An allosteric mechanism to displace nuclear export cargo from CRM1 and RanGTP by RanBP1

The karyopherin CRM1 mediates nuclear export of proteins and ribonucleoproteins bearing a leucine‐rich nuclear export signal (NES). To elucidate the precise mechanism by which NES‐cargos are dissociated from CRM1 in the cytoplasm, which is important for transport directionality, we determined a 2.0‐Å resolution crystal structure of yeast CRM1:RanBP1:RanGTP complex, an intermediate in the disassembly of the CRM1 nuclear export complex. The structure shows that on association of Ran‐binding domain (RanBD) of RanBP1 with CRM1:NES‐cargo:RanGTP complex, RanBD and the C‐terminal acidic tail of Ran induce a large movement of the intra‐HEAT9 loop of CRM1. The loop moves to the CRM1 inner surface immediately behind the NES‐binding site and causes conformational rearrangements in HEAT repeats 11 and 12 so that the hydrophobic NES‐binding cleft on the CRM1 outer surface closes, squeezing out the NES‐cargo. This allosteric mechanism accelerates dissociation of NES by over two orders of magnitude. Structure‐based mutagenesis indicated that the HEAT9 loop also functions as an allosteric autoinhibitor to stabilize CRM1 in a conformation that is unable to bind NES‐cargo in the absence of RanGTP.     

Supraphysiological nuclear export signals bind CRM1 independently of RanGTP and arrest at Nup358

Leucine-rich nuclear export signals (NESs) mediate rapid nuclear export of proteins via interaction with CRM1. This interaction is stimulated by RanGTP but remains of a relatively low affinity. In order to identify strong signals, we screened a 15-mer random peptide library for CRM1 binding, both in the presence and absence of RanGTP. Under each condition, strikingly similar signals were enriched, conforming to the NES consensus sequence. A derivative of an NES selected in the absence of RanGTP exhibits very high affinity for CRM1 in vitro and stably binds without the requirement of RanGTP. Localisation studies and RNA interference demonstrate inefficient CRM1-mediated export and accumulation of CRM1 complexed with the high-affinity NES at nucleoporin Nup358. These results provide in vivo evidence for a nuclear export reaction intermediate. They suggest that NESs have evolved to maintain low affinity for CRM1 to allow efficient export complex disassembly and release from Nup358.     

核孔复合体的动态组装

核孔是介导所有大分子入核出核的唯一通道。在整个生命活动中,核孔复合体的组成蛋白总是处于动态变化中。核孔复合体的动态组装改变了核质转运状态,并最终改变了细胞的功能。     

Functional Analysis of Tpr: Identification of Nuclear Pore Complex Association and Nuclear Localization Domains and a Role in mRNA Export

Tpr is a 270-kD coiled-coil protein localized to intranuclear filaments of the nuclear pore complex (NPC). The mechanism by which Tpr contributes to the structure and function of the nuclear pore is currently unknown. To gain insight into Tpr function, we expressed the full-length protein and several subdomains in mammalian cell lines and examined their effects on nuclear pore function. Through this analysis, we identified an NH₂-terminal domain that was sufficient for association with the nucleoplasmic aspect of the NPC. In addition, we unexpectedly found that the acidic COOH terminus was efficiently transported into the nuclear interior, an event that was apparently mediated by a putative nuclear localization sequence. Ectopic expression of the full-length Tpr caused a dramatic accumulation of poly(A)⁺ RNA within the nucleus. Similar results were observed with domains that localized to the NPC and the nuclear interior. In contrast, expression of these proteins did not appear to affect nuclear import. These data are consistent with a model in which Tpr is tethered to intranuclear filaments of the NPC by its coiled coil domain leaving the acidic COOH terminus free to interact with soluble transport factors and mediate export of macromolecules from the nucleus.     

Solution structure of the Ran-binding domain 2 of RanBP2 and its interaction with the C terminus of Ran

The termination of export processes from the nucleus to the cytoplasm in higher eukaryotes is mediated by binding of the small GTPase Ran as part of the export complexes to the Ran-binding domains (RanBD) of Ran-binding protein 2 (RanBP2) of the nuclear pore complex. So far, the structures of the first RanBD of RanBP2 and of RanBP1 in complexes with Ran have been known from X-ray crystallographic studies. Here we report the NMR solution structure of the uncomplexed second RanBD of RanBP2. The structure shows a pleckstrin homology (PH) fold featuring two almost orthogonal beta-sheets consisting of three and four strands and an alpha-helix sitting on top. This is in contrast to the RanBD in the crystal structure complexes in which one beta-strand is missing. That is probably due to the binding of the C-terminal alpha-helix of Ran to the RanBD in these complexes. To analyze the interaction between RanBD2 and the C terminus of Ran, NMR-titration studies with peptides comprising the six or 28 C-terminal residues of Ran were performed. While the six-residue peptide alone does not bind to RanBD2 in a specific manner, the 28-residue peptide, including the entire C-terminal helix of Ran, binds to RanBD2 in a manner analogous to the crystal structures. By solving the solution structure of the 28mer peptide alone, we confirmed that it adopts a stable alpha-helical structure like in native Ran and therefore serves as a valid model of the Ran C terminus. These results support current models that assume recognition of the transport complexes by the RanBDs through the Ran C terminus that is exposed in these complexes.     

Ran-binding protein 3 is a cofactor for Crm1-mediated nuclear protein export.

Crm1 is a member of the karyopherin family of nucleocytoplasmic transport receptors and mediates the export of proteins from the nucleus by forming a ternary complex with cargo and Ran:GTP. This complex translocates through the nuclear pores and dissociates in the cytosol. The yeast protein Yrb2p participates in this pathway and binds Crm1, but its mechanism of action has not been established. We show that the human orthologue of Yrb2p, Ran-binding protein 3 (RanBP3), acts as a cofactor for Crm1-mediated export in a permeabilized cell assay. RanBP3 binds directly to Crm1, and the complex possesses an enhanced affinity for both Ran:GTP and cargo. RanBP3 shuttles between the nucleus and the cytoplasm by a Crm1-dependent mechanism, and the Crm1--RanBP3-NES-Ran:GTP quarternary complex can associate with nucleoporins. We infer that this complex translocates through the nuclear pore to the cytoplasm where it is disassembled by RanBP1 and Ran GTPase--activating protein.     

Sumoylation of the GTPase Ran by the RanBP2 SUMO E3 Ligase Complex

The SUMO E3 ligase complex RanBP2/RanGAP1*SUMO1/Ubc9 localizes at cytoplasmic nuclear pore complex (NPC) filaments and is a docking site in nucleocytoplasmic transport. RanBP2 has four Ran binding domains (RBDs), two of which flank RanBP2''s E3 ligase region. We thus wondered whether the small GTPase Ran is a target for RanBP2-dependent sumoylation. Indeed, Ran is sumoylated both by a reconstituted and the endogenous RanBP2 complex in semi-permeabilized cells. Generic inhibition of SUMO isopeptidases or depletion of the SUMO isopeptidase SENP1 enhances sumoylation of Ran in semi-permeabilized cells. As Ran is typically associated with transport receptors, we tested the influence of Crm1, Imp β, Transportin, and NTF2 on Ran sumoylation. Surprisingly, all inhibited Ran sumoylation. Mapping Ran sumoylation sites revealed that transport receptors may simply block access of the E2-conjugating enzyme Ubc9, however the acceptor lysines are perfectly accessible in Ran/NTF2 complexes. Isothermal titration calorimetry revealed that NTF2 prevents sumoylation by reducing RanGDP''s affinity to RanBP2''s RBDs to undetectable levels. Taken together, our findings indicate that RanGDP and not RanGTP is the physiological target for the RanBP2 SUMO E3 ligase complex. Recognition requires interaction of Ran with RanBP2''s RBDs, which is prevented by the transport factor NTF2.     

Quantitative Analysis of Membrane Protein Transport Across the Nuclear Pore Complex

Nuclear transport of the Saccharomyces cerevisiae membrane proteins Src1/Heh1 and Heh2 across the NPC is facilitated by a long intrinsically disordered linker between the nuclear localization signal (NLS) and the transmembrane domain. The import of reporter proteins derived from Heh2 is dependent on the FG‐Nups in the central channel, and the linker can position the transport factor‐bound NLS in the vicinity of the FG‐Nups in the central channel, while the transmembrane segment resides in the pore membrane. Here, we present a quantitative analysis of karyopherin‐mediated import and passive efflux of reporter proteins derived from Heh2, including data on the mobility of the reporter proteins in different membrane compartments. We show that membrane proteins with extralumenal domains up to 174 kDa, terminal to the linker and NLS, passively leak out of the nucleus via the NPC, albeit at a slow rate. We propose that also during passive efflux, the unfolded linker facilitates the passage of extralumenal domains through the central channel of the NPC .     

CRM1 controls the composition of nucleoplasmic pre-snoRNA complexes to licence them for nucleolar transport

Transport of C/D snoRNPs to nucleoli involves nuclear export factors. In particular, CRM1 binds nascent snoRNPs, but its precise role remains unknown. We show here that both CRM1 and nucleocytoplasmic trafficking are required to transport snoRNPs to nucleoli, but the snoRNPs do not transit through the cytoplasm. Instead, CRM1 controls the composition of nucleoplasmic pre-snoRNP complexes. We observed that Tgs1 long form (Tgs1 LF), the long isoform of the cap hypermethylase, contains a leucine-rich nuclear export signal, shuttles in a CRM1-dependent manner, and binds to the nucleolar localization signal (NoLS) of the core snoRNP protein Nop58. In vitro data indicate that CRM1 binds Tgs1 LF and promotes its dissociation from Nop58 NoLS, and immunoprecipitation experiments from cells indicate that the association of Tgs1 LF with snoRNPs increases upon CRM1 inhibition. Thus, CRM1 appears to promote nucleolar transport of snoRNPs by removing Tgs1 LF from the Nop58 NoLS. Microarray/IP data show that this occurs on most snoRNPs, from both C/D and H/ACA families, and on the telomerase RNA. Hence, CRM1 provides a general molecular link between nuclear events and nucleocytoplasmic trafficking.     

Nucleoporin FG Domains Facilitate mRNP Remodeling at the Cytoplasmic Face of the Nuclear Pore Complex

Directional export of messenger RNA (mRNA) protein particles (mRNPs) through nuclear pore complexes (NPCs) requires multiple factors. In Saccharomyces cerevisiae, the NPC proteins Nup159 and Nup42 are asymmetrically localized to the cytoplasmic face and have distinct functional domains: a phenylalanine-glycine (FG) repeat domain that docks mRNP transport receptors and domains that bind the DEAD-box ATPase Dbp5 and its activating cofactor Gle1, respectively. We speculated that the Nup42 and Nup159 FG domains play a role in positioning mRNPs for the terminal mRNP-remodeling steps carried out by Dbp5. Here we find that deletion (Δ) of both the Nup42 and Nup159 FG domains results in a cold-sensitive poly(A)+ mRNA export defect. The nup42ΔFG nup159ΔFG mutant also has synthetic lethal genetic interactions with dbp5 and gle1 mutants. RNA cross-linking experiments further indicate that the nup42ΔFG nup159ΔFG mutant has a reduced capacity for mRNP remodeling during export. To further analyze the role of these FG domains, we replaced the Nup159 or Nup42 FG domains with FG domains from other Nups. These FG “swaps” demonstrate that only certain FG domains are functional at the NPC cytoplasmic face. Strikingly, fusing the Nup42 FG domain to the carboxy-terminus of Gle1 bypasses the need for the endogenous Nup42 FG domain, highlighting the importance of proximal positioning for these factors. We conclude that the Nup42 and Nup159 FG domains target the mRNP to Gle1 and Dbp5 for mRNP remodeling at the NPC. Moreover, these results provide key evidence that character and context play a direct role in FG domain function and mRNA export.     

CRM1 Is a Cellular Target of Curcumin: New Insights for the Myriad of Biological Effects of an Ancient Spice

Curcumin is the major constituent of turmeric plant, an ancient spice widely used in Indian cuisine and traditional herbal medicine. Recently, the potential medical use of curcumin as anti‐cancer and anti‐inflammatory agent has set off an upsurge in research into the mechanism for its broad biological effects. We showed that CRM1, an important nuclear exportin, is a cellular target of curcumin by serious experimental and theoretical investigation. Using a nuclear export functional assay, we observed a clear and rapid shift of cargo proteins from a cytoplasmic localization to the nucleus when treated with curcumin or its structural analogue dibenzylideneacetone (DBA). We demonstrated that curcumin could specifically target the conserved Cys⁵²⁸ of CRM1 through mass spectrometric analysis and in vivo experiments. Furthermore, computational modeling has revealed that curcumin could be correctly docked into the hydrophobic pocket of CRM1 judged from shape complementarity and putative molecular interactions. The Michael acceptor moiety on curcumin is within the appropriate distance to enable Michael reaction with Cys residue of CRM1. More importantly, we showed that nuclear retention of FOXO1 could be observed in the presence of Leptomycin B (LMB) or curcumin whereas in cells expressing the CRM1‐Cys⁵²⁸ mutant, only a cytoplasmic localization was observed. The inhibition of nuclear traffic by curcumin may account for its myriad of biological effects, particularly for its therapeutic properties in cancer and inflammatory diseases. Our findings may have important implications for further clinical investigation of curcumin .     

Scyl1 Facilitates Nuclear tRNA Export in Mammalian Cells by Acting at the Nuclear Pore Complex

Scyl1 is an evolutionarily conserved N-terminal protein kinase-like domain protein that plays a role in COP1-mediated retrograde protein trafficking in mammalian cells. Furthermore, loss of Scyl1 function has been shown to result in neurodegenerative disorders in mice. Here, we report that Scyl1 is also a cytoplasmic component of the mammalian nuclear tRNA export machinery. Like exportin-t, overexpression of Scyl1 restored export of a nuclear export-defective serine amber suppressor tRNA mutant in COS-7 cells. Scyl1 binds tRNA saturably, and associates with the nuclear pore complex by interacting, in part, with Nup98. Scyl1 copurifies with the nuclear tRNA export receptors exportin-t and exportin-5, the RanGTPase, and the eukaryotic elongation factor eEF-1A, which transports aminoacyl-tRNAs to the ribosomes. Scyl1 interacts directly with exportin-t and RanGTP but not with eEF-1A or RanGDP in vitro. Moreover, exportin-t containing tRNA, Scyl1, and RanGTP form a quaternary complex in vitro. Biochemical characterization also suggests that the nuclear aminoacylation-dependent pathway is primarily responsible for tRNA export in mammalian cells. These findings together suggest that Scyl1 participates in the nuclear aminoacylation-dependent tRNA export pathway and may unload aminoacyl-tRNAs from the nuclear tRNA export receptor at the cytoplasmic side of the nuclear pore complex and channels them to eEF-1A.     

Cryo-electron Microscopy Reveals the Structure of the Nuclear Pore Complex

The nuclear pore complex (NPC) is a giant protein assembly that penetrates the double layers of the nuclear membrane. The overall structure of the NPC has approximately eightfold symmetry and is formed by approximately 30 nucleoporins. The great size and complexity of the NPC have hindered the study of its structure for many years until recent breakthroughs were achieved by integrating the latest high-resolution cryo-electron microscopy (cryo-EM), the emerging artificial intelligence-based modeling and all other available structural information from crystallography and mass spectrometry. Here, we review our latest knowledge of the NPC architecture and the history of its structural study from in vitro to in situ with progressively improved resolutions by cryo-EM, with a particular focus on the latest subnanometer-resolution structural studies. The future directions for structural studies of NPCs are also discussed.     

GTP酶Ran在细胞核运输、核分裂与重建中的作用

小分子的单体G蛋白Ran具有鸟苷三磷酸酶活性,其结合形式Ran-GTP作为区分间期细胞的核质和胞质的一个分子标记,并参与调控核质运输、指导纺锤体形成以及引导核膜解体与装配。现就Ran在真核细胞核质运输、有丝分裂纺锤体组装与核膜动力学中的功能作一综述。     

Structural Analysis of a Metazoan Nuclear Pore Complex Reveals a Fused Concentric Ring Architecture

The sole gateway for molecular exchange between the cytoplasm and the nucleus is the nuclear pore complex (NPC). This large supramolecular assembly mediates transport of cargo into and out of the nucleus and fuse the inner and outer nuclear membranes to form an aqueous translocation channel. The NPC is composed of eight proteinaceous asymmetric units forming a pseudo-8-fold symmetric passage. Due to its shear size, complexity, and plastic nature, dissecting the high-resolution three-dimensional structure of the NPC in its hydrated state is a formidable challenge. Toward this goal, we applied cryo-electron tomography to spread nuclear envelopes from Xenopus oocytes. To compensate for perturbations of the 8-fold symmetry of individual NPCs, we performed symmetry-independent asymmetric unit averaging of three-dimensional tomographic NPC volumes to eventually yield a refined model at 6.4 nm resolution. This approach revealed novel structural features, particularly in the spoke-ring complex and luminal domains. Fused concentric ring architecture of the spoke-ring complex was found along the translocation channel. Additionally, a comparison of the refined Xenopus model to that of its Dictyostelium homologue yielded similar pore diameters at the level of the three canonical rings, although the Xenopus NPC was found to be 30% taller than the Dictyostelium pore. This discrepancy is attributed primarily to the relatively low homology and different organization of some nucleoporins in the Dictyostelium genome as compared to that of vertebrates. Nevertheless, the experimental conditions impose a preferred axial orientation of the NPCs within spread Xenopus oocyte nuclear envelopes. This may at least in part explain the increased height of the reconstructed vertebrate NPCs compared to those obtained from tomographic reconstruction of intact Dictyostelium nuclei.     

Ty1 Gag Enhances the Stability and Nuclear Export of Ty1 mRNA

Retrotransposon and retroviral RNA delivery to particle assembly sites is essential for their replication. mRNA and Gag from the Ty1 retrotransposon colocalize in cytoplasmic foci, which are required for transposition and may be the sites for virus‐like particle (VLP) assembly. To determine which Ty1 components are required to form mRNA/Gag foci, localization studies were performed in a Ty1‐less strain expressing galactose‐inducible Ty1 plasmids (pGTy1) containing mutations in GAG or POL. Ty1 mRNA/Gag foci remained unaltered in mutants defective in Ty1 protease (PR) or deleted for POL. However, Ty1 mRNA containing a frameshift mutation (Ty1fs) that prevents the synthesis of all proteins accumulated in the nucleus. Ty1fs RNA showed a decrease in stability that was mediated by the cytoplasmic exosome, nonsense‐mediated decay (NMD) and the processing body. Localization of Ty1fs RNA remained unchanged in an nmd2Δ mutant. When Gag and Ty1fs mRNA were expressed independently, Gag provided in trans increased Ty1fs RNA level and restored localization of Ty1fs RNA in cytoplasmic foci. Endogenously expressed Gag also localized to the nuclear periphery independent of RNA export. These results suggest that Gag is required for Ty1 mRNA stability, efficient nuclear export and localization into cytoplasmic foci.     

A Second CRM1-Dependent Nuclear Export Signal in the Influenza A Virus NS2 Protein Contributes to the Nuclear Export of Viral Ribonucleoproteins

Influenza A virus NS2 protein, also called nuclear export protein (NEP), is crucial for the nuclear export of viral ribonucleoproteins. However, the molecular mechanisms of NEP mediation in this process remain incompletely understood. A leucine-rich nuclear export signal (NES2) in NEP, located at the predicted N2 helix of the N-terminal domain, was identified in the present study. NES2 was demonstrated to be a transferable NES, with its nuclear export activity depending on the nuclear export receptor chromosome region maintenance 1 (CRM1)-mediated pathway. The interaction between NEP and CRM1 is coordinately regulated by both the previously reported NES (NES1) and now the new NES2. Deletion of the NES1 enhances the interaction between NEP and CRM1, and deletion of the NES1 and NES2 motifs completely abolishes this interaction. Moreover, NES2 interacts with CRM1 in the mammalian two-hybrid system. Mutant viruses containing NES2 alterations generated by reversed genetics exhibit reduced viral growth and delay in the nuclear export of viral ribonucleoproteins (vRNPs). The NES2 motif is highly conserved in the influenza A and B viruses. The results demonstrate that leucine-rich NES2 is involved in the nuclear export of vRNPs and contributes to the understanding of nucleocytoplasmic transport of influenza virus vRNPs.     

玉米根细胞发育过程中核孔复合物的变化(英文)

以玉米根部不同区域(分生区、伸长区和分化区)为材料,进行冷冻蚀刻,对不同生长阶段的细胞核大小、核孔复合物数量及孔径进行了测量,证明根尖细胞在整个发育周期中,细胞核膜孔总量几乎不变旭核孔复合物的孔径差异颇大。分生区核孔复合物的孔径平均约50 nm;伸长区有80％核孔直径为80 nm;分化区核孔直径恢复到50 um。这些复合物孔径的变化周期与Jordan等(1980)由玉米根部观察到三个区域的染色质变化相符,在细胞伸长区常染色质多,合成RNA旺盛,核孔复合物开大,以利RNA进入细胞质合成蛋白质。     

Nuclear shuttling and TRAF2-mediated retention in the cytoplasm regulate the subcellular localization of cIAP1 and cIAP2

Dynamic subcellular localization is an important regulatory mechanism for many proteins. cIAP1 and cIAP2 are two closely related members of inhibitor of apoptosis (IAP) family that play a role both as caspase inhibitors and as mediators of tumor necrosis factor (TNF) receptor signaling. Here, we report that cIAP1 and cIAP2 are nuclear shuttling proteins, whose subcellular localization is mediated by the CRM1-dependent nuclear export pathway. Blocking export with leptomycin B induces accumulation of both endogenous cIAP1 and epitope-tagged cIAP1 and cIAP2 in the nucleus of human cancer cells. We have identified a new CRM1-dependent leucine-rich nuclear export signal (NES) in the linker region between cIAP1 BIR2 and BIR3 repeats. Mutational inactivation of the NES, which is not conserved in cIAP2, reduces cIAP1 nuclear export. Forced relocation of cIAP1 to the nucleus did not significantly alter its ability to prevent apoptosis. Interestingly, co-expression experiments showed that the cIAP1 and cIAP2-interacting protein TNF receptor-associated factor 2 (TRAF2) plays an important role as regulator of IAP nucleocytoplasmic localization, by preventing nuclear translocation of cIAP1 and cIAP2. TRAF2-mediated cytoplasmic retention of cIAP1 was reduced upon TNFalpha treatment. Our results identify molecular mechanisms that contribute to regulate the subcellular localization of cIAP1 and cIAP2. Translocation between different cell compartments may add a further level of control for cIAP1 and cIAP2 activity.