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Thomas U. Schwartz - One of the best experts on this subject based on the ideXlab platform.
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Yeast Nup84-NUP133 complex structure details flexibility and reveals conservation of the membrane anchoring ALPS motif.
Nature communications, 2020Co-Authors: Sarah A Nordeen, Daniel L Turman, Thomas U. SchwartzAbstract:The hallmark of the eukaryotic cell is the complex endomembrane system that compartmentalizes cellular functions. Transport into and out of the nucleus occurs through the nuclear pore complex (NPC). The heptameric Nup84 or Y complex is an essential scaffolding component of the NPC. Here we report two nanobody-bound structures: the full-length Nup84-NUP133 C-terminal domain complex and the NUP133 N-terminal domain, both from S. cerevisiae. Together with previously published structures, this work enables the structural description of the entire 575 kDa Y complex from one species. The structure of Nup84-NUP133CTD details the high flexibility of this dimeric unit of the Y complex. Further, the NUP133NTD contains a structurally conserved amphipathic lipid packing sensor motif, confirmed by liposome interaction studies. The presented structures reveal important details about the function of the Y complex that affect our understanding of NPC structure and assembly.
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Structure of the yeast Nup84-NUP133 complex details flexibility and reveals universal conservation of the membrane anchoring ALPS motif
2020Co-Authors: Sarah A Nordeen, Daniel L Turman, Thomas U. SchwartzAbstract:Abstract The hallmark of the eukaryotic cell is the complex endomembrane system that compartmentalizes cellular functions. Transport into and out of the nucleus, occurs through the Nuclear Pore Complex (NPC). The heptameric Nup84 or Y complex is an essential scaffolding component of the NPC. Here we report two nanobody-bound structures: the full-length Nup84-NUP133 C-terminal domain complex and the NUP133 N-terminal domain, both from S. cerevisiae. Together with previously published structures, this work enables the structural description of the entire 575 kDa Y complex, from one species. The structure of Nup84-Nup1 33CTD details the high flexibility of this dimeric unit of the Y complex. Further, the NUP133NTD contains a structurally conserved amphipathic lipid packing sensor (ALPS) motif, confirmed by liposome interaction studies. The new structures reveal important details about the function of the Y complex that affect our understanding of NPC structure and assembly.
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architectural nucleoporins nup157 170 and NUP133 are structurally related and descend from a second ancestral element
Journal of Biological Chemistry, 2009Co-Authors: James R.r. Whittle, Thomas U. SchwartzAbstract:Abstract The nuclear pore complex (NPC) constitutes one of the largest protein assemblies in the eukaryotic cell and forms the exclusive gateway to the nucleus. The stable, ∼15–20-MDa scaffold ring of the NPC is built from two multiprotein complexes arranged around a central 8-fold axis. Here we present crystal structures of two large architectural units, yNup170979–1502 and hNup107658–925·hNUP133517–1156, each a constituent of one of the two multiprotein complexes. Conservation of domain arrangement and of tertiary structure suggests that Nup157/170 and NUP133 derived from a common ancestor. Together with the previously established ancestral coatomer element (ACE1), these two elements constitute the major α-helical building blocks of the NPC scaffold and define its branched, lattice-like architecture, similar to vesicle coats like COPII. We hypothesize that the extant NPC evolved early during eukaryotic evolution from a rudimentary structure composed of several identical copies of a few ancestral elements, later diversified and specified by gene duplication.
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Architectural Nucleoporins Nup157/170 and NUP133 Are Structurally Related and Descend from a Second Ancestral Element
Journal of Biological Chemistry, 2009Co-Authors: James R.r. Whittle, Thomas U. SchwartzAbstract:Abstract The nuclear pore complex (NPC) constitutes one of the largest protein assemblies in the eukaryotic cell and forms the exclusive gateway to the nucleus. The stable, ∼15–20-MDa scaffold ring of the NPC is built from two multiprotein complexes arranged around a central 8-fold axis. Here we present crystal structures of two large architectural units, yNup170979–1502 and hNup107658–925·hNUP133517–1156, each a constituent of one of the two multiprotein complexes. Conservation of domain arrangement and of tertiary structure suggests that Nup157/170 and NUP133 derived from a common ancestor. Together with the previously established ancestral coatomer element (ACE1), these two elements constitute the major α-helical building blocks of the NPC scaffold and define its branched, lattice-like architecture, similar to vesicle coats like COPII. We hypothesize that the extant NPC evolved early during eukaryotic evolution from a rudimentary structure composed of several identical copies of a few ancestral elements, later diversified and specified by gene duplication.
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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.
Valérie Doye - One of the best experts on this subject based on the ideXlab platform.
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Regulation of Cenp-F localization to nuclear pores and kinetochores.
Cell cycle (Georgetown Tex.), 2018Co-Authors: Alessandro Berto, Valérie DoyeAbstract:In metazoans, the assembly of kinetochores on centrometric chromatin and the dismantling of nuclear pore complexes are processes that have to be tightly coordinated to ensure the proper assembly of the mitotic spindle and a successful mitosis. It is therefore noteworthy that these two macromolecular assemblies share a subset of constituents. One of these multifaceted components is Cenp-F, a protein implicated in cancer and developmental pathologies. During the cell cycle, Cenp-F localizes in multiple cellular structures including the nuclear envelope in late G2/early prophase and kinetochores throughout mitosis. We recently characterized the molecular determinants of Cenp-F interaction with NUP133, a structural nuclear pore constituent. In parallel with two other independent studies, we further elucidated the mechanisms governing Cenp-F kinetochore recruitment that mainly relies on its interaction with Bub1, with redundant contribution of Cenp-E upon acute microtubule depolymerisation. Here we synthesize the current literature regarding the dual location of Cenp-F at nuclear pores and kinetochores and extend our discussion to the regulation of these NPC and kinetochore localizations by mitotic kinase and spindle microtubules.
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Nucleoporin 133 deficiency leads to glomerular damage in zebrafish
2018Co-Authors: Chiara Cianciolo Cosentino, Alessandro Berto, Michelle Hari, Johannes Loffing, Stephan C.f. Neuhauss, Valérie DoyeAbstract:Although structural nuclear pore proteins (nucleoporins) are seemingly required in every cell type to assemble a functional nuclear transport machinery, mutations or deregulation of a subset of them have been associated with specific human hereditary diseases. In particular, previous genetic studies of patients with nephrotic syndrome identified mutations in Nup107 that impaired the expression or the localization of its direct partner at nuclear pores, NUP133. In the present study, we characterized the zebrafish NUP133 orthologous gene and its expression pattern during larval development. Morpholino-mediated gene knockdown revealed that NUP133 depletion in zebrafish larvae leads to the formation of kidney cysts, a phenotype that can be rescued by co-injection of wild type mRNA. Analysis of different markers for tubular and glomerular development shows that the overall kidney development is not affected by NUP133 knockdown. On the other hand, we demonstrate that NUP133 is essential for the organization and functional integrity of the pronephric glomerular filtration barrier, as its downregulation results in proteinuria and moderate foot process effacement, mimicking some of the abnormalities typically featured by patients with nephrotic syndrome. These data indicate that NUP133 is a new gene required for proper glomerular structure and function in zebrafish.
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NUP133 Is Required for Proper Nuclear Pore Basket Assembly and Dynamics in Embryonic Stem Cells
Cell reports, 2018Co-Authors: Benoit Souquet, Ellen Freed, Alessandro Berto, Vedrana Andric, Nicolas Audugé, Bernardo Reina-san-martin, Elizabeth Lacy, Valérie DoyeAbstract:Summary NUP133 belongs to the Y-complex, a key component of the nuclear pore complex (NPC) scaffold. Studies on a null mutation in mice previously revealed that NUP133 is essential for embryonic development but not for mouse embryonic stem cell (mESC) proliferation. Using single-pore detection and average NE-fluorescence intensity, we find that NUP133 is dispensable for interphase and postmitotic NPC scaffold assembly in pluripotent mESCs. However, loss of NUP133 specifically perturbs the formation of the nuclear basket as manifested by the absence of Tpr in about half of the NPCs combined with altered dynamics of Nup153. We further demonstrate that its central domain mediates NUP133's role in assembling Tpr and Nup153 into a properly configured nuclear basket. Our findings thus revisit the role of the Y-complex in pore biogenesis and provide insights into the interplay between NPC scaffold architecture, nuclear basket assembly, and the generation of heterogeneity among NPCs.
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Disentangling the molecular determinants for Cenp‐F localization to nuclear pores and kinetochores
EMBO reports, 2018Co-Authors: Alessandro Berto, Stéphanie Morchoisne-bolhy, Chiara Bertipaglia, Julien Dumont, Françoise Ochsenbein, Raphaël Guérois, Richard B. Vallee, Valérie DoyeAbstract:Abstract Cenp‐F is a multifaceted protein implicated in cancer and developmental pathologies. The Cenp‐F C‐terminal region contains overlapping binding sites for numerous proteins that contribute to its functions throughout the cell cycle. Here, we focus on the nuclear pore protein NUP133 that interacts with Cenp‐F both at nuclear pores in prophase and at kinetochores in mitosis, and on the kinase Bub1, known to contribute to Cenp‐F targeting to kinetochores. By combining in silico structural modeling and yeast two‐hybrid assays, we generate an interaction model between a conserved helix within the NUP133 β‐propeller and a short leucine zipper‐containing dimeric segment of Cenp‐F. We thereby create mutants affecting the NUP133/Cenp‐F interface and show that they prevent Cenp‐F localization to the nuclear envelope, but not to kinetochores. Conversely, a point mutation within an adjacent leucine zipper affecting the kinetochore targeting of Cenp‐F KT‐core domain impairs its interaction with Bub1, but not with NUP133, identifying Bub1 as the direct KT‐core binding partner of Cenp‐F. Finally, we show that Cenp‐E redundantly contributes together with Bub1 to the recruitment of Cenp‐F to kinetochores.
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Disentangling the molecular determinants for Cenp-F localization to nuclear pores and kinetochores
EMBO Reports, 2018Co-Authors: Alessandro Berto, Stéphanie Morchoisne-bolhy, Chiara Bertipaglia, Richard Vallee, Julien Dumont, Françoise Ochsenbein, Raphaël Guérois, Valérie DoyeAbstract:Cenp-F is a multifaceted protein implicated in cancer and developmental pathologies. The Cenp-F C-terminal region contains overlapping binding sites for numerous proteins that contribute to its functions throughout the cell cycle. Here, we focus on the nuclear pore protein NUP133 that interacts with Cenp-F both at nuclear pores in prophase and at kinetochores in mitosis, and on the kinase Bub1, known to contribute to Cenp-F targeting to kinetochores. By combining in silico structural modeling and yeast two-hybrid assays, we generate an interaction model between a conserved helix within the NUP133 β-propeller and a short leucine zipper-containing dimeric segment of Cenp-F. We thereby create mutants affecting the NUP133/Cenp-F interface and show that they prevent Cenp-F localization to the nuclear envelope, but not to kinetochores. Conversely, a point mutation within an adjacent leucine zipper affecting the kinetochore targeting of Cenp-F KT-core domain impairs its interaction with Bub1, but not with NUP133, identifying Bub1 as the direct KT-core binding partner of Cenp-F. Finally, we show that Cenp-E redundantly contributes together with Bub1 to the recruitment of Cenp-F to kinetochores.
Thomas Boehmer - 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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Purification, crystallization and preliminary X-ray analysis of a Nup107-NUP133 heterodimeric nucleoporin complex.
Acta Crystallographica Section F Structural Biology and Crystallization Communications, 2007Co-Authors: Thomas Boehmer, Thomas U. SchwartzAbstract:The nuclear pore complex (NPC), the sole gateway of traffic between the nucleus and the cytoplasm, is built up from multiple copies of about 30 proteins collectively termed nucleoporins (nups). Nups are organized into distinct subcomplexes. Nup107 and NUP133 are members of the essential Nup107–160 subcomplex, a component of the central NPC architecture. A dimeric complex of the C-terminal domains of human Nup107 and NUP133 was expressed from a bicistronic vector in Escherichia coli, purified and crystallized in two different crystal forms. Crystals grown in the presence of 18–22% PEG 3350 belong to space group P212121 and diffracted to 2.9 A. Native and seleno-l-methionine-derivative crystals grown in the presence of 1.1 M sodium malonate belong to space group C2 and diffracted to 2.55 and 2.9 A, respectively. Structure determination of this complex will give the first insights into the protein–protein interactions within a core module of the NPC.
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Cell-cycle-dependent phosphorylation of the nuclear pore Nup107-160 subcomplex.
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Joseph S. Glavy, Gunter Blobel, Thomas Boehmer, Ian C. Berke, Andrew N. Krutchinsky, Ileana M. Cristea, Brian T. ChaitAbstract:Abstract The nuclear pore complex (NPC) mediates macromolecular transport between the nucleus and the cytoplasm. Many NPC proteins (nucleoporins, Nups) are modified by phosphorylation. It is believed that phosphorylation regulates the breakdown of the nuclear envelope at mitosis and the disassembly of the NPC into different subcomplexes. In this study, we examined the cell-cycle-dependent phosphorylation of the Nup107–160 subcomplex, a core building block of the NPC. Using in vivo 32P labeling in HeLa cells, we found that Nup107, Nup96, and NUP133 are phosphorylated during mitosis. To precisely map the phosphorylation sites within the complex, we used a comprehensive multiple-stage MS approach (MS, MS2, and MS3), establishing that Nup160, NUP133, Nup96, and Nup107 are all targets of phosphorylation. We determined that the phosphorylation sites are clustered mainly at the N-terminal regions of these proteins, which are predicted to be natively disordered. In addition, we determined the cell-cycle dependence of the phosphorylation of these sites by using stable isotope labeling and MS2 analysis. Measurement of the site-specific phosphorylation ratios between mitotic and G1 cells led us to conclude that several phosphorylation events of the subcomplex are mainly mitotic. Based on these results and our finding that the entire Nup107–160 subcomplex is stable throughout the cell cycle, we propose that phosphorylation does not affect interactions within the Nup107–160 subcomplex, but regulates the association of the subcomplex with the NPC and other proteins. mitosis nucleoporin mass spectrometry nuclear pore complex mammalian
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A general amphipathic alpha-helical motif for sensing membrane curvature.
Nature Structural and Molecular Biology, 2007Co-Authors: Guillaume Drin, Thomas U. Schwartz, Thomas Boehmer, Jean-françois Casella, Romain Gautier, Bruno AntonnyAbstract:The Golgi-associated protein ArfGAP1 has an unusual membrane-adsorbing amphipathic alpha-helix: its polar face is weakly charged, containing mainly serine and threonine residues. We show that this feature explains the specificity of ArfGAP1 for curved versus flat lipid membranes. We built an algorithm to identify other potential amphipathic alpha-helices rich in serine and threonine residues in protein databases. Among the identified sequences, we show that three act as membrane curvature sensors. In the golgin GMAP-210, the sensor may serve to trap small vesicles at the end of a long coiled coil. In Osh4p/Kes1p, which transports sterol between membranes, the sensor controls access to the sterol-binding pocket. In the nucleoporin NUP133, the sensor corresponds to an exposed loop of a beta-propeller structure. Ser/Thr-rich amphipathic helices thus define a general motif used by proteins of various functions for sensing membrane curvature.
Bruno Antonny - One of the best experts on this subject based on the ideXlab platform.
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Membrane Curvature Sensing by Amphipathic Helices Is Modulated by the Surrounding Protein Backbone
PloS one, 2015Co-Authors: Christine Doucet, Nina Esmery, Maud De Saint-jean, Bruno AntonnyAbstract:Membrane curvature is involved in numerous biological pathways like vesicle trafficking, endocytosis or nuclear pore complex assembly. In addition to its topological role, membrane curvature is sensed by specific proteins, enabling the coordination of biological processes in space and time. Amongst membrane curvature sensors are the ALPS (Amphipathic Lipid Packing Sensors). ALPS motifs are short peptides with peculiar amphipathic properties. They are found in proteins targeted to distinct curved membranes, mostly in the early secretory pathway. For instance, the ALPS motif of the golgin GMAP210 binds trafficking vesicles, while the ALPS motif of NUP133 targets nuclear pores. It is not clear if, besides curvature sensitivity, ALPS motifs also provide target specificity, or if other domains in the surrounding protein backbone are involved. To elucidate this aspect, we studied the subcellular localization of ALPS motifs outside their natural protein context. The ALPS motifs of GMAP210 or NUP133 were grafted on artificial fluorescent probes. Importantly, ALPS motifs are held in different positions and these contrasting architectures were mimicked by the fluorescent probes. The resulting chimeras recapitulated the original proteins localization, indicating that ALPS motifs are sufficient to specifically localize proteins. Modulating the electrostatic or hydrophobic content of NUP133 ALPS motif modified its avidity for cellular membranes but did not change its organelle targeting properties. In contrast, the structure of the backbone surrounding the helix strongly influenced targeting. In particular, introducing an artificial coiled-coil between ALPS and the fluorescent protein increased membrane curvature sensitivity. This coiled-coil domain also provided membrane curvature sensitivity to the amphipathic helix of Sar1. The degree of curvature sensitivity within the coiled-coil context remains correlated to the natural curvature sensitivity of the helices. This suggests that the chemistry of ALPS motifs is a key parameter for membrane curvature sensitivity, which can be further modulated by the surrounding protein backbone.
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A general amphipathic alpha-helical motif for sensing membrane curvature.
Nature Structural and Molecular Biology, 2007Co-Authors: Guillaume Drin, Thomas U. Schwartz, Thomas Boehmer, Jean-françois Casella, Romain Gautier, Bruno AntonnyAbstract:The Golgi-associated protein ArfGAP1 has an unusual membrane-adsorbing amphipathic alpha-helix: its polar face is weakly charged, containing mainly serine and threonine residues. We show that this feature explains the specificity of ArfGAP1 for curved versus flat lipid membranes. We built an algorithm to identify other potential amphipathic alpha-helices rich in serine and threonine residues in protein databases. Among the identified sequences, we show that three act as membrane curvature sensors. In the golgin GMAP-210, the sensor may serve to trap small vesicles at the end of a long coiled coil. In Osh4p/Kes1p, which transports sterol between membranes, the sensor controls access to the sterol-binding pocket. In the nucleoporin NUP133, the sensor corresponds to an exposed loop of a beta-propeller structure. Ser/Thr-rich amphipathic helices thus define a general motif used by proteins of various functions for sensing membrane curvature.
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A general amphipathic α-helical motif for sensing membrane curvature
Nature Structural & Molecular Biology, 2007Co-Authors: Guillaume Drin, Thomas U. Schwartz, Thomas Boehmer, Jean-françois Casella, Romain Gautier, Bruno AntonnyAbstract:The Golgi-associated protein ArfGAP1 has an unusual membrane-adsorbing amphipathic α-helix: its polar face is weakly charged, containing mainly serine and threonine residues. We show that this feature explains the specificity of ArfGAP1 for curved versus flat lipid membranes. We built an algorithm to identify other potential amphipathic α-helices rich in serine and threonine residues in protein databases. Among the identified sequences, we show that three act as membrane curvature sensors. In the golgin GMAP-210, the sensor may serve to trap small vesicles at the end of a long coiled coil. In Osh4p/Kes1p, which transports sterol between membranes, the sensor controls access to the sterol-binding pocket. In the nucleoporin NUP133, the sensor corresponds to an exposed loop of a β-propeller structure. Ser/Thr-rich amphipathic helices thus define a general motif used by proteins of various functions for sensing membrane curvature.
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A general amphipathic |[alpha]|-helical motif for sensing membrane curvature
Nature structural & molecular biology, 2007Co-Authors: Guillaume Drin, Thomas U. Schwartz, Thomas Boehmer, Jean-françois Casella, Romain Gautier, Bruno AntonnyAbstract:The Golgi-associated protein ArfGAP1 has an unusual membrane-adsorbing amphipathic α-helix: its polar face is weakly charged, containing mainly serine and threonine residues. We show that this feature explains the specificity of ArfGAP1 for curved versus flat lipid membranes. We built an algorithm to identify other potential amphipathic α-helices rich in serine and threonine residues in protein databases. Among the identified sequences, we show that three act as membrane curvature sensors. In the golgin GMAP-210, the sensor may serve to trap small vesicles at the end of a long coiled coil. In Osh4p/Kes1p, which transports sterol between membranes, the sensor controls access to the sterol-binding pocket. In the nucleoporin NUP133, the sensor corresponds to an exposed loop of a β-propeller structure. Ser/Thr-rich amphipathic helices thus define a general motif used by proteins of various functions for sensing membrane curvature.
Gunter Blobel - One of the best experts on this subject based on the ideXlab platform.
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Structural and Functional Analysis of Nup120 Suggests Ring Formation of the Nup84 Complex
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Hyuk-soo Seo, Gunter Blobel, Erik W. Debler, Daniel Wacker, Stephan Kutik, André HoelzAbstract:The Nup84 complex constitutes a key building block in the nuclear pore complex (NPC). Here we present the crystal structure of one of its 7 components, Nup120, which reveals a β propeller and an α-helical domain representing a novel fold. We discovered a previously unidentified interaction of Nup120 with NUP133 and confirmed the physiological relevance in vivo. As mapping of the individual components in the Nup84 complex places Nup120 and NUP133 at opposite ends of the heptamer, our findings indicate a head-to-tail arrangement of elongated Nup84 complexes into a ring structure, consistent with a fence-like coat for the nuclear pore membrane. The attachment site for NUP133 lies at the very end of an extended unstructured region, which allows for flexibility in the diameter of the Nup84 complex ring. These results illuminate important roles of terminal unstructured segments in nucleoporins for the architecture, function, and assembly of the NPC.
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Cell-cycle-dependent phosphorylation of the nuclear pore Nup107-160 subcomplex.
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Joseph S. Glavy, Gunter Blobel, Thomas Boehmer, Ian C. Berke, Andrew N. Krutchinsky, Ileana M. Cristea, Brian T. ChaitAbstract:Abstract The nuclear pore complex (NPC) mediates macromolecular transport between the nucleus and the cytoplasm. Many NPC proteins (nucleoporins, Nups) are modified by phosphorylation. It is believed that phosphorylation regulates the breakdown of the nuclear envelope at mitosis and the disassembly of the NPC into different subcomplexes. In this study, we examined the cell-cycle-dependent phosphorylation of the Nup107–160 subcomplex, a core building block of the NPC. Using in vivo 32P labeling in HeLa cells, we found that Nup107, Nup96, and NUP133 are phosphorylated during mitosis. To precisely map the phosphorylation sites within the complex, we used a comprehensive multiple-stage MS approach (MS, MS2, and MS3), establishing that Nup160, NUP133, Nup96, and Nup107 are all targets of phosphorylation. We determined that the phosphorylation sites are clustered mainly at the N-terminal regions of these proteins, which are predicted to be natively disordered. In addition, we determined the cell-cycle dependence of the phosphorylation of these sites by using stable isotope labeling and MS2 analysis. Measurement of the site-specific phosphorylation ratios between mitotic and G1 cells led us to conclude that several phosphorylation events of the subcomplex are mainly mitotic. Based on these results and our finding that the entire Nup107–160 subcomplex is stable throughout the cell cycle, we propose that phosphorylation does not affect interactions within the Nup107–160 subcomplex, but regulates the association of the subcomplex with the NPC and other proteins. mitosis nucleoporin mass spectrometry nuclear pore complex mammalian
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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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depletion of a single nucleoporin nup107 prevents the assembly of a subset of nucleoporins into the nuclear pore complex
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Thomas Boehmer, Gunter Blobel, Jost Enninga, Samuel Dales, Hualin ZhongAbstract:The nuclear pore complex (NPC) is a protein assembly that contains several distinct subcomplexes. The mammalian nucleoporin (Nup)-107 is part of a hetero-oligomeric complex, that also contains Nup160, NUP133, Nup96, and the mammalian homolog of yeast Sec13p. We used transfection of HeLa cells with small interfering RNAs to specifically deplete mRNA for Nup107. In a domino effect, Nup107 depletion caused codepletion of a subset of other Nups on their protein but not on their mRNA level. Among the affected Nups was a member of the Nup107 subcomplex, NUP133, whereas two other tested members of this complex, Nup96 and Sec13, were unaffected and assembled into Nup107/NUP133-deficient NPCs. We also tested several phenylalanine-glycine repeat-containing Nups that serve as docking sites for karyopherins. Some of these, such as Nup358, Nup214 on the cytoplasmic, and Nup153 on the nucleoplasmic side of the NPC, failed to assemble into Nup107/NUP133-depleted NPCs, whereas p62, a Nup at the center of the NPC, was unaffected. Interestingly, the filamentous, NPC-associated protein Tpr also failed to assemble into the NPCs of Nup107-depleted cells. These data indicate that Nup107 functions as a keystone Nup that is required for the assembly of a subset of Nups into the NPC. Despite the depletion of Nup107 and the accompanying effects on other Nups, there was no significant effect on the growth rate of these cells and only a partial inhibition of mRNA export. These data indicate redundancy of Nups in the function of the mammalian NPC.
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RanGTP-mediated nuclear export of karyopherin α involves its interaction with the nucleoporin Nup153
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Junona Moroianu, Gunter Blobel, Aurelian RaduAbstract:Abstract Using binding assays, we discovered an interaction between karyopherin α2 and the nucleoporin Nup153 and mapped their interacting domains. We also isolated a 15-kDa tryptic fragment of karyopherin β1, termed β1*, that contains a determinant for binding to the peptide repeat containing nucleoporin Nup98. In an in vitro assay in which export of endogenous nuclear karyopherin α from nuclei of digitonin-permeabilized cells was quantitatively monitored by indirect immunofluorescence with anti-karyopherin α antibodies, we found that karyopherin α export was stimulated by added GTPase Ran, required GTP hydrolysis, and was inhibited by wheat germ agglutinin. RanGTP-mediated export of karyopherin α was inhibited by peptides representing the interacting domains of Nup153 and karyopherin α2, indicating that the binding reactions detected in vitro are physiologically relevant and verifying our mapping data. Moreover, β1*, although it inhibited import, did not inhibit export of karyopherin α. Hence, karyopherin α import into and export from nuclei are asymmetric processes.
