The C-terminal domain of Nup93 is essential for assembly of the structural backbone of nuclear pore complexes

Nuclear pore complexes (NPCs) are large macromolecular assemblies that control all transport across the nuclear envelope. They are formed by about 30 nucleoporins (Nups), which can be roughly categorized into those forming the structural skeleton of the pore and those creating the central channel and thus providing the transport and gating properties of the NPC. Here we show that the conserved nucleoporin Nup93 is essential for NPC assembly and connects both portions of the NPC. Although the C-terminal domain of the protein is necessary and sufficient for the assembly of a minimal structural backbone, full-length Nup93 is required for the additional recruitment of the Nup62 complex and the establishment of transport-competent NPCs.     

蛋白质入核转运的机制和研究进展

细胞核膜是由外膜和内膜组成的磷脂双分子层结构,同时镶嵌一些核孔复合体（NPC）.核孔复合体是胞浆和胞核之间主动和被动转运的生理屏障.核内功能蛋白在胞浆内合成后通过核孔复合体进入胞核,这个过程除了需要NPC上核孔蛋白、胞浆内核转运受体和RanGTP等蛋白的参与外, 货物蛋白本身的结构特征在其入核转运过程中亦发挥重要作用.本文着重就蛋白入核转运的机制及近年来取得的相关进展进行综述.     

From the trap to the basket: getting to the bottom of the nuclear pore complex

Nuclear pore complexes (NPCs) are large supramolecular assemblies that perforate the double-membraned nuclear envelope and serve as the sole gateways of molecular exchange between the cytoplasm and the nucleus in interphase cells. Combining novel specimen preparation regimes with innovative use of high-resolution scanning electron microscopy, Hans Ris produced in the late eighties stereo images of the NPC with unparalleled clarity and structural detail, thereby setting new standards in the field. Since that time, efforts undertaken to resolve the molecular structure and architecture, and the numerous interactions that occur between NPC proteins (nucleoporins), soluble transport receptors, and the small GTPase Ran, have led to a deeper understanding of the functional role of NPCs in nucleocytoplasmic transport. In spite of these breakthroughs, getting to the bottom of the actual cargo translocation mechanism through the NPC remains elusive and controversial. Here, we review recent insights into NPC function by correlating structural findings with biochemical data. By introducing new experimental and computational results, we reexamine how NPCs can discriminate between receptor-mediated and passive cargo to promote vectorial translocation in a highly regulated manner. Moreover, we comment on the importance and potential benefits of identifying and experimenting with individual key components implicated in the translocation mechanism. We conclude by dwelling on questions that we feel are pertinent to a more rational understanding of the physical aspects governing NPC mechanics. Last but not least, we substantiate these uncertainties by boldly suggesting a new direction in NPC research as a means to verify such novel concepts, for example, a de novo designed ‘minimalist’ NPC. This article is dedicated to the memory of Hans Ris.     

Aggregation,Phase Separation and Spatial Morphologies of the Assemblies of FG Nucleoporins

Nuclear pore complex (NPC) is a biomolecular “nanomachine” that controls nucleocytoplasmic transport in eukaryotic cells. The key component of the functional architecture of the NPC is the assembly of intrinsically disordered proteins that line its passageway and play a central role in the NPC transport mechanism. Due to paucity of experimental methods capable to directly probe the morphology of this assembly in intact NPCs, much of our knowledge about its properties derives from in vitro experiments augmented by theoretical and computational modeling. I review the major insights into the biophysics of the assemblies of the intrinsically disordered proteins of the NPC arising from the theoretical analysis of the recent in vitro experimental results, with the emphasis on the phase separation and aggregation phenomena.     

Model Inspired by Nuclear Pore Complex Suggests Possible Roles for Nuclear Transport Receptors in Determining Its Structure

Nuclear transport receptors (NTRs) mediate nucleocytoplasmic transport via their affinity for unstructured proteins (polymers) in the nuclear pore complex (NPC). Here, we have modeled the effect of NTRs on polymeric structure in the nanopore confinement of the NPC central conduit. The model explicitly takes into account inter- and intramolecular interactions, as well as the finite size of the NTRs (∼20% of the NPC channel diameter). It reproduces various proposed scenarios for the channel structure, ranging from a central polymer condensate (selective phase) to brushlike polymer arrangements localized at the channel wall (virtual gate, reduction of dimensionality), with the transport receptors lining the polymer surface. In addition, it predicts a new structure in which NTRs become an integral part of the transport barrier by forming a cross-linked network with the unstructured proteins stretching across the pore. The model provides specific and distinctive predictions for the equilibrium spatial distributions of NTRs for these different scenarios that can be experimentally verified by, e.g., superresolution fluorescence microscopy. Moreover, it suggests mechanisms by which globular macromolecules (colloidal particles) can cause polymer-coated nanopores to switch between open and closed configurations, a possible explanation of the biological function of the NPC, and suggests potential technological applications for filtration and single-molecule sensing.     

Large cargo transport by nuclear pores: implications for the spatial organization of FG‐nucleoporins

Nuclear pore complexes (NPCs) mediate cargo traffic between the nucleus and the cytoplasm of eukaryotic cells. Nuclear transport receptors (NTRs) carry cargos through NPCs by transiently binding to phenylalanine‐glycine (FG) repeats on intrinsically disordered polypeptides decorating the NPCs. Major impediments to understand the transport mechanism are the thousands of FG binding sites on each NPC, whose spatial distribution is unknown, and multiple binding sites per NTR, which leads to multivalent interactions. Using single molecule fluorescence microscopy, we show that multiple NTR molecules are required for efficient transport of a large cargo, while a single NTR promotes binding to the NPC but not transport. Particle trajectories and theoretical modelling reveal a crucial role for multivalent NTR interactions with the FG network and indicate a non‐uniform FG repeat distribution. A quantitative model is developed wherein the cytoplasmic side of the pore is characterized by a low effective concentration of free FG repeats and a weak FG‐NTR affinity, and the centrally located dense permeability barrier is overcome by multivalent interactions, which provide the affinity necessary to permeate the barrier.     

Correction

Nuclear transport receptors (NTRs) mediate nucleocytoplasmic transport via their affinity for unstructured proteins (polymers) in the nuclear pore complex (NPC). Here, we have modeled the effect of NTRs on polymeric structure in the nanopore confinement of the NPC central conduit. The model explicitly takes into account inter- and intramolecular interactions, as well as the finite size of the NTRs (∼20% of the NPC channel diameter). It reproduces various proposed scenarios for the channel structure, ranging from a central polymer condensate (selective phase) to brushlike polymer arrangements localized at the channel wall (virtual gate, reduction of dimensionality), with the transport receptors lining the polymer surface. In addition, it predicts a new structure in which NTRs become an integral part of the transport barrier by forming a cross-linked network with the unstructured proteins stretching across the pore. The model provides specific and distinctive predictions for the equilibrium spatial distributions of NTRs for these different scenarios that can be experimentally verified by, e.g., superresolution fluorescence microscopy. Moreover, it suggests mechanisms by which globular macromolecules (colloidal particles) can cause polymer-coated nanopores to switch between open and closed configurations, a possible explanation of the biological function of the NPC, and suggests potential technological applications for filtration and single-molecule sensing.     

Charge as a Selection Criterion for Translocation through the Nuclear Pore Complex

Nuclear pore complexes (NPCs) are highly selective filters that control the exchange of material between nucleus and cytoplasm. The principles that govern selective filtering by NPCs are not fully understood. Previous studies find that cellular proteins capable of fast translocation through NPCs (transport receptors) are characterized by a high proportion of hydrophobic surface regions. Our analysis finds that transport receptors and their complexes are also highly negatively charged. Moreover, NPC components that constitute the permeability barrier are positively charged. We estimate that electrostatic interactions between a transport receptor and the NPC result in an energy gain of several k _B T, which would enable significantly increased translocation rates of transport receptors relative to other cellular proteins. We suggest that negative charge is an essential criterion for selective passage through the NPC.     

Esophageal cancer alters the expression of nuclear pore complex binding protein Hsc70 and eIF5A-1

Nuclear pore complex (NPC) is the only corridor for macromolecules exchange between nucleus and cytoplasm. NPC and its components, nucleoporins, play important role in the diverse physiological processes including macromolecule exchange, chromosome segregation, apoptosis and gene expression. Recent reports also suggest involvement of nucleoporins in carcinogenesis. Applying proteomics, we analyzed expression pattern of the NPC components in a newly established esophageal cancer cell line from Persia (Iran), the high-risk region for esophageal cancer. Our results indicate overexpression of Hsc70 and downregulation of subunit alpha type-3 of proteasome, calpain small subunit 1, and eIF5A-1. Among these proteins, Hsc70 and eIF5A-1 are in direct interaction with NPC and involved in the nucleocytoplasmic exchange. Hsc70 plays a critical role as a chaperone in the formation of a cargo–receptor complex in nucleocytoplasmic transport. On the other hand, it is an NPC-associated protein that binds to nucleoporins and contributes in recycling of the nucleocytoplasmic transport receptors in mammals and affects transport of proteins between nucleus and cytoplasm. The other nuclear pore interacting protein: eIF5A-1 binds to the several nucleoporins and participates in nucleocytoplasmic transport. Altered expression of Hsc70 and eIF5A-1 may cause defects in nucleocytoplasmic transport and play a role in esophageal carcinogenesis.     

Exceptional structural and mechanical flexibility of the nuclear pore complex

Nuclear pore complexes (NPCs) mediate all transport between the cytosol and the nucleus and therefore take centre stage in physiology. While transport through NPCs has been extensively investigated little is known about their structural and barley anything about their mechanical flexibility. Structural and mechanical flexibility of NPCs, however, are presumably of key importance. Like the cell and the cell nucleus, NPCs themselves are regularly exposed to physiological mechanical forces. Besides, NPCs reveal striking transport properties which are likely to require fairly high structural flexibility. The NPC transports up to 1,000 molecules per second through a physically 9 nm wide channel which repeatedly opens to accommodate macromolecules significantly larger than its physical diameter. We hypothesised that NPCs possess remarkable structural and mechanical stability. Here, we tested this hypothesis at the single NPC level using the nano‐imaging and probing approach atomic force microscopy (AFM). AFM presents the NPC as a highly flexible structure. The NPC channel dilates by striking 35% on exposure to trans‐cyclohexane‐1,2‐diol (TCHD), which is known to transiently collapse the hydrophobic phase in the NPC channel like receptor–cargo complexes do in transit. It constricts again to its initial size after TCHD removal. AFM‐based nano‐indentation measurements show that the 50 nm long NPC basket can astonishingly be squeezed completely into the NPC channel on exposure to incremental mechanical loads but recovers its original vertical position within the nuclear envelope plane when relieved. We conclude that the NPC possesses exceptional structural and mechanical flexibility which is important to fulfilling its functions. J. Cell. Physiol. 226: 675–682, 2011. © 2010 Wiley‐Liss, Inc.     

Reticulon-like REEP4 at the inner nuclear membrane promotes nuclear pore complex formation

Nuclear pore complexes (NPCs) are channels within the nuclear envelope that mediate nucleocytoplasmic transport. NPCs form within the closed nuclear envelope during interphase or assemble concomitantly with nuclear envelope reformation in late stages of mitosis. Both interphase and mitotic NPC biogenesis require coordination of protein complex assembly and membrane deformation. During early stages of mitotic NPC assembly, a seed for new NPCs is established on chromatin, yet the factors connecting the NPC seed to the membrane of the forming nuclear envelope are unknown. Here, we report that the reticulon homology domain protein REEP4 not only localizes to high-curvature membrane of the cytoplasmic endoplasmic reticulum but is also recruited to the inner nuclear membrane by the NPC biogenesis factor ELYS. This ELYS-recruited pool of REEP4 promotes NPC assembly and appears to be particularly important for NPC formation during mitosis. These findings suggest a role for REEP4 in coordinating nuclear envelope reformation with mitotic NPC biogenesis.     

Multiscale dynamics in nucleocytoplasmic transport

The nuclear pore complex (NPC) has long been viewed as a point-like entry and exit channel between the nucleus and the cytoplasm. New data support a different view whereby the complex displays distinct spatial dynamics of variable duration ranging from milliseconds to events spanning the entire cell cycle. Discrete interaction sites outside the central channel become apparent, and transport regulation at these sites seems to be of greater importance than currently thought. Nuclear pore components are highly active outside the NPC or impact the fate of cargo transport away from the nuclear pore. The NPC is a highly dynamic, crowded environment-constantly loaded with cargo while providing selectivity based on unfolded proteins. Taken together, this comprises a new paradigm in how we view import/export dynamics and emphasizes the multiscale nature of NPC-mediated cellular transport.     

Nuclear pore complex is able to transport macromolecules with diameters of about 39 nm

Bidirectional transport of macromolecules between the nucleus and the cytoplasm occurs through the nuclear pore complexes (NPCs) by a signal-mediated mechanism that is directed by targeting signals (NLSs) residing on the transported molecules or "cargoes." Nuclear transport starts after interaction of the targeting signal with soluble cellular receptors. After the formation of the cargo-receptor complex in the cytosol, this complex crosses the NPC. Herein, we use gold particles of various sizes coated with cargo-receptor complexes to determine precisely how large macromolecules crossing the NPC by the signal-mediated transport mechanism could be. We found that cargo-receptor-gold complexes with diameter close to 39 nm could be translocated by the NPC. This implies that macromolecules much larger than the assumed functional NPC diameter of 26 nm can be transported into the karyoplasm. The physiological relevance of this finding was supported by the observation that intact nucleocapsids of human hepatitis B virus with diameters of 32 and 36 nm are able to cross the nuclear pore without disassembly.     

The Role of Cohesiveness in the Permeability of the Spatial Assemblies of FG Nucleoporins

Nuclear pore complexes (NPCs) conduct selective, bidirectional transport across the nuclear envelope. The NPC passageway is lined by intrinsically disordered proteins that contain hydrophobic phenylalanine-glycine (FG) motifs, known as FG nucleoporins (FG nups), that play the key role in the NPC transport mechanism. Cohesive interactions among the FG nups, which arise from the combination of hydrophobic, electrostatic, and other forces, have been hypothesized to control the morphology of the assemblies of FG nups in the NPC, as well as their permeability with respect to the transport proteins. However, the role of FG nup cohesiveness is still vigorously debated. Using coarse-grained polymer theory and numerical simulations, we study the effects of cohesiveness on the selective permeability of in vitro FG nup assemblies in different geometries that have served as proxies for the morphological and transport properties of the NPC. We show that in high-density FG nup assemblies, increase in cohesiveness leads to the decrease in their permeability, in accordance with the accepted view. On the other hand, the permeability of low-density assemblies is a nonmonotonic function of the cohesiveness, and a moderate increase in cohesiveness can enhance permeability. The density- and cohesiveness-dependent effects on permeability are explained by considering the free-energy cost associated with penetrating the FG nup assemblies. We discuss the implications of these findings for the organization and function of the NPC.     

Nup62‐mediated nuclear import of p63 in squamous cell carcinoma

Nuclear pore complexes (NPCs) are multiprotein channels that bridge the nucleus with the cytoplasm and regulate all nucleo‐cytoplasmic traffic. NPCs are built by the repetition of ~30 different proteins known as nucleoporins (Nups). Accumulating evidence has revealed a diversity in NPC composition that is critical for cell‐specific functionality and fate determination. A new report by Hazawa et al 1 now identifies the central transport channel nucleoporin Nup62 as a novel regulator of cell proliferation and differentiation in squamous cell carcinoma (SCC), via modulation of p63 nucleo‐cytoplasmic transport. These findings provide further evidence on how alterations in NPC composition might be utilized to determine cell fate.     

Structural and functional insights into nucleocytoplasmic transport

The cell nucleus is surrounded by a double membrane system, the nuclear envelope (NE), with the outer nuclear membrane being continuous with the endoplasmic reticulum. Nuclear pore complexes (NPCs) fuse the inner and outer nuclear membranes, forming aqueous channels that allow free diffusion of small molecules but that also mediate the energy-dependent transport of large macromolecules. The NPC represents the largest known molecular complex and is composed of about 30 different proteins, termed nucleoporins (Nups). Here, we review recent studies that provide novel insight into the structural and functional organization of nucleocytoplasmic transport. In addition, prospects towards a high resolution model of the nuclear pore are discussed.     

Transport of macromolecules between the nucleus and the cytoplasm.

Nuclear transport is an energy-dependent process mediated by saturable receptors. Import and export receptors are thought to recognize and bind to nuclear localization signals or nuclear export signals, respectively, in the transported molecules. The receptor-substrate interaction can be direct or mediated by an additional adapter protein. The transport receptors dock their cargoes to the nuclear pore complexes (NPC) and facilitate their translocation through the NPC. After delivering their cargoes, the receptors are recycled to initiate additional rounds of transport. Because a transport event for a cargo molecule is unidirectional, the transport receptors engage in asymmetric cycles of translocation across the NPC. The GTPase Ran acts as a molecular switch for receptor-cargo interaction and imparts directionality to the transport process. Recently, the combined use of different in vitro and in vivo approaches has led to the characterization of novel import and export signals and to the identification of the first nuclear import and export receptors.     

Mediators of Nuclear Protein Import Target Karyophilic Proteins to Pore Complexes of Cytoplasmic Annulate Lamellae

Nuclear pore complexes are constitutive structures of the nuclear envelope in eukaryotic cells and represent the sites where transport of molecules between nucleus and cytoplasm takes place. However, pore complexes of similar structure, but with largely unknown functional properties, are long known to occur also in certain cytoplasmic cisternae that have been termed annulate lamellae (AL). To analyze the capability of the AL pore complex to interact with the soluble mediators of nuclear protein import and their karyophilic protein substrates, we have performed a microinjection study in stage VI oocytes ofXenopus laevis.In these cells AL are especially abundant and can easily be identified by light and electron microscopy. Following injection into the cytoplasm, fluorochrome-labeled mediators of two different nuclear import pathways, importin β and transportin, not only associate with the nuclear envelope but also with AL. Likewise, nuclear localization signals (NLS) of the basic and M9 type, but not nuclear export signals, confer targeting and transient binding of fluorochrome-labeled proteins to cytoplasmic AL. Mutation or deletion of the NLS signals prevents these interactions. Furthermore, binding to AL is abolished by dominant negative inhibitors of nuclear protein import. Microinjections of gold-coupled NLS-bearing proteins reveal specific gold decoration at distinct sites within the AL pore complex. These include such at the peripheral pore complex-attached fibrils and at the central “transporter” and closely resemble those of “transport intermediates” found in electron microscopic studies of the nuclear pore complex (NPC). These data demonstrate that AL can represent distinct sites within the cytoplasm of transient accumulation of nuclear proteins and that the AL pore complex shares functional binding properties with the NPC.     

Nuclear protein accumulation by facilitated transport and intranuclear binding

Nuclear proteins are transported from the cytoplasm into the nucleus via nuclear envelope pore complexes (NPCs). At the molecular level, the mechanisms responsible for this transport remain obscure. However, it is known that, for many proteins, the process requires ATP and proceeds against formidable nucleocytoplasmic concentration gradients. Therefore, the NPC is often thought of as an active transport site. In this article, Philip Paine presents the alternative hypothesis that, on current evidence, protein translocation across the nuclear envelope and accumulation in the nucleus can equally well be explained by facilitated transport through the NPC and subsequent intranuclear binding.