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53BP1 (TP53-binding protein 1), a key player in DNA double-strand break repair, has a classical bipartite nuclear localization signal (NLS) of sequence 1666-GKRKLITSEEERSPAKRGRKS-1686 that binds to importin-α, a nuclear import adaptor protein. Nucleoporin Nup153 is involved in nuclear import of 53BP1, and the binding of Nup153 to importin-α has been proposed to promote efficient import of classical NLS-containing proteins. Here, the ARM-repeat domain of human importin-α3 bound to 53BP1 NLS was crystallized in the presence of a synthetic peptide corresponding to the extreme C-terminus of Nup153 (sequence: 1459-GTSFSGRKIKTAVRRRK-1475). The crystal belonged to space group I2, with unit-cell parameters a = 95.70, b = 79.60, c = 117.44 Å, β = 95.57°. The crystal diffracted X-rays to 1.9 Å resolution, and the structure was solved by molecular replacement. The asymmetric unit contained two molecules of importin-α3 and two molecules of 53BP1 NLS. Although no convincing density was observed for the Nup153 peptide, the electron density corresponding to 53BP1 NLS was unambiguous and continuous along the entire length of the bipartite NLS. The structure revealed a novel dimer of importin-α3, in which two protomers of importin-α3 are bridged by the bipartite NLS of 53BP1. In this structure, the upstream basic cluster of the NLS is bound to the minor NLS-binding site of one protomer of importin-α3, whereas the downstream basic cluster of the same chain of NLS is bound to the major NLS-binding site of another protomer of importin-α3. This quaternary structure is distinctly different from the previously determined crystal structure of mouse importin-α1 bound to the 53BP1 NLS. The atomic coordinates and structure factors have been deposited in the Protein Data Bank (accession code 8HKW).

Summary: Plant intracellular nucleotide‐binding leucine‐rich repeat immune receptors (NLRs) perceive the activity of pathogen‐secreted effector molecules that, when undetected, promote colonisation of hosts. Signalling from activated NLRs converges with and potentiates downstream responses from activated pattern recognition receptors (PRRs) that sense microbial signatures at the cell surface. Efficient signalling of both receptor branches relies on the host cell nucleus as an integration point for transcriptional reprogramming, and on the macromolecular transport processes that mediate the communication between cytoplasm and nucleoplasm. Studies on nuclear pore complexes (NPCs), the nucleoporin proteins (NUPs) that compose NPCs, and nuclear transport machinery constituents that control nucleocytoplasmic transport, have revealed that they play important roles in regulating plant immune responses. Here, we discuss the contributions of nucleoporins and nuclear transport receptor (NTR)‐mediated signal transduction in plant immunity with an emphasis on NLR immune signalling across the nuclear compartment boundary and within the nucleus. We also highlight and discuss cytoplasmic and nuclear functions of NLRs and their signalling partners and further consider the potential implications of NLR activation and resistosome formation in both cellular compartments for mediating plant pathogen resistance and programmed host cell death. [ABSTRACT FROM AUTHOR]

Nuclear pore complexes (NPCs) are the only transport channels that cross the nuclear envelope. Constructed from ~500–1000 nucleoporin proteins each, they are among the largest macromolecular assemblies in eukaryotic cells. Thanks to advances in structural analysis approaches, the construction principles and architecture of the NPC have recently been revealed at submolecular resolution. Although the overall structure and inventory of nucleoporins are conserved, NPCs exhibit significant compositional and functional plasticity even within single cells and surprising variability in their assembly pathways. Once assembled, NPCs remain seemingly unexchangeable in post-mitotic cells. There are a number of as yet unresolved questions about how the versatility of NPC assembly and composition is established, how cells monitor the functional state of NPCs or how they could be renewed. Here, we review current progress in our understanding of the key aspects of NPC architecture and lifecycle. [ABSTRACT FROM AUTHOR]

Defects in nucleocytoplasmic transport have been associated with several neurodegenerative disorders and, in particular, the formation of pathological protein aggregates characteristic for the respective disease. However, whether impaired nucleocytoplasmic transport is a consequence of such aggregates or rather contributes to their formation is still mostly unclear. In this review, we summarize recent findings how both soluble and stationary components of the nucleocytoplasmic transport machinery are altered in neurodegenerative diseases, in particular amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Alzheimer's disease (AD) and Huntington's disease (HD). We discuss the functional significance of the observed defects for nucleocytoplasmic transport of proteins and mRNAs. Moreover, we highlight interesting parallels observed in physiological ageing and the premature ageing syndrome progeria and propose that they that might provide mechanistic insights also for neurodegenerative processes. [ABSTRACT FROM AUTHOR]

In Arabidopsis, EXPORTIN1A (HIT2/XPO1A) and EXPORTIN1B (XPO1B) mediate the translocation of nuclear export sequence (NES)-bearing proteins from nucleus to cytoplasm. However, a mutation in HIT2/XPO1A but not in XPO1B induces sensitivity to high irradiance (HI). Arabidopsis thaliana heat stress elements A4a and A5 (AtHsfA4a and AtHsfA5) are involved in plant responses to HI and possess NESs; therefore, their nucleo-cytoplasmic partitioning was analyzed. In wild-type and xpo1b mutant cells, AtHsfA4a normally remained in the cytoplasm but became concentrated in the nucleus following exposure to HI, whereas AtHsfA5 was constitutively distributed in both cytoplasm and nucleus. However, in hit2/xpo1a mutant, AtHsfA4a and AtHsfA5 were always confined to the nucleus, regardless of the irradiance. Although AtHsfA4a can enhance the ability of plants to scavenge H2O2, and AtHsfA5 is a repressor of AtHsfA4a, athsfa5 but not athsfa4a mutant plants exhibited HI sensitivity. Additionally, athsfa4a plants expressing AtHsfA4aΔNES were sensitive to HI, but athsfa5 plants expressing AtHsfA5ΔNES were not. Meanwhile, hit2/athsfa4a double mutant was more tolerant to HI than hit2. These results indicate that both AtHsfA4a and AtHsfA5 were HIT2/XPO1A-specific substrates. Long-term accumulation of AtHsfA4a contributed to the hit2 HI-sensitive phenotype independent of the scavenging ability of H2O2, and the presence of AtHsfA5 could mitigate this adverse effect.

In eukaryotic cells, RNA binding proteins (RBPs) play critical roles in regulating almost every aspect of gene expression, often shuttling between the nucleus and the cytoplasm. They are also key determinants in cell fate via controlling the target mRNAs under the regulation of various signaling pathways in response to environmental stresses. Therefore, understanding the mechanisms that couple the location of mRNA and RBPs is a major challenge in the field of gene expression and signal responses. In fission yeast, a KH-type RBP Rnc1 negatively regulates MAPK signaling activation via mRNA stabilization of the dual-specificity MAPK phosphatase Pmp1, which dephosphorylates MAPK Pmk1. Rnc1 also serves as a target of MAPK phosphorylation, which makes a feedback loop mediated by an RBP. We recently discovered that the nuclear export of Rnc1 requires mRNA-binding ability and the mRNA export factor Rae1. This strongly suggested the presence of an mRNA-export system, which recognizes the mRNA/RBP complex and dictates the location and post-transcriptional regulation of mRNA cargo. Here, we briefly review the known mechanisms of general nuclear transporting systems, with an emphasis on our recent findings on the spatial regulation of Rnc1 and its impact on the regulation of the MAPK signal transduction cascade. [ABSTRACT FROM AUTHOR]

The nucleocytoplasmic exchange is of fundamental importance to eukaryotic life and is mediated by karyopherins, a superfamily of nuclear transport receptors. However, the function and cargo spectrum of plant karyopherins are largely obscure. Here, we report proximity-labeling-based proteomic profiling of in vivo substrates of KA120, a karyopherin-β required for suppressing autoimmune induction in Arabidopsis. We identify multiple components of the MOS4-associated complex (MAC), a conserved splicing regulatory protein complex. Surprisingly, we find that KA120 does not affect the nucleocytoplasmic distribution of MAC proteins but rather prevents their protein condensation in the nucleus. Furthermore, we demonstrate that MAC condensation is robustly induced by pathogen infection, which is sufficient to activate defense gene expression, possibly by sequestrating negative immune regulators via phase transition. Our study reveals a noncanonical chaperoning activity of a plant karyopherin, which modulates the nuclear condensation of an evolutionarily conserved splicing regulatory complex to coordinate plant immune activation. [Display omitted] • Loss of KA120 leads to MAC-dependent autoimmune activation in plants • KA120 interacts with MAC proteins and prevents spontaneous nuclear condensation of MAC • MAC condensation is robustly induced by pathogen-triggered immune activation • The assembly of MAC condensates is sufficient to activate defense response in plants Jia et al. report that KA120, a conserved nuclear transport receptor, processes a noncanonical chaperoning activity to prevent spontaneous nuclear condensation of the MAC protein complex, an essential regulator of pre-mRNA splicing. However, pathogen infection triggers MAC condensate assembly in plants, sequestering negative immune regulators to coordinate host defense response. [ABSTRACT FROM AUTHOR]

In Arabidopsis, EXPORTIN1A (HIT2/XPO1A) and EXPORTIN1B (XPO1B) mediate the translocation of nuclear export sequence (NES)-bearing proteins from nucleus to cytoplasm. However, a mutation in HIT2/XPO1A but not in XPO1B induces sensitivity to high irradiance (HI). Arabidopsis thaliana heat stress elements A4a and A5 (AtHsfA4a and AtHsfA5) are involved in plant responses to HI and possess NESs; therefore, their nucleo-cytoplasmic partitioning was analyzed. In wild-type and xpo1b mutant cells, AtHsfA4a normally remained in the cytoplasm but became concentrated in the nucleus following exposure to HI, whereas AtHsfA5 was constitutively distributed in both cytoplasm and nucleus. However, in hit2/xpo1a mutant, AtHsfA4a and AtHsfA5 were always confined to the nucleus, regardless of the irradiance. Although AtHsfA4a can enhance the ability of plants to scavenge H2O2, and AtHsfA5 is a repressor of AtHsfA4a, athsfa5 but not athsfa4a mutant plants exhibited HI sensitivity. Additionally, athsfa4a plants expressing AtHsfA4aΔNES were sensitive to HI, but athsfa5 plants expressing AtHsfA5ΔNES were not. Meanwhile, hit2/athsfa4a double mutant was more tolerant to HI than hit2. These results indicate that both AtHsfA4a and AtHsfA5 were HIT2/XPO1A-specific substrates. Long-term accumulation of AtHsfA4a contributed to the hit2 HI-sensitive phenotype independent of the scavenging ability of H2O2, and the presence of AtHsfA5 could mitigate this adverse effect. [ABSTRACT FROM AUTHOR]

The permeability barrier of nuclear pore complexes (NPCs) controls bulk nucleocytoplasmic exchange. It consists of nucleoporin domains rich in phenylalanine-glycine motifs (FG domains). As a bottom-up nanoscale model for the permeability barrier, we have used planar films produced with three different end-grafted FG domains, and quantitatively analyzed the binding of two different nuclear transport receptors (NTRs), NTF2 and Importin β, together with the concomitant film thickness changes. NTR binding caused only moderate changes in film thickness; the binding isotherms showed negative cooperativity and could all be mapped onto a single master curve. This universal NTR binding behavior – a key element for the transport selectivity of the NPC – was quantitatively reproduced by a physical model that treats FG domains as regular, flexible polymers, and NTRs as spherical colloids with a homogeneous surface, ignoring the detailed arrangement of interaction sites along FG domains and on the NTR surface.

53BP1 (TP53-binding protein 1), a key player in DNA double-strand break repair, has a classical bipartite nuclear localization signal (NLS) of sequence 1666-GKRKLITSEEERSPAKRGRKS-1686 that binds to importin-α, a nuclear import adaptor protein. Nucleoporin Nup153 is involved in nuclear import of 53BP1, and the binding of Nup153 to importin-α has been proposed to promote efficient import of classical NLS-containing proteins. Here, the ARM-repeat domain of human importin-α3 bound to 53BP1 NLS was crystallized in the presence of a synthetic peptide corresponding to the extreme C-terminus of Nup153 (sequence: 1459-GTSFSGRKIKTAVRRRK-1475). The crystal belonged to space group I 2, with unit-cell parameters a  = 95.70, b  = 79.60, c  = 117.44 Å, β  = 95.57°. The crystal diffracted X-rays to 1.9 Å resolution, and the structure was solved by molecular replacement. The asymmetric unit contained two molecules of importin-α3 and two molecules of 53BP1 NLS. Although no convincing density was observed for the Nup153 peptide, the electron density corresponding to 53BP1 NLS was unambiguous and continuous along the entire length of the bipartite NLS. The structure revealed a novel dimer of importin-α3, in which two protomers of importin-α3 are bridged by the bipartite NLS of 53BP1. In this structure, the upstream basic cluster of the NLS is bound to the minor NLS-binding site of one protomer of importin-α3, whereas the downstream basic cluster of the same chain of NLS is bound to the major NLS-binding site of another protomer of importin-α3. This quaternary structure is distinctly different from the previously determined crystal structure of mouse importin-α1 bound to the 53BP1 NLS. The atomic coordinates and structure factors have been deposited in the Protein Data Bank (accession code 8HKW)., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:, (© 2023 The Author(s).)

Hikeshi is a nuclear transport receptor required for cell survival after stress. It mediates heat-shock-induced nuclear import of 70 kDa heat-shock proteins (Hsp70s) through interactions with FG-nucleoporins (FG-Nups), which are proteins in nuclear pore complexes (NPCs). Here, the crystal structure of human Hikeshi is presented at 1.8 Å resolution. Hikeshi forms an asymmetric homodimer that is responsible for the interaction with Hsp70s. The asymmetry of Hikeshi arises from the distinct conformation of the C-terminal domain (CTD) and the flexibility of the linker regions of each monomer. Structure-guided mutational analyses showed that both the flexible linker region and the CTD are important for nuclear import of Hsp70. Pull-down assays revealed that only full-length Hsp70s can interact with Hikeshi. The N-terminal domain (NTD) consists of a jelly-roll/β-sandwich fold structure which contains hydrophobic pockets involved in FG-Nup recognition. A unique extended loop (E-loop) in the NTD is likely to regulate the interactions of Hikeshi with FG-Nups. The crystal structure of Hikeshi explains how Hikeshi participates in the regulation of nuclear import through the recognition of FG-Nups and which part of Hikeshi affects its binding to Hsp70. This study is the first to yield structural insight into this highly unique import receptor. [ABSTRACT FROM AUTHOR]

Nucleocytoplasmic trafficking in eukaryotic cells is a highly regulated and coordinated process which involves an increasing variety of soluble nuclear transport receptors. Generally, transport receptors specifically bind their cargo and facilitate its transition through nuclear pore complexes, aqueous channels connecting the two compartments. Directionality of such transport events by receptors of the importin β superfamily requires the interaction with the small GTPase Ras-related nuclear antigen (Ran). While importins need RanGTP to release their cargo in the nucleus and thus to terminate import, exportins recruit cargo in the RanGTP-bound state. The exportin chromosome region maintenance 1 (CRM1) is a highly versatile transport receptor that exports a plethora of different protein and RNP cargoes. Moreover, binding of RanGTP and of cargo to CRM1 are highly cooperative events despite the fact that cargo and RanGTP do not interact directly in crystal structures of assembled export complexes. Integrative approaches have recently unraveled the individual steps of the CRM1 transport cycle at a structural level and explained how the HEAT-repeat architecture of CRM1 provides a framework for the key elements to mediate allosteric interactions with RanGTP, Ran binding proteins and cargo. Moreover, during the last decade, CRM1 has become a more and more appreciated target for anti-cancer drugs. Hence, detailed understanding of the flexibility, the regulatory features and the positive binding cooperativity between CRM1, Ran and cargo is a prerequisite for the development of highly effective drugs. Here we review recent structural advances in the characterization of CRM1 and CRM1-containing complexes with a special emphasis on X-ray crystallographic studies. [ABSTRACT FROM AUTHOR]

Abstract: It is generally accepted that transport through the nuclear pore complex (NPC) involves an abundance of phenylalanine–glycine rich protein domains (FG-domains) that serve as docking sites for soluble nuclear transport receptors (NTRs) and their cargo complexes. But the precise mechanism of translocation through the NPC allowing for high speed and selectivity is still vividly debated. To ultimately decipher the underlying gating mechanism it is indispensable to shed more light on the molecular arrangement of FG-domains and the distribution of NTR-binding sites within the central channel of the NPC. In this review we revisit current transport models, summarize recent results regarding translocation through the NPC obtained by super-resolution microscopy and finally discuss the status and potential of optical methods in the analysis of the NPC. [Copyright &y& Elsevier]

•The Arabidopsis heat-intolerant 2 ( hit2) mutant was isolated on the basis of its impaired ability to withstand moderate heat stress (37°C). Determination of the genetic mutation that underlies the hit2 thermosensitive phenotype allowed better understanding of the mechanisms by which plants cope with heat stress. •Genetic analysis revealed that hit2 is a single recessive mutation. Map-based cloning was used to identify the hit2 locus. The response of hit2 to other types of heat stress was also investigated to characterize the protective role of HIT2. • hit2 was defective in basal but not in acquired thermotolerance. hit2 was sensitive to methyl viologen-induced oxidative stress, and the survival of hit2 seedlings in response to heat stress was affected by light conditions. The mutated locus was located at the EXPORTIN1A ( XPO1A) gene, which encodes a nuclear transport receptor. Two T-DNA insertion lines, xpo1a-1 and xpo1a-3, exhibited the same phenotypes as hit2. •The results provide evidence that Arabidopsis XPO1A is dispensable for normal plant growth and development but is essential for thermotolerance, in part by mediating the protection of plants against heat-induced oxidative stress. [ABSTRACT FROM AUTHOR]

Transport from and into the nucleus is essential to all eukaryotic life and occurs through the nuclear pore complex (NPC). There are a multitude of data supporting a role for nuclear transport in neurodegenerative diseases, but actual transport assays in disease models have provided diverse outcomes. In this review, we summarize how nuclear transport works, which transport assays are available, and what matters complicate the interpretation of their results. Taking a specific type of ALS caused by mutations in C9orf72 as an example, we illustrate these complications, and discuss how the current data do not firmly answer whether the kinetics of nucleocytoplasmic transport are altered. Answering this open question has far-reaching implications, because a positive answer would imply that widespread mislocalization of proteins occurs, far beyond the reported mislocalization of transport reporters, and specific proteins such as FUS, or TDP43, and thus presents a challenge for future research. [ABSTRACT FROM AUTHOR]

Hikeshi is a new nuclear transport receptor that plays an important role in the nuclear import of Hsp70 heat-shock proteins under thermal stress. Wild-type human Hikeshi and its Phe97Ala mutant were overproduced and purified using an Escherichia coli expression system. The purified proteins were crystallized using the hanging-drop vapour-diffusion technique. Wild-type crystals grew in space group C2221, with unit-cell parameters a = 61.1, b = 137.8, c = 97.9 Å, α = 90.0, β = 90.0, γ = 90.0°. Phe97Ala mutant crystals were obtained in space group P32, with unit-cell parameters a = 85.7, b = 85.7, c = 69.1 Å, α = 90.0, β = 90.0, γ = 120.0°. These crystals diffracted to 1.8 and 2.5 Å resolution, respectively. This study is the first to yield structural insight into this highly unusual fourth import receptor after importins, NTF2 and TAP. [ABSTRACT FROM AUTHOR]

Macromolecular cargoes are asymmetrically partitioned in the nucleus or cytoplasm by nucleocytoplasmic transport (NCT). At the center of this activity lies the nuclear pore complex (NPC), through which soluble factors circulate to orchestrate NCT. These include cargo-carrying importin and exportin receptors from the ß-karyopherin (Kapß) family and the small GTPase Ran, which switches between guanosine triphosphate (GTP)- and guanosine diphosphate (GDP)-bound forms to regulate cargo delivery and compartmentalization. Ongoing efforts have shed considerable light on how these soluble factors traverse the NPC permeability barrier to sustain NCT. However, this does not explain how importins and exportins are partitioned in the cytoplasm and nucleus, respectively, nor how a steep RanGTP-RanGDP gradient is maintained across the nuclear envelope. In this Review, we peel away the multiple layers of control that regulate NCT and juxtapose unresolved features against known aspects of NPC function. Finally, we discuss how NPCs might function synergistically with Kapßs, cargoes and Ran to establish the asymmetry of NCT. [ABSTRACT FROM AUTHOR]

Macromolecular cargoes are asymmetrically partitioned in the nucleus or cytoplasm by nucleocytoplasmic transport (NCT). At the center of this activity lies the nuclear pore complex (NPC), through which soluble factors circulate to orchestrate NCT. These include cargo-carrying importin and exportin receptors from the ß-karyopherin (Kapß) family and the small GTPase Ran, which switches between guanosine triphosphate (GTP)- and guanosine diphosphate (GDP)-bound forms to regulate cargo delivery and compartmentalization. Ongoing efforts have shed considerable light on how these soluble factors traverse the NPC permeability barrier to sustain NCT. However, this does not explain how importins and exportins are partitioned in the cytoplasm and nucleus, respectively, nor how a steep RanGTP-RanGDP gradient is maintained across the nuclear envelope. In this Review, we peel away the multiple layers of control that regulate NCT and juxtapose unresolved features against known aspects of NPC function. Finally, we discuss how NPCs might function synergistically with Kapßs, cargoes and Ran to establish the asymmetry of NCT. [ABSTRACT FROM AUTHOR]

Nucleocytoplasmic transport of proteins with molar masses of larger than 60 000 is mediated by transport receptors. The transport receptor transportin1 (Trn1) transports various kinds of RNA-binding proteins such as JKTBP, hnRNP D and TAP. Trn1 was successfully cocrystallized with nucleocytoplasmic shuttling fragments of JKTBP and hnRNP D and a nuclear localization fragment of TAP. The crystal of the Trn1–JKTBP fragment complex belongs to space group P21212, with unit-cell parameters a = 131.5, b =171.5, c = 68.2 Å. The crystals of Trn1 in complex with hnRNP D and TAP fragments are orthorhombic, space group P212121, with unit-cell parameters a = 69.1, b = 119.1, c = 151.1 Å and a = 69.0, b = 119.1, c = 146.0 Å, respectively. The crystals diffracted to beyond 3.0, 3.2 and 2.4 Å resolution, respectively, using synchrotron radiation at SPring-­8. [ABSTRACT FROM AUTHOR]

Nuclear import receptors, also called importins, mediate nuclear import of proteins and chaperone aggregation-prone cargoes (e.g., neurodegeneration-linked RNA-binding proteins [RBPs]) in the cytoplasm. Importins were identified as modulators of cellular toxicity elicited by arginine-rich dipeptide repeat proteins (DPRs), an aberrant protein species found in C9orf72-linked amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Mechanistically, the link between importins and arginine-rich DPRs remains unclear. Here, we show that arginine-rich DPRs (poly-GR and poly-PR) bind directly to multiple importins and, in excess, promote their insolubility and condensation. In cells, poly-GR impairs Impα/β-mediated nuclear import, including import of TDP-43, an RBP that aggregates in C9orf72-ALS/FTD patients. Arginine-rich DPRs promote phase separation and insolubility of TDP-43 in vitro and in cells, and this pathological interaction is suppressed by elevating importin concentrations. Our findings suggest that importins can decrease toxicity of arginine-rich DPRs by suppressing their pathological interactions., Competing Interests: Declaration of Interests J.S. is a consultant for Dewpoint Therapeutics., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Phenylalanine-glycine-rich nucleoporins (FG-Nups) are intrinsically disordered proteins, constituting the selective barrier of the nuclear pore complex. They are highly dynamic under physiological conditions and studying their interaction with nuclear transport receptors (NTRs) is key to understanding the molecular mechanism of nucleocytoplasmic transport. Distinct conformational features of FG-Nups interacting with diverse NTRs can be detected by multiparameter single-molecule fluorescence energy transfer (smFRET), which is a powerful technique for studying the dynamics and interactions of biomolecules in solution. Here we provide a detailed protocol utilizing smFRET to reveal differential binding mechanisms of FG-Nups to NTRs, with a focus on practical considerations on sample preparation of unglycosylated and glycosylated FG-Nups, site-specific dual-labeling, smFRET measurements, and data analysis., (© 2018 Elsevier Inc. All rights reserved.)

The permeability barrier of nuclear pore complexes (NPCs) controls bulk nucleocytoplasmic exchange. It consists of nucleoporin domains rich in phenylalanine-glycine motifs (FG domains). As a bottom-up nanoscale model for the permeability barrier, we have used planar films produced with three different end-grafted FG domains, and quantitatively analyzed the binding of two different nuclear transport receptors (NTRs), NTF2 and Importin β, together with the concomitant film thickness changes. NTR binding caused only moderate changes in film thickness; the binding isotherms showed negative cooperativity and could all be mapped onto a single master curve. This universal NTR binding behavior - a key element for the transport selectivity of the NPC - was quantitatively reproduced by a physical model that treats FG domains as regular, flexible polymers, and NTRs as spherical colloids with a homogeneous surface, ignoring the detailed arrangement of interaction sites along FG domains and on the NTR surface.

Nucleocytoplasmic transport is an essential cellular activity and occurs via nuclear pore complexes (NPCs) that reside in the double membrane of the nuclear envelope. Significant progress has been made during the past few years in unravelling the ultrastructural organization of NPCs and their constituents, the nucleoporins, by cryo-electron tomography and X-ray crystallography. Mass spectrometry and genomic approaches have provided deeper insight into the specific regulation and fine tuning of individual nuclear transport pathways. Recent research has also focused on the roles nucleoporins play in health and disease, some of which go beyond nucleocytoplasmic transport. Here we review emerging results aimed at understanding NPC architecture and nucleocytoplasmic transport at the atomic level, elucidating the specific function individual nucleoporins play in nuclear trafficking, and finally lighting up the contribution of nucleoporins and nuclear transport receptors in human diseases, such as cancer and certain genetic disorders., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The nuclear pore complex (NPC) is composed of ∼30 unique proteins, collectively referred to as nucleoporins or Nups. While metazoan Nups are known to be phosphorylated during mitosis to cause disassembly of the NPC, what is less clear is whether Nups are phosphorylated and regulated by extracellular stimuli in interphase cells. Our multi-step phosphoproteomic approach revealed a number of physiologically relevant extracellular signal-regulated kinase (ERK) targets, including Nups containing FG repeats (FG Nups) that provide binding sites for nuclear transport receptors (NTRs) during the NPC passage. The phosphorylation of FG Nups by ERK does not affect the overall architecture of the NPC but directly inhibits their interactions with NTRs and regulates the permeability barrier properties of the NPC. Such regulation at the levels of transport machinery is expected to have a broad impact on cellular physiology through the spatiotemporal control of signaling events. Until recently, many studies have focused on cellular signaling-mediated phosphorylation of individual cargo proteins, such as transcription factors. An understanding of the effects of signaling pathways on nucleocytoplasmic transport machinery is only beginning to emerge.