Cell Cycle-Dependent Phosphorylation of Nucleoporins and Nuclear Pore Membrane Protein Gp210
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During mitosis in higher eukaryotic cells, the nuclear envelope membranes break down into distinct populations of vesicles and the proteins of the nuclear lamina and the nuclear pore complexes disperse in the cytoplasm. Since phosphorylation can alter protein−protein interactions and membrane traffic, we have examined the cell cycle-dependent phosphorylation of nuclear pore complex proteins. Nonmembrane nucleoporins Nup153, Nup214, and Nup358 that are modified by O-linked N-acetylglucosamine and recognized by a monoclonal antibody were phosphorylated throughout the cell cycle and hyperphosphorylated during M phase. Pore membrane glycoprotein gp210, that has a cytoplasmic, carboxyl-terminal domain facing the pore, was not phosphorylated in interphase but specifically phosphorylated in mitosis. Mutant and wild-type fusion proteins containing the cytoplasmic domain of gp210 were phosphorylated in vitro and their phosphopeptide maps compared to that of mitotic gp210. This analysis showed that Ser1880 of gp210 was phosphorylated in mitosis, possibly by cyclin B-p34cdc2 or a related kinase. Several nuclear pore complex proteins are therefore differentially phosphorylated during mitosis when pore complexes disassemble and reassemble.Keywords:
Nucleoporin
Nuclear pore
Nuclear lamina
Nucleoporins containing phenylalanine glycine (FG) repeats play an important role in nucleocytoplasmic transport as they bind to transport receptors and mediate translocation of transport complexes across the nuclear pore complex (NPC). Nup214/CAN, a nucleoporin that is found at the cytoplasmic side of the NPC, interacts with both import and export receptors. In functional assays, dominant-negative fragments of Nup214 inhibited CRM1-dependent nuclear export, as the export receptor became rate-limiting. Several nuclear import pathways, by contrast, were not affected by the Nup214 fragments. We now characterize the CRM1-binding region of Nup214 in detail and identify several FG motives that are required for this interaction. Our results support a model where CRM1, like other transport receptors, contacts FG-Nups via multiple binding sites.Background: Nup214 interacts with the nuclear export receptor CRM1 and promotes export of certain cargos.Results: Several FG motives in the C-terminal region participate in CRM1 binding.Conclusion: CRM1, like other transport receptors, makes multiple contacts to nucleoporins.Significance: Elucidation of the details of nucleoporin-receptor interactions is essential for our understanding of the transition process of transport complexes through the nuclear pore. Nucleoporins containing phenylalanine glycine (FG) repeats play an important role in nucleocytoplasmic transport as they bind to transport receptors and mediate translocation of transport complexes across the nuclear pore complex (NPC). Nup214/CAN, a nucleoporin that is found at the cytoplasmic side of the NPC, interacts with both import and export receptors. In functional assays, dominant-negative fragments of Nup214 inhibited CRM1-dependent nuclear export, as the export receptor became rate-limiting. Several nuclear import pathways, by contrast, were not affected by the Nup214 fragments. We now characterize the CRM1-binding region of Nup214 in detail and identify several FG motives that are required for this interaction. Our results support a model where CRM1, like other transport receptors, contacts FG-Nups via multiple binding sites. Background: Nup214 interacts with the nuclear export receptor CRM1 and promotes export of certain cargos. Results: Several FG motives in the C-terminal region participate in CRM1 binding. Conclusion: CRM1, like other transport receptors, makes multiple contacts to nucleoporins. Significance: Elucidation of the details of nucleoporin-receptor interactions is essential for our understanding of the transition process of transport complexes through the nuclear pore.
Nucleoporin
Nuclear pore
Nuclear export signal
Ran
Importin
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Author(s): Tang, Jeffrey Hsin Nien | Advisor(s): Liphardt, Jan | Abstract: The nuclear pore complex (NPC) is one of the largest known protein structures in the cell. Evolutionarily conserved in eukaryotes ranging from fungi to plants and animals, the NPC is the main transporter of molecules between the cell cytoplasm and nucleus. Maintaining the proper compartment-specific localization of proteins and RNA is crucial for normal cell function, and the nuclear pore accomplishes this task both robustly and efficiently. Over the past several decades, insight into the composition, organization, structure, and mechanism of the NPC has been gradually teased out through careful experimentation. However, many questions about the pore's function remain unanswered. In this dissertation, I describe efforts aimed at elucidating several aspects of the NPC. First, I investigate the transport properties of the pore, specifically looking at how the nuclear transport receptor importin-β and the Ran GTPase interact not only with each other but also how they may affect the pore itself. The nucleoporin Nup153 is identified as an important player in the nuclear transport process which binds strongly to importin-β in a Ran-sensitive manner. Using multiple experimental techniques, the properties of importin-β, and Nup153's interactions are characterized and shown to be capable of modulating the selective permeability barrier of the NPC.Next, I examine how members of a major class of nuclear pore proteins, the scaffold nucleoporins, are both structurally and functionally similar to the karyopherin family of soluble nuclear transport receptors. Structures of the proteins Nup188 and Nup192 are analyzed and shown to resemble those of karyopherins. Furthermore, in vitro assays indicate that at least a subset of the scaffold nucleoporins behave functionally as transport receptors, hinting at an evolutionary relationship between these two important classes of proteins.Finally, a calcium-mediated phenomenon affecting the permeability of the NPC is explored. I show that certain cytosolic proteases are activated by millimolar concentrations of calcium ion which leads irreversibly to an increase in the nuclear pore's permeability to large molecules. A model for physiological pathways implicated in this effect is proposed.
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Nuclear pore
Importin
Karyopherin
Ran
Transport protein
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Nuclear pore complex (NPC) is a large macromolecular assembly with an estimated size of 110 MDa in vertebrates and 60 MDa in yeast. Y nucleoporins (Nups) are contained within the Y-complex. Nup153 and Nup50, along with translocated promoter region protein, make up the nuclear basket and provide the surface to utilize binding areas for transport. When the nuclear envelope (NE) breaks down during mitosis, the nuclear lamina also disassembles, which occurs when lamins are phosphorylated by protein kinase C. Because the mitotic spindles form outside the NE in higher eukaryotes, the whole NE must disintegrate to progress through the cell cycle. Micronuclei are small nuclei that form separately from the primary nucleus following the formation of an NE around mis-segregated chromosomes. Several types of cancer are associated with intricate mechanisms known as chromothripsis and kataegis that occur in a single event and catastrophically alter the genome.
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Nuclear lamina
Chromothripsis
Inner membrane
Interphase
Mitotic exit
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J. Tetenbaum-Novatt and M.P. Rout⇓ Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, New York 10065 Correspondence: rout{at}rockefeller.edu
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Transport protein
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The rapid (<1 ms) transport of biological material to and from the cell nucleus is regulated by the nuclear pore complex (NPC). At the core of the NPC is a permeability barrier consisting of intrinsically disordered phenylalanine-glycine nucleoporins (FG Nups). Various types of nuclear transport receptors (NTRs) facilitate transport by partitioning in the FG Nup assembly, overcoming the barrier by their affinity to the FG Nups, and comprise a significant fraction of proteins in the NPC barrier. In previous work (Zahn et al., 2016), we revealed a universal physical behaviour in the experimentally observed binding of two well-characterised NTRs, Nuclear Transport Factor 2 (NTF2) and the larger Importin-β (Imp-β), to different planar assemblies of FG Nups, with the binding behaviour defined by negative cooperativity. This was further validated by a minimal physical model that treated the FG Nups as flexible homopolymers and the NTRs as uniformly cohesive spheres. Here, we build upon our original study by first parametrising our model to experimental data, and next predicting the effects of crowding by different types of NTRs. We show how varying the amounts of one type of NTR modulates how the other NTR penetrates the FG Nup assembly. Notably, at similar and physiologically relevant NTR concentrations, our model predicts demixed phases of NTF2 and Imp-β within the FG Nup assembly. The functional implication of NTR phase separation is that NPCs may sustain separate transport pathways that are determined by inter-NTR competition.
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Intrinsically Disordered Proteins
Importin
Cooperativity
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The bidirectional nucleocytoplasmic transport of macromolecules is mediated by the nuclear pore complex (NPC) which, in yeast, is composed of ∼30 different proteins (nucleoporins). Pre-embedding immunogold-electron microscopy revealed that Nic96p, an essential yeast nucleoporin, is located about the cytoplasmic and the nuclear periphery of the central channel, and near or at the distal ring of the yeast NPC. Genetic approaches further implicated Nic96p in nuclear protein import. To more specifically explore the potential role of Nic96p in nuclear protein import, we performed a two-hybrid screen withNIC96 as the bait against a yeast genomic library to identify transport factors and/or nucleoporins involved in nuclear protein import interacting with Nic96p. By doing so, we identified the yeast nucleoporin Nup53p, which also exhibits multiple locations within the yeast NPC and colocalizes with Nic96p in all its locations. Whereas Nup53p is directly involved in NLS-mediated protein import by its interaction with the yeast nuclear import receptor Kap95p, it appears not to participate in NES-dependent nuclear export.
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Importin
Transport protein
Nuclear export signal
Fungal protein
Immunogold labelling
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Nucleoporin
Nuclear pore
Nuclear lamina
Importin
Inner membrane
Immunogold labelling
Transport protein
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This chapter contains sections titled: Introduction The Structure of the Nuclear Pore Complex (NPC) Nucleoporins (NUPS) Nuclear Envelope and Membrane Proteins Nuclear Lamina Nuclear Transport Cycle Genes, Transport, and the NPC Nuclear Envelope Breakdown (NEBD) The NPC and Diseases References
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Nuclear lamina
Inner membrane
Envelope (radar)
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Nucleoporin
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Proteome
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A major question in nuclear import concerns the identity of the nucleoporin(s) that interact with the nuclear localization sequences (NLS) receptor and its cargo as they traverse the nuclear pore. Ligand blotting and solution binding studies of isolated proteins have attempted to gain clues to the identities of these nucleoporins, but the studies have from necessity probed binding events far from an in vivo context. Here we have asked what binding events occur in the more physiological context of a Xenopus egg extract, which contains nuclear pore subcomplexes in an assembly competent state. We have then assessed our conclusions in the context of assembled nuclear pores themselves. We have used immunoprecipitation to identify physiologically relevant complexes of nucleoporins and importin subunits. In parallel, we have demonstrated that it is possible to obtain immunofluorescence localization of nucleoporins to subregions of the nuclear pore and its associated structures. By immunoprecipitation, we find the nucleoporin Nup153 and the pore-associated filament protein Tpr, previously shown to reside at distinct sites on the intranuclear side of assembled pores, are each in stable subcomplexes with importin alpha and beta in Xenopus egg extracts. Importin subunits are not in stable complexes with nucleoporins Nup62, Nup93, Nup98, or Nup214/CAN, either in egg extracts or in extracts of assembled nuclear pores. In characterizing the Nup153 complex, we find that Nup153 can bind to a complete import complex containing importin alpha, beta, and an NLS substrate, consistent with an involvement of this nucleoporin in a terminal step of nuclear import. Importin beta binds directly to Nup153 and in vitro can do so at multiple sites in the Nup153 FXFG repeat region. Tpr, which has no FXFG repeats, binds to importin beta and to importin alpha/beta heterodimers, but only to those that do not carry an NLS substrate. That the complex of Tpr with importin beta is fundamentally different from that of Nup153 is additionally demonstrated by the finding that recombinant beta or beta45-462 fragment freely exchanges with the endogenous importin beta/Nup153 complex, but cannot displace endogenous importin beta from a Tpr complex. However, the GTP analogue GMP-PNP is able to disassemble both Nup153- and Tpr-importin beta complexes. Importantly, analysis of extracts of isolated nuclei indicates that Nup153- and Tpr-importin beta complexes exist in assembled nuclear pores. Thus, Nup153 and Tpr are major physiological binding sites for importin beta. Models for the roles of these interactions are discussed.
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Nuclear pore
Importin
Karyopherin
Immunoprecipitation
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