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.     

Nuclear export signal consensus sequences defined using a localization-based yeast selection system

Proteins bearing nuclear export signals (NESs) are translocated to the cytoplasm from the nucleus mainly through the CRM1-dependent pathway. However, the NES consensus sequence remains poorly defined, and there are currently no high-throughput methods for identifying NESs. In this study, we report the development of an efficient yeast selection system for detecting nuclear export activity as well as several reliable NES consensus sequences identified using this method. Our selection system is based on the nuclear export-dependent rescue of Tys1p, an essential cytoplasmic protein that has been artificially localized to the nucleus in a haploid Delta tys1 knockout strain. A screen of a random peptide library revealed 101 distinct CRM1-dependent NESs, which were classified into six patterns according to the conserved hydrophobic spacing. By combining mutational analyses, we have defined new NES consensus sequences with more specific and redundant residues than the traditional consensus sequence, which are consistent with most experimentally confirmed NESs. These NES consensus sequences should help identify functional NESs, and our selection system can be used to identify other targeting signals or proteins imported to specific subcellular compartments.     

Nuclear export of MAP kinase (ERK) involves a MAP kinase kinase (MEK)-dependent active transport mechanism

In response to extracellular stimuli, mitogen-activated protein kinase (MAPK, also known as ERK), which localizes to the cytoplasm in quiescent cells, translocates to the nucleus and then relocalizes to the cytoplasm again. The relocalization of nuclear MAPK to the cytoplasm was not inhibited by cycloheximide, confirming that the relocalization is achieved by nuclear export, but not synthesis, of MAPK. The nuclear export of MAPK was inhibited by leptomycin B (LMB), a specific inhibitor of the nuclear export signal (NES)-dependent transport. We have then shown that MAP kinase kinase (MAPKK, also known as MEK), which mostly localizes to the cytoplasm because of its having NES, is able to shuttle between the cytoplasm and the nucleus constantly. MAPK, when injected into the nucleus, was rapidly exported from the nucleus by coinjected wild-type MAPKK, but not by the NES-disrupted MAPKK. In addition, injection of the fragment corresponding to the MAPK-binding site of MAPKK into the nucleus, which would disrupt the binding of MAPK to MAPKK in the nucleus, significantly inhibited the nuclear export of endogenous MAPK. Taken together, these results suggest that the relocalization of nuclear MAPK to the cytoplasm involves a MAPKK-dependent, active transport mechanism.     

SUMOylation mediates the disassembly of the Smad4 nuclear export complex via RanGAP1 in KELOIDS

Sentrin/small ubiquitin-like modifier (SUMO) has emerged as a powerful mediator regulating biological processes and participating in pathophysiological processes that cause human diseases, such as cancer, myocardial fibrosis and neurological disorders. Sumoylation has been shown to play a positive regulatory role in keloids. However, the sumoylation mechanism in keloids remains understudied. We proposed that sumoylation regulates keloids via a complex. RanGAP1 acted as a synergistic, functional partner of SUMOs in keloids. Nuclear accumulation of Smad4, a TGF-β/Smad pathway member, was associated with RanGAP1 after SUMO1 inhibition. RanGAP1*SUMO1 mediated the nuclear accumulation of Smad4 due to its impact on nuclear export and reduction in the dissociation of Smad4 and CRM1. We clarified a novel mechanism of positive regulation of sumoylation in keloids and demonstrated the function of sumoylation in Smad4 nuclear export. The NPC-associated RanGAP1*SUMO1 complex functions as a disassembly machine for the export receptor CRM1 and Smad4. Our research provides new perspectives for the mechanisms of keloids and nucleocytoplasmic transport.     

Ratjadones inhibit nuclear export by blocking CRM1/exportin 1

In addition to previously isolated ratjadone A we describe three new members of this family, ratjadones B, C, and D, from another strain of the myxobacterium Sorangium cellulosum. We have investigated the properties of these ratjadones with respect to their activity on mammalian cell lines. We found IC(50) values in the picomolar range and a significant increase in the size of nuclei. A further examination showed that they inhibit the export of the leucine-rich nuclear export signal (LR-NES) containing proteins in different cell lines. Ratjadones are able to inhibit the formation of the nuclear export complex composed of the CRM1, RanGTP, and the cargo protein, as shown by two different in vitro assays. Finally, the binding of ratjadone C to CRM1 was demonstrated. These ratjadone activities are in the same concentration range as described for the polyketide leptomycin B (LMB) from Streptomyces sp. Like LMB, it seems that the ratjadones covalently bind to CRM1, inhibit cargo protein binding via LR-NES, and thereby block nuclear export. Thus, the ratjadones represent a new class of natural compounds which inhibit proliferation in eukaryotes by blocking nuclear export.     

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.     

Characterization of nuclear localization and nuclear export signals of yeast actin-binding protein Pan1

Pan1 is an actin patch-associated protein involved in endocytosis. Our studies revealed that in oleate-grown cells Pan1 is located in the nucleus as well as in patches. One of three putative nuclear localization signals (NLS) of Pan1, NLS2, directed beta-galactosidase (beta-gal) to the nucleus. However, GFP-Pan1(886-1219), containing NLS2, was found in the cytoplasm indicating that it may contain a nuclear export signal (NES). A putative Pan1 NES, overlapping with NLS3, re-addressed NLS(H2B)-NES/NLS3-beta-gal from the nucleus to the cytoplasm. Inactivation of the NES allowed NLS3 to be effective. Thus, Pan1 contains functional NLSs and a NES and appears to shuttle in certain circumstances.     

Nucleocytoplasmic shuttling and the biological activity of mouse survivin are regulated by an active nuclear export signal

Survivin appears to function as a regulator of cell division and as an apoptosis inhibitor in many species. Here, we characterized the nucleocytoplasmic transport of mouse survivin(140), and its splice variants survivin(121) and survivin(40). We show that the dynamic intracellular localization of survivin(140) is mediated by a Crm1-dependent nuclear export signal (NES) present also in survivin(121), but absent in survivin(40). In contrast, neither survivin nor survivin splice variants contain an active nuclear import signal and seem to enter the nucleus by passive diffusion. The activity of the NES is required for survivin-mediated protection against cell death inducing stimuli and influences protein degradation. During mitosis, NES-deficient survivin variants fail to correctly localize to the mitotic machinery and promote proper cell division. In vivo and in vitro protein interaction assays show that survivin(140) and survivin(121) as well as their export-deficient mutants are able to form homo- as well as heterodimers. The trans-dominant negative phenotype observed upon expression of export-deficient survivin appears, therefore, to be mediated by the formation of inactive survivin heterodimers. The survivin-Crm1 axis is essential for the biological activities of murine survivin, and mouse models will allow investigating its functional implications during development and tumorigenesis.     

Mechanisms of receptor-mediated nuclear import and nuclear export

Nuclear transport of proteins and RNA occurs through the nuclear pore complex and is mediated by a superfamily of transport receptors known collectively as karyopherins. Karyopherins bind to their cargoes by recognition of specific nuclear localization signals or nuclear export signals. Transport through the nuclear pore complex is facilitated by transient interactions between the karyopherins and the nuclear pore complex. The interactions of karyopherins with their cargoes are regulated by the Ras-related GTPase Ran. Ran is assisted in this process by proteins that regulate its GTPase cycle and subcellular localization. In this review, we describe several of the major transport pathways that are conserved in higher and lower eukaryotes, with particular emphasis on the role of Ran. We highlight the latest advances in the structure and function of transport receptors and discuss recent examples of steroid hormone receptor import and regulation by signal transduction pathways. Understanding the molecular basis of nuclear transport may provide insight into human diseases by revealing how nucleocytoplasmic trafficking regulates protein activity.     

Engineering chromosome region maintenance 1 fragments that bind to nuclear export signals

Chromosome region maintenance 1 (CRM1) exports nuclear export signal (NES) containing cargos from nucleus to cytoplasm and plays critical roles in cancer and viral infections. Biochemical and biophysical studies on this protein were often obstructed by its low purification yield and stability. With the help of PROSS server and NES protection strategy, we successfully designed three small fragments of CRM1, each made of four HEAT repeats and capable of binding to NESs in the absence of RanGTP. One of the fragments, C7, showed dramatically improved purification yield, thermostability, mechanostability, and resistance to protease digestion. We showed by isothermal titration that the protein kinase inhibitor NES binds to C7 at 1.18 μM affinity. Direct binding to C7 by several reported CRM1 inhibitors derived from plants were verified using pull‐down assays. These fragments might be useful for the development of CRM1 inhibitors towards treatment of related diseases. The strategy applied here might help to tackle similar problems encountered in different fields.     

昆虫中的核输出现象

核质转运是真核细胞生命活动重要的组成部分,细胞核内蛋白的平衡及转录出的RNA出核成熟过程都依赖于核输出,核输出与真核生物的生命活动密切相关;病原物在侵染昆虫时也会利用宿主的核输出机制干扰宿主的免疫反应或劫持宿主的核输出蛋白以利于病毒的组装及复制.本文主要介绍真核细胞中核输出的基本机制、蚊子和果蝇等模式昆虫中核输出通路;...     

Cofactor requirements for nuclear export of Rev response element (RRE)- and constitutive transport element (CTE)-containing retroviral RNAs. An unexpected role for actin

Nuclear export of proteins containing leucine-rich nuclear export signals (NESs) is mediated by the export receptor CRM1/exportin1. However, additional protein factors interacting with leucine-rich NESs have been described. Here, we investigate human immunodeficiency virus type 1 (HIV-1) Rev-mediated nuclear export and Mason-Pfizer monkey virus (MPMV) constitutive transport element (CTE)-mediated nuclear export in microinjected Xenopus laevis oocytes. We show that eukaryotic initiation factor 5A (eIF-5A) is essential for Rev and Rev-mediated viral RNA export, but not for nuclear export of CTE RNA. In vitro binding studies demonstrate that eIF-5A is required for efficient interaction of Rev-NES with CRM1/exportin1 and that eIF-5A interacts with the nucleoporins CAN/nup214, nup153, nup98, and nup62. Quite unexpectedly, nuclear actin was also identified as an eIF-5A binding protein. We show that actin is associated with the nucleoplasmic filaments of nuclear pore complexes and is critically involved in export processes. Finally, actin- and energy-dependent nuclear export of HIV-1 Rev is reconstituted by using a novel in vitro egg extract system. In summary, our data provide evidence that actin plays an important functional role in nuclear export not only of retroviral RNAs but also of host proteins such as protein kinase inhibitor (PKI).     

Leptomycin B-sensitive nuclear export of MAPKAP kinase 2 is regulated by phosphorylation.

To study the intracellular localization of MAPKAP kinase 2 (MK2), which carries a putative bipartite nuclear localization signal (NLS), we constructed a green fluorescent protein-MAPKAP kinase 2 fusion protein (GFP-MK2). In transfected cells, this protein is located predominantly in the nucleus; unexpectedly, upon stress, it rapidly translocates to the cytoplasm. This translocation can be blocked by the p38 MAP kinase inhibitor SB203580, indicating its regulation by phosphorylation. Molecular mimicry of MK2 phosphorylation at T317 in GFP-MK2 led to a mutant which is located almost exclusively in the cytoplasm of the cell, whereas the mutant T317A shows no stress-induced redistribution. Since leptomycin B, which inhibits the interaction of exportin 1 with the Rev-type leucine-rich nuclear export signal (NES), blocks stress-dependent translocation of GFP-MK2, it is supposed that phosphorylation-induced export of the protein causes the translocation. We have identified the region responsible for nuclear export in MK2 which is partially overlapping with and C-terminal to the autoinhibitory motif. This region contains a cluster of hydrophobic amino acids in the characteristic spacing of a leucine-rich Rev-type NES which is necessary to direct GFP-MK2 to the cytoplasm. However, unlike the Rev-type NES, this region alone is not sufficient for nuclear export. The data obtained indicate that MK2 contains a constitutively active NLS and a stress-regulated signal for nuclear export. Keywords: nuclear export/nuclear import/protein phosphorylation/signal transduction/stress response     

Comprehensive analysis of nuclear export of herpes simplex virus type 1 tegument proteins and their Epstein‐Barr virus orthologs

Morphogenesis of herpesviral virions is initiated in the nucleus but completed in the cytoplasm. Mature virions contain more than 25 tegument proteins many of which perform both nuclear and cytoplasmic functions suggesting they shuttle between these compartments. While nuclear import of herpesviral proteins was shown to be crucial for viral propagation, active nuclear export and its functional impact are still poorly understood. To systematically analyze nuclear export of tegument proteins present in virions of Herpes simplex virus type 1 (HSV1) and Epstein‐Barr virus (EBV), the Nuclear EXport Trapped by RAPamycin (NEX‐TRAP) was applied. Nine of the 22 investigated HSV1 tegument proteins including pUL4, pUL7, pUL11, pUL13, pUL21, pUL37d11, pUL47, pUL48 and pUS2 as well as 2 out of 6 EBV orthologs harbor nuclear export activity. A functional leucine‐rich nuclear export sequence (NES) recognized by the export factor CRM1/Xpo1 was identified in six of them. The comparison between experimental and bioinformatic data indicates that experimental validation of predicted NESs is required. Mutational analysis of the pUL48/VP16 NES revealed its importance for herpesviral propagation. Together our data suggest that nuclear export is an important feature of the herpesviral life cycle required to co‐ordinate nuclear and cytoplasmic processes.      

Transport of galectin-3 between the nucleus and cytoplasm. II. Identification of the signal for nuclear export

Galectin-3, a factor involved in the splicing of pre-mRNA, shuttles between the nucleus and the cytoplasm. Previous studies have shown that incubation of fibroblasts with leptomycin B resulted in the accumulation of galectin-3 in the nucleus, suggesting that the export of galectin-3 from the nucleus may be mediated by the CRM1 receptor. A candidate nuclear export signal fitting the consensus sequence recognized by CRM1 can be found between residues 240 and 255 of the murine galectin-3 sequence. This sequence was engineered into the pRev(1.4) reporter system, in which candidate sequences can be tested for nuclear export activity in terms of counteracting the nuclear localization signal present in the Rev(1.4) protein. Rev(1.4)-galectin-3(240-255) exhibited nuclear export activity that was sensitive to inhibition by leptomycin B. Site-directed mutagenesis of Leu247 and Ile249 in the galectin-3 nuclear export signal decreased nuclear export activity, consistent with the notion that these two positions correspond to the critical residues identified in the nuclear export signal of the cAMP-dependent protein kinase inhibitor. The nuclear export signal activity was also analyzed in the context of a full-length galectin-3 fusion protein; galectin-3(1-263; L247A) showed more nuclear localization than wild-type, implicating Leu247 as critical to the function of the nuclear export signal. These results indicate that residues 240-255 of the galectin-3 polypeptide contain a leucine-rich nuclear export signal that overlaps with the region (residues 252-258) identified as important for nuclear localization.