from Saccharomyces cerevisiae and found to be essential. The protein p10 can bind directly to several peptide repeat-containing Nucleoporins. It also binds to the guanosine triphosphatase (GTPase) Ran in its guanosine diphosphate (GDP)-bound form and to karyopherin β. Assembly of the karyopherin heterodimer on immobilized Nucleoporin yielded cooperative binding of p10 and Ran-GDP. Addition of GTP to this pentameric complex led to dissociation of karyopherin α, presumably via in situ formation of Ran-GTP from Ran-GDP. Thus, p10 appears to coordinate the Ran-dependent association and dissociation reactions underlying nuclear import.
Michael Rexach - One of the best experts on this subject based on the ideXlab platform.
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intramolecular cohesion of coils mediated by phenylalanine glycine motifs in the natively unfolded domain of a Nucleoporin
PLOS Computational Biology, 2008Co-Authors: Viswanathan V Krishnan, Samir S. Patel, Daniel P. Denning, Edmond Y Lau, Justin Yamada, Michael E Colvin, Michael RexachAbstract:The nuclear pore complex (NPC) provides the sole aqueous conduit for macromolecular exchange between the nucleus and the cytoplasm of cells. Its diffusion conduit contains a size-selective gate formed by a family of NPC proteins that feature large, natively unfolded domains with phenylalanine–glycine repeats (FG domains). These domains of Nucleoporins play key roles in establishing the NPC permeability barrier, but little is known about their dynamic structure. Here we used molecular modeling and biophysical techniques to characterize the dynamic ensemble of structures of a representative FG domain from the yeast Nucleoporin Nup116. The results showed that its FG motifs function as intramolecular cohesion elements that impart order to the FG domain and compact its ensemble of structures into native premolten globular configurations. At the NPC, the FG motifs of Nucleoporins may exert this cohesive effect intermolecularly as well as intramolecularly to form a malleable yet cohesive quaternary structure composed of highly flexible polypeptide chains. Dynamic shifts in the equilibrium or competition between intra- and intermolecular FG motif interactions could facilitate the rapid and reversible structural transitions at the NPC conduit needed to accommodate passing karyopherin–cargo complexes of various shapes and sizes while simultaneously maintaining a size-selective gate against protein diffusion.
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Natively Unfolded Nucleoporins Gate Protein Diffusion across the Nuclear Pore Complex
Cell, 2007Co-Authors: Samir S. Patel, Brian J. Belmont, Joshua Sante, Michael RexachAbstract:Nuclear pore complexes (NPCs) form aqueous conduits in the nuclear envelope and gate the diffusion of large proteins between the cytoplasm and nucleoplasm. NPC proteins (Nucleoporins) that contain phenylalanine-glycine motifs in filamentous, natively unfolded domains (FG domains) line the diffusion conduit of the NPC, but their role in the size-selective barrier is unclear. We show that deletion of individual FG domains in yeast relaxes the NPC permeability barrier. At the molecular level, the FG domains of five Nucleoporins anchored at the NPC center form a cohesive meshwork of filaments through hydrophobic interactions, which involve phenylalanines in FG motifs and are dispersed by aliphatic alcohols. In contrast, the FG domains of four peripherally anchored Nucleoporins are generally noncohesive. The results support a two-gate model of NPC architecture featuring a central diffusion gate formed by a meshwork of cohesive FG Nucleoporin filaments and a peripheral gate formed by repulsive FG Nucleoporin filaments.
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Increased nuclear envelope permeability and Pep4p-dependent degradation of Nucleoporins during hydrogen peroxide-induced cell death
FEMS yeast research, 2005Co-Authors: D. Adam Mason, Michael Rexach, Nataliya Shulga, Satyen Undavai, Elisa Ferrando-may, David S. GoldfarbAbstract:The death of yeast treated with hydrogen peroxide (H(2)O(2)) shares a number of morphological and biochemical features with mammalian apoptosis. In this study, we report that the permeability of yeast nuclear envelopes (NE) increased during H(2)O(2)-induced cell death. Similar phenomena have been observed during apoptosis in mammalian tissue culture cells. Increased NE permeability in yeast was temporally correlated with an increase in the production of reactive-oxygen species (ROS). Later, after ROS levels began to decline and viability was lost, specific nuclear pore complex (NPC) proteins (Nucleoporins) were degraded. Although caspases are responsible for the degradation of mammalian Nucleoporins during apoptosis, the deletion of the metacaspase gene YCA1 had no effect on the stability of yeast Nucleoporins. Instead, Pep4p, a vacuolar cathepsin D homolog, was responsible for the proteolysis of Nucleoporins. Coincident with Nucleoporin degradation, a Pep4p-EGFP reporter migrated out of the vacuole in H(2)O(2)-treated cells. We conclude that increases in ROS and NPC permeability occur relatively early during H(2)O(2)-induced cell death. Later, Pep4p migrates out of vacuoles and degrades Nucleoporins after the cells are effectively dead.
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The Saccharomyces cerevisiae Nucleoporin Nup2p is a natively unfolded protein.
The Journal of biological chemistry, 2002Co-Authors: Daniel P. Denning, Samir S. Patel, Vladimir N. Uversky, Anthony L. Fink, Michael RexachAbstract:Little is known about the structure of the individual Nucleoporins that form eukaryotic nuclear pore complexes (NPCs). We report here in vitro physical and structural characterizations of a full-length Nucleoporin, the Saccharomyces cerevisiae protein Nup2p. Analyses of the Nup2p structure by far-UV circular dichroism (CD) spectroscopy, Fourier transform infrared (FTIR) spectroscopy, protease sensitivity, gel filtration, and sedimentation velocity experiments indicate that Nup2p is a "natively unfolded protein," belonging to a class of proteins that exhibit little secondary structure, high flexibility, and low compactness. Nup2p possesses a very large Stokes radius (79 A) in gel filtration columns, sediments slowly in sucrose gradients as a 2.9 S particle, and is highly sensitive to proteolytic digestion by proteinase K; these characteristics suggest a structure of low compactness and high flexibility. Spectral analyses (CD and FTIR spectroscopy) provide additional evidence that Nup2p contains extensive regions of structural disorder with comparatively small contributions of ordered secondary structure. We address the possible significance of natively unfolded Nucleoporins in the mechanics of nucleocytoplasmic trafficking across NPCs.
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Proteomic Analysis of Nucleoporin Interacting Proteins
The Journal of biological chemistry, 2001Co-Authors: Nadia P.c. Allen, Lan Huang, Al Burlingame, Michael RexachAbstract:The Saccharomyces cerevisiae nuclear pore complex is a supramolecular assembly of 30 Nucleoporins that cooperatively facilitate nucleocytoplasmic transport. Thirteen Nucleoporins that contain FG peptide repeats (FG Nups) are proposed to function as stepping stones in karyopherin-mediated transport pathways. Here, protein interactions that occur at individual FG Nups were sampled using immobilized Nucleoporins and yeast extracts. We find that many proteins bind to FG Nups in highly reproducible patterns. Among 135 proteins identified by mass spectrometry, most were karyopherins and Nucleoporins. The PSFG Nucleoporin Nup42p and the GLFG Nucleoporins Nup49p, Nup57p, Nup100p, and Nup116p exhibited generic interactions with karyopherins; each bound 6--10 different karyopherin betas, including importins as well as exportins. Unexpectedly, the same Nups also captured the hexameric Nup84p complex and Nup2p. In contrast, the FXFG Nucleoporins Nup1p, Nup2p, and Nup60p were more selective and captured mostly the Kap95p.Kap60p heterodimer. When the concentration of Gsp1p-GTP was elevated in the extracts to mimic the nucleoplasmic environment, the patterns of interacting proteins changed; exportins exhibited enhanced binding to FG Nups, and importins exhibited reduced binding. The results demonstrate a global role for Gsp1p-GTP on karyopherin-Nucleoporin interactions and provide a rudimentary map of the routes that karyopherins take as they cross the nuclear pore complex.
Michael J Matunis - One of the best experts on this subject based on the ideXlab platform.
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a conserved biogenesis pathway for Nucleoporins proteolytic processing of a 186 kilodalton precursor generates nup98 and the novel Nucleoporin nup96
Journal of Cell Biology, 1999Co-Authors: Beatriz M A Fontoura, Gunter Blobel, Michael J MatunisAbstract:The mammalian nuclear pore complex (NPC) is comprised of ∼50 unique proteins, collectively known as Nucleoporins. Through fractionation of rat liver nuclei, we have isolated >30 potentially novel Nucleoporins and have begun a systematic characterization of these proteins. Here, we present the characterization of Nup96, a novel Nucleoporin with a predicted molecular mass of 96 kD. Nup96 is generated through an unusual biogenesis pathway that involves synthesis of a 186-kD precursor protein. Proteolytic cleavage of the precursor yields two Nucleoporins: Nup98, a previously characterized GLFG-repeat containing Nucleoporin, and Nup96. Mutational and functional analyses demonstrate that both the Nup98-Nup96 precursor and the previously characterized Nup98 (synthesized independently from an alternatively spliced mRNA) are proteolytically cleaved in vivo. This biogenesis pathway for Nup98 and Nup96 is evolutionarily conserved, as the putative Saccharomyces cerevisiae homologues, N-Nup145p and C-Nup145p, are also produced through proteolytic cleavage of a precursor protein. Using immunoelectron microscopy, Nup96 was localized to the nucleoplasmic side of the NPC, at or near the nucleoplasmic basket. The correct targeting of both Nup96 and Nup98 to the nucleoplasmic side of the NPC was found to be dependent on proteolytic cleavage, suggesting that the cleavage process may regulate NPC assembly. Finally, by biochemical fractionation, a complex containing Nup96, Nup107, and at least two Sec13- related proteins was identified, revealing that a major sub-complex of the NPC is conserved between yeast and mammals.
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A Conserved Biogenesis Pathway for Nucleoporins: Proteolytic Processing of a 186-Kilodalton Precursor Generates Nup98 and the Novel Nucleoporin, Nup96
The Journal of cell biology, 1999Co-Authors: Beatriz M A Fontoura, Gunter Blobel, Michael J MatunisAbstract:The mammalian nuclear pore complex (NPC) is comprised of approximately 50 unique proteins, collectively known as Nucleoporins. Through fractionation of rat liver nuclei, we have isolated >30 potentially novel Nucleoporins and have begun a systematic characterization of these proteins. Here, we present the characterization of Nup96, a novel Nucleoporin with a predicted molecular mass of 96 kD. Nup96 is generated through an unusual biogenesis pathway that involves synthesis of a 186-kD precursor protein. Proteolytic cleavage of the precursor yields two Nucleoporins: Nup98, a previously characterized GLFG-repeat containing Nucleoporin, and Nup96. Mutational and functional analyses demonstrate that both the Nup98-Nup96 precursor and the previously characterized Nup98 (synthesized independently from an alternatively spliced mRNA) are proteolytically cleaved in vivo. This biogenesis pathway for Nup98 and Nup96 is evolutionarily conserved, as the putative Saccharomyces cerevisiae homologues, N-Nup145p and C-Nup145p, are also produced through proteolytic cleavage of a precursor protein. Using immunoelectron microscopy, Nup96 was localized to the nucleoplasmic side of the NPC, at or near the nucleoplasmic basket. The correct targeting of both Nup96 and Nup98 to the nucleoplasmic side of the NPC was found to be dependent on proteolytic cleavage, suggesting that the cleavage process may regulate NPC assembly. Finally, by biochemical fractionation, a complex containing Nup96, Nup107, and at least two Sec13- related proteins was identified, revealing that a major sub-complex of the NPC is conserved between yeast and mammals.
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nup358 a cytoplasmically exposed Nucleoporin with peptide repeats ran gtp binding sites zinc fingers a cyclophilin a homologous domain and a leucine rich region
Journal of Biological Chemistry, 1995Co-Authors: Michael J Matunis, Gunter Blobel, Doris Kraemer, Elias CoutavasAbstract:Abstract The Ras-related nuclear protein, Ran, has been implicated in nuclear transport. By screening a HeLa cell expression library with Ran-GTP and sequencing overlapping cDNA clones, we have obtained the derived primary structure of a protein with a calculated molecular mass of 358 kDa. Using antibodies raised against an expressed segment of this protein, we obtained punctate nuclear surface staining by immunofluorescence microscopy that is characteristic for Nucleoporins. Electron microscopy of immunogold-decorated rat liver nuclear envelopes sublocalized the 358-kDa protein at (or near) the tip of the cytoplasmic fibers of the nuclear pore complex (NPC). In agreement with current convention, this protein was therefore termed Nup358 (for Nucleoporin of 358 kDa). Nup358 contains a leucine-rich region, four potential Ran binding sites (i.e. Ran binding protein 1 homologous domains) flanked by Nucleoporin-characteristic FXFG or FG repeats, eight zinc finger motifs, and a C-terminal cyclophilin A homologous domain. Consistent with the location of Nup358 at the cytoplasmic fibers of the NPC, we found decoration with Ran-gold at only the cytoplasmic side of the NPC. Thus, Nup358 is the first Nucleoporin shown to contain binding sites for two of three soluble nuclear transport factors so far isolated, namely karyopherin and Ran-GTP.
