Showing papers on "Importin published in 2014"
TL;DR: It is proposed that the β-catenin nuclear import pathway may provide an opportunity for identification of specific drug targets and inhibition of β-catsin nuclear function, much like the current screening of drugs that block binding ofβ- catenin to LEF-1/TCFs.
122 citations
TL;DR: It is reported that importin 8 (IPO8), a member of the karyopherin β (also named the protein import receptor importin β) family, plays a critical role in mediating the cytoplasm-to-nucleus transport of mature miRNAs.
115 citations
TL;DR: The structural bases for nuclear import of splicing factors and the Tnpo3–CPSF6 nexus in HIV-1 biology are elucidated and a potential route of pharmacological intervention in the treatment of AIDS is suggested.
Abstract: Transportin 3 (Tnpo3, Transportin-SR2) is implicated in nuclear import of splicing factors and HIV-1 replication. Herein, we show that the majority of cellular Tnpo3 binding partners contain arginine-serine (RS) repeat domains and present crystal structures of human Tnpo3 in its free as well as GTPase Ran- and alternative splicing factor/splicing factor 2 (ASF/SF2)-bound forms. The flexible β-karyopherin fold of Tnpo3 embraces the RNA recognition motif and RS domains of the cargo. A constellation of charged residues on and around the arginine-rich helix of Tnpo3 HEAT repeat 15 engage the phosphorylated RS domain and are critical for the recognition and nuclear import of ASF/SF2. Mutations in the same region of Tnpo3 impair its interaction with the cleavage and polyadenylation specificity factor 6 (CPSF6) and its ability to support HIV-1 replication. Steric incompatibility of the RS domain and RanGTP engagement by Tnpo3 provides the mechanism for cargo release in the nucleus. Our results elucidate the structural bases for nuclear import of splicing factors and the Tnpo3–CPSF6 nexus in HIV-1 biology.
115 citations
TL;DR: Over the past few years, significant progress has been made in the characterization of the nuclear localization signals (NLSs) that Transportin‐1 recognizes, thereby providing the molecular basis of its diversified repertoire of cargoes.
92 citations
TL;DR: When the G-actin pool is depleted, CCG-1423 binds specifically to the NLS of MRTF-A/B and prevents the interaction between MRTFs A and B and importin α/β1, resulting in inhibition of the nuclear import of MR TFs, suggesting that the specific binding of CCG -1423 to theNLSs of RPEL-containing proteins is suggested.
Abstract: Epithelial–msenchymal transition (EMT) is closely associated with cancer and tissue fibrosis. The nuclear accumulation of myocardin-related transcription factor A (MRTF-A/MAL/MKL1) plays a vital role in EMT. In various cells treated with CCG-1423, a novel inhibitor of Rho signaling, the nuclear accumulation of MRTF-A is inhibited. However, the molecular target of this inhibitor has not yet been identified. In this study, we investigated the mechanism of this effect of CCG-1423. The interaction between MRTF-A and importin α/β1 was inhibited by CCG-1423, but monomeric G-actin binding to MRTF-A was not inhibited. We coupled Sepharose with CCG-1423 (CCG-1423 Sepharose) to investigate this mechanism. A pull-down assay using CCG-1423 Sepharose revealed the direct binding of CCG-1423 to MRTF-A. Furthermore, we found that the N-terminal basic domain (NB) of MRTF-A, which acts as a functional nuclear localization signal (NLS) of MRTF-A, was the binding site for CCG-1423. G-actin did not bind to CCG-1423 Sepharose, but the interaction between MRTF-A and CCG-1423 Sepharose was reduced in the presence of G-actin. We attribute this result to the high binding affinity of MRTF-A for G-actin and the proximity of NB to G-actin-binding sites (RPEL motifs). Therefore, when MRTF-A forms a complex with G-actin, the binding of CCG-1423 to NB is expected to be blocked. NF-E2 related factor 2, which contains three distinct basic amino acid-rich NLSs, did not bind to CCG-1423 Sepharose, but other RPEL-containing proteins such as MRTF-B, myocardin, and Phactr1 bound to CCG-1423 Sepharose. These results suggest that the specific binding of CCG-1423 to the NLSs of RPEL-containing proteins. Our proposal to explain the inhibitory action of CCG-1423 is as follows: When the G-actin pool is depleted, CCG-1423 binds specifically to the NLS of MRTF-A/B and prevents the interaction between MRTF-A/B and importin α/β1, resulting in inhibition of the nuclear import of MRTF-A/B.
74 citations
TL;DR: This code, experimentally tested in 17 ARPs, predicts the nuclear-cytoplasmic localization of over 150 annotated human ARPs with high accuracy and is acquired by the most common familial melanoma-associated CDKN2A mutation, leading to nuclear accumulation of mutant p16ink4a.
68 citations
TL;DR: The discovery of a conserved nuclear carrier Tsr2 that coordinates transfer of the r-protein eS26 to the earliest assembling pre-ribosome, the 90S is reported, and it is anticipated similar carriers—termed here escortins—to securely connect the nuclear import machinery with pathways that deposit r-proteins onto developing pre- ribosomal particles.
Abstract: Within a single generation time a growing yeast cell imports ∼14 million ribosomal proteins (r-proteins) into the nucleus for ribosome production. After import, it is unclear how these intrinsically unstable and aggregation-prone proteins are targeted to the ribosome assembly site in the nucleolus. Here, we report the discovery of a conserved nuclear carrier Tsr2 that coordinates transfer of the r-protein eS26 to the earliest assembling pre-ribosome, the 90S. In vitro studies revealed that Tsr2 efficiently dissociates importin:eS26 complexes via an atypical RanGTP-independent mechanism that terminates the import process. Subsequently, Tsr2 binds the released eS26, shields it from proteolysis, and ensures its safe delivery to the 90S pre-ribosome. We anticipate similar carriers-termed here escortins-to securely connect the nuclear import machinery with pathways that deposit r-proteins onto developing pre-ribosomal particles.
68 citations
TL;DR: Nup50 is a mobile nucleoporin with a pronounced presence both at the nuclear pore complex and in the nucleoplasm that can move between these different localizations, dependent on active transcription by RNA polymerase II.
Abstract: The nuclear pore complex (NPC) plays a critical role in gene expression by mediating import of transcription regulators into the nucleus and export of RNA transcripts to the cytoplasm. Emerging evidence suggests that in addition to mediating transport, a subset of nucleoporins (Nups) engage in transcriptional activation and elongation at genomic loci that are not associated with NPCs. The underlying mechanism and regulation of Nup mobility on and off nuclear pores remain unclear. Here we show that Nup50 is a mobile Nup with a pronounced presence both at the NPC and in the nucleoplasm that can move between these different localizations. Strikingly, the dynamic behavior of Nup50 in both locations is dependent on active transcription by RNA polymerase II and requires the N-terminal half of the protein, which contains importin α- and Nup153-binding domains. However, Nup50 dynamics are independent of importin α, Nup153, and Nup98, even though the latter two proteins also exhibit transcription-dependent mobility. Of interest, depletion of Nup50 from C2C12 myoblasts does not affect cell proliferation but inhibits differentiation into myotubes. Taken together, our results suggest a transport-independent role for Nup50 in chromatin biology that occurs away from the NPC.
62 citations
TL;DR: A previously unidentified mechanism for Oxtr action on bone is defined and possibilities for direct transcriptional modulation by nuclear G protein-coupled receptors are open.
Abstract: We report that oxytocin (Oxt) receptors (Oxtrs), on stimulation by the ligand Oxt, translocate into the nucleus of osteoblasts, implicating this process in the action of Oxt on osteoblast maturation. Sequential immunocytochemistry of intact cells or isolated nucleoplasts stripped of the outer nuclear membrane showed progressive nuclear localization of the Oxtr; this nuclear translocation was confirmed by monitoring the movement of Oxtr–EGFP as well as by immunogold labeling. Nuclear Oxtr localization was conclusively shown by Western immunoblotting and MS of nuclear lysate proteins. We found that the passage of Oxtrs into the nucleus was facilitated by successive interactions with β-arrestins (Arrbs), the small GTPase Rab5, importin-β (Kpnb1), and transportin-1 (Tnpo1). siRNA-mediated knockdown of Arrb1, Arrb2, or Tnpo1 abrogated Oxt-induced expression of the osteoblast differentiation genes osterix (Sp7), Atf4, bone sialoprotein (Ibsp), and osteocalcin (Bglap) without affecting Erk phosphorylation. Likewise and again, without affecting pErk, inhibiting Arrb recruitment by mutating Ser rich clusters of the nuclear localization signal to Ala abolished nuclear import and Oxtr-induced gene expression. These studies define a previously unidentified mechanism for Oxtr action on bone and open possibilities for direct transcriptional modulation by nuclear G protein-coupled receptors.
58 citations
TL;DR: It can be concluded that α-syn can accumulate in nucleus, which is mediated by importin α, and promote neurotoxicity by accelerating the cell cycle.
51 citations
TL;DR: A strong body of evidence is emerging implicating Ran as a key protein in the metastatic progression of cancer, and these perturbed levels are associated with local invasion, metastasis and reduced patient survival.
Abstract: Ran is a small ras-related GTPase that controls the nucleocytoplasmic exchange of macromolecules across the nuclear envelope. It binds to chromatin early during nuclear formation and has important roles during the eukaryotic cell cycle, where it regulates mitotic spindle assembly, nuclear envelope formation and cell cycle checkpoint control. Like other GTPases, Ran relies on the cycling between GTP-bound and GDP-bound conformations to interact with effector proteins and regulate these processes. In nucleocytoplasmic transport, Ran shuttles across the nuclear envelope through nuclear pores. It is concentrated in the nucleus by an active import mechanism where it generates a high concentration of RanGTP by nucleotide exchange. It controls the assembly and disassembly of a range of complexes that are formed between Ran-binding proteins and cellular cargo to maintain rapid nuclear transport. Ran also has been identified as an essential protein in nuclear envelope formation in eukaryotes. This mechanism is dependent on importin-β, which regulates the assembly of further complexes important in this process, such as Nup107-Nup160. A strong body of evidence is emerging implicating Ran as a key protein in the metastatic progression of cancer. Ran is overexpressed in a range of tumors, such as breast and renal, and these perturbed levels are associated with local invasion, metastasis and reduced patient survival. Furthermore, tumors with oncogenic KRAS or PIK3CA mutations are addicted to Ran expression, which yields exciting future therapeutic opportunities.
TL;DR: The results suggest that β-like importins are central mediators of stimulated nuclear translocation of signaling proteins and therefore add a central level of regulation to stimulated transcription.
Abstract: The rapid nuclear translocation of signaling proteins upon stimulation is important for the regulation of de novo gene expression. We have studied the stimulated nuclear shuttling of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinases (MAPKs) and found that they translocate into the nucleus in a Ran-dependent, but NLS- or NTS-independent, manner, unrelated to their catalytic activity. We show that this translocation involves three β-like importins, importins 3, 7, and 9 (Imp3/7/9). Knockdown of these importins inhibits the nuclear translocation of the MAPKs and, thereby, activation of their transcription factor targets. We further demonstrate that the translocation requires the stimulated formation of heterotrimers composed of Imp3/Imp7/MAPK or Imp3/Imp9/MAPK. JNK1/2 and p38α/β bind to either Imp7 or Imp9 upon stimulated posttranslational modification of the two Imps, while Imp3 joins the complex after its stimulation-induced phosphorylation. Once formed, these heterotrimers move to the nuclear envelope, where importin 3 remains, while importins 7 and 9 escort the MAPKs into the nucleus. These results suggest that β-like importins are central mediators of stimulated nuclear translocation of signaling proteins and therefore add a central level of regulation to stimulated transcription.
TL;DR: It is demonstrated that the BEFV α1 ORF encodes a protein which has the structural and functional characteristics of a viroporin, and it is shown that α1 localizes in the Golgi complex and increases cellular permeability.
Abstract: Bovine ephemeral fever virus (BEFV) is an arthropod-borne rhabdovirus that is classified as the type species of the genus Ephemerovirus. In addition to the five canonical rhabdovirus structural proteins (N, P, M, G, and L), the large and complex BEFV genome contains several open reading frames (ORFs) between the G and L genes (α1, α2/α3, β, and γ) encoding proteins of unknown function. We show that the 10.5-kDa BEFV α1 protein is expressed in infected cells and, consistent with previous predictions based on its structure, has the properties of a viroporin. Expression of a BEFV α1-maltose binding protein (MBP) fusion protein in Escherichia coli was observed to inhibit cell growth and increase membrane permeability to hygromycin B. Increased membrane permeability was also observed in BEFV-infected mammalian cells (but not cells infected with an α1-deficient BEFV strain) and in cells expressing a BEFV α1-green fluorescent protein (GFP) fusion protein, which was shown by confocal microscopy to localize to the Golgi complex. Furthermore, the predicted C-terminal cytoplasmic domain of α1, which contains a strong nuclear localization signal (NLS), was translocated to the nucleus when expressed independently, and in an affinity chromatography assay employing a GFP trap, the full-length α1 was observed to interact specifically with importin β1 and importin 7 but not with importin α3. These data suggest that, in addition to its function as a viroporin, BEFV α1 may modulate components of nuclear trafficking pathways, but the specific role thereof remains unclear. Although rhabdovirus accessory genes occur commonly among arthropod-borne rhabdoviruses, little is known of their functions. Here, we demonstrate that the BEFV α1 ORF encodes a protein which has the structural and functional characteristics of a viroporin. We show that α1 localizes in the Golgi complex and increases cellular permeability. We also show that BEFV α1 binds importin β1 and importin 7, suggesting that it may have a yet unknown role in modulating nuclear trafficking. This is the first functional analysis of an ephemerovirus accessory protein and of a rhabdovirus viroporin.
TL;DR: 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.
Abstract: 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.
TL;DR: This work delineates the entire life cycle of HCV and the active involvement of NLS sequences during HCV replication and assembly and supports the previous hypothesis that NPC-like structures and nuclear transport factors function in the membranous web to create an environment conducive to viral replication.
Abstract: The hepatitis C virus (HCV) is a positive strand RNA virus of the Flavivirus family that replicates in the cytoplasm of infected hepatocytes. Previously, several nuclear localization signals (NLS) and nuclear export signals (NES) have been identified in HCV proteins, however, there is little evidence that these proteins travel into the nucleus during infection. We have recently shown that nuclear pore complex (NPC) proteins (termed nucleoporins or Nups) are present in the membranous web and are required during HCV infection. In this study, we identify a total of 11 NLS and NES sequences in various HCV proteins. We show direct interactions between HCV proteins and importin α5 (IPOA5/kapα1), importin β3 (IPO5/kap β3), and exportin 1 (XPO1/CRM1) both in-vitro and in cell culture. These interactions can be disrupted using peptides containing the specific NLS or NES sequences of HCV proteins. Moreover, using a synchronized infection system, we show that these peptides inhibit HCV infection during distinct phases of the HCV life cycle. The inhibitory effects of these peptides place them in two groups. The first group binds IPOA5 and inhibits infection during the replication stage of HCV life cycle. The second group binds IPO5 and is active during both early replication and early assembly. This work delineates the entire life cycle of HCV and the active involvement of NLS sequences during HCV replication and assembly. Given the abundance of NLS sequences within HCV proteins, our previous finding that Nups play a role in HCV infection, and the relocation of the NLS double-GFP reporter in HCV infected cells, this work supports our previous hypothesis that NPC-like structures and nuclear transport factors function in the membranous web to create an environment conducive to viral replication.
TL;DR: Results show that conformational changes in the flexible amphiphilic motifs play a critical role in translocation through the NPC.
TL;DR: Findings demonstrate that rotavirus probably employs a novel strategy to inhibit IFN-induced STAT signalling, which acts after STAT activation and binding to the nuclear import machinery.
Abstract: The importance of innate immunity to rotaviruses is exemplified by the range of strategies evolved by rotaviruses to interfere with the IFN response. We showed previously that rotaviruses block gene expression induced by type I and II IFNs, through a mechanism allowing activation of signal transducer and activator of transcription (STAT) 1 and STAT2 but preventing their nuclear accumulation. This normally occurs through activated STAT1/2 dimerization, enabling an interaction with importin α5 that mediates transport into the nucleus. In rotavirus-infected cells, STAT1/2 inhibition may limit the antiviral actions of IFN produced early in infection. Here we further analysed the block to STAT1/2 nuclear accumulation, showing that activated STAT1 accumulates in the cytoplasm in rotavirus-infected cells. STAT1/2 nuclear accumulation was inhibited by rotavirus even in the presence of the nuclear export inhibitor Leptomycin B, demonstrating that enhanced nuclear export is not involved in STAT1/2 cytoplasmic retention. The ability to inhibit STAT nuclear translocation was completely conserved amongst the group A rotaviruses tested, including a divergent avian strain. Analysis of mutant rotaviruses indicated that residues after amino acid 47 of NSP1 are dispensable for STAT inhibition. Furthermore, expression of any of the 12 Rhesus monkey rotavirus proteins did not inhibit IFN-stimulated STAT1 nuclear translocation. Finally, co-immunoprecipitation experiments from transfected epithelial cells showed that STAT1/2 binds importin α5 normally following rotavirus infection. These findings demonstrate that rotavirus probably employs a novel strategy to inhibit IFN-induced STAT signalling, which acts after STAT activation and binding to the nuclear import machinery.
TL;DR: The role and regulation of specific importin and exportin involved in the transport of activated transcription factors in allergic diseases, the underlying molecular mechanisms, and the potential target sites for developing better therapeutic approaches are critically reviewed.
Abstract: Nucleocytoplasmic shuttling of macromolecules is a well-controlled process involving importins and exportins. These karyopherins recognize and bind to receptor-mediated intracellular signals through specific signal sequences that are present on cargo proteins and transport into and out of the nucleus through nuclear pore complexes. Nuclear localization signals (NLS) present on cargo molecules to be imported while nuclear export signals (NES) on the molecules to be exported are recognized by importins and exportins, respectively. The classical NLS are found on many transcription factors and molecules that are involved in the pathogenesis of allergic diseases. In addition, several immune modulators, including corticosteroids and vitamin D, elicit their cellular responses by regulating the expression and activity of importin molecules. In this review article, we provide a comprehensive list of importin and exportin molecules and their specific cargo that shuttled between cytoplasm and the nucleus. We also critically review the role and regulation of specific importin and exportin involved in the transport of activated transcription factors in allergic diseases, the underlying molecular mechanisms, and the potential target sites for developing better therapeutic approaches.
TL;DR: Studies on the regulation of nucleocytoplasmic transport under several cellular stresses should provide new insights into the fundamental principles of protein homeostasis (proteostasis) in both compartments, the nucleus and cy toplasm.
TL;DR: Two classic nuclear localization signals are reported in the minor capsid protein (VP1) of porcine parvovirus, which can be used as a strategy by the virus to avoid transport of incorrectly folded proteins and to selectively import assembled trimers into the nucleus.
Abstract: Nuclear targeting of capsid proteins (VPs) is important for genome delivery and precedes assembly in the replication cycle of porcine parvovirus (PPV). Clusters of basic amino acids, corresponding to potential nuclear localization signals (NLS), were found only in the unique region of VP1 (VP1up, for VP1 unique part). Of the five identified basic regions (BR), three were important for nuclear localization of VP1up: BR1 was a classic Pat7 NLS, and the combination of BR4 and BR5 was a classic bipartite NLS. These NLS were essential for viral replication. VP2, the major capsid protein, lacked these NLS and contained no region with more than two basic amino acids in proximity. However, three regions of basic clusters were identified in the folded protein, assembled into a trimeric structure. Mutagenesis experiments showed that only one of these three regions was involved in VP2 transport to the nucleus. This structural NLS, termed the nuclear localization motif (NLM), is located inside the assembled capsid and thus can be used to transport trimers to the nucleus in late steps of infection but not for virions in initial infection steps. The two NLS of VP1up are located in the N-terminal part of the protein, externalized from the capsid during endosomal transit, exposing them for nuclear targeting during early steps of infection. Globally, the determinants of nuclear transport of structural proteins of PPV were different from those of closely related parvoviruses.
IMPORTANCE Most DNA viruses use the nucleus for their replication cycle. Thus, structural proteins need to be targeted to this cellular compartment at two distinct steps of the infection: in early steps to deliver viral genomes to the nucleus and in late steps to assemble new viruses. Nuclear targeting of proteins depends on the recognition of a stretch of basic amino acids by cellular transport proteins. This study reports the identification of two classic nuclear localization signals in the minor capsid protein (VP1) of porcine parvovirus. The major protein (VP2) nuclear localization was shown to depend on a complex structural motif. This motif can be used as a strategy by the virus to avoid transport of incorrectly folded proteins and to selectively import assembled trimers into the nucleus. Structural nuclear localization motifs can also be important for nuclear proteins without a classic basic amino acid stretch, including multimeric cellular proteins.
TL;DR: The X-ray structure reveals that the four ZFs of Snails1 are required for tight binding to importin β in the nuclear import of Snail1, and is reminiscent of a round snail.
Abstract: Snail contributes to the epithelial–mesenchymal transition by suppressing E-cadherin in transcription processes. The Snail C2H2-type zinc-finger (ZF) domain functions both as a nuclear localization signal which binds to importin β directly and as a DNA-binding domain. Here, a 2.5 A resolution structure of four ZF domains of Snail1 complexed with importin β is presented. The X-ray structure reveals that the four ZFs of Snail1 are required for tight binding to importin β in the nuclear import of Snail1. The shape of the ZFs in the X-ray structure is reminiscent of a round snail, where ZF1 represents the head, ZF2–ZF4 the shell, showing a novel interaction mode, and the five C-terminal residues the tail. Although there are many kinds of C2H2-type ZFs which have the same fold as Snail, nuclear import by direct recognition of importin β is observed in a limited number of C2H2-type ZF proteins such as Snail, Wt1, KLF1 and KLF8, which have the common feature of terminating in ZF domains with a short tail of amino acids.
TL;DR: The p53-induced protein Ei24 can bind specifically to importin-β1 and importin -α2 to impede their normal role in nuclear import.
Abstract: The etoposide-induced protein Ei24 was initially identified as a p53-responsive, proapoptotic factor, but no clear function has been described. Here, we use a nonbiased proteomics approach to identify members of the importin (IMP) family of nuclear transporters as interactors of Ei24 and characterize an IMPβ-binding-like (IBBL) domain within Ei24. We show that Ei24 can bind specifically to IMPβ1 and IMPα2, but not other IMPs, and use a mutated IMPβ1 derivative to show that Ei24 binds to the same site on IMPβ1 as the IMPα IBB. Ectopic expression of Ei24 reduced the extent of IMPβ1- or IMPα/β1-dependent nuclear protein import specifically, whereas specific alanine substitutions within the IBBL abrogated this activity. Induction of endogenous Ei24 expression through etoposide treatment similarly inhibited nuclear import in a mouse embryonic fibroblast model. Thus, Ei24 can bind specifically to IMPβ1 and IMPα2 to impede their normal role in nuclear import, shedding new light on the cellular functions of Ei24 and its tumor suppressor role.
TL;DR: A major nuclear localization signal (NLS) is confirmed in the N-terminal region of hTERT and a novel one in the C- terminal part is described, which sheds light on the nuclear import pathway of h TERT.
Abstract: In actively dividing eukaryotic cells, chromosome ends (telomeres) are subject to progressive shortening, unless they are maintained by the action of telomerase, a dedicated enzyme that adds DNA sequence repeats to chromosomal 3′end. For its enzymatic function on telomeres, telomerase requires nuclear import of its protein component (hTERT in human cells) and assembly with the RNA component, TERC. We now confirm a major nuclear localization signal (NLS) in the N-terminal region of hTERT and describe a novel one in the C-terminal part. Using an siRNA approach to deplete several import receptors, we identify importin 7 as a soluble nuclear transport factor that is required for efficient import. At the level of the nuclear pore complex (NPC), Nup358, a nucleoporin that forms the cytoplasmic filaments of the NPC, plays an important role in nuclear import of hTERT. A structure-function analysis of Nup358 revealed that the zinc finger region of the nucleoporin is of particular importance for transport of hTERT. Together, our study sheds light on the nuclear import pathway of hTERT.
20 Oct 2014
TL;DR: Three models of how proteasomes are transported from the cytoplasm into the nucleus of yeast cells are reported, based on the canonical pathway using classical nuclear localization sequences of proteasomal subcomplexes and the classical import receptor importin/karyopherin αβ.
Abstract: Proteasomes are conserved protease complexes enriched in the nuclei of dividing yeast cells, a major site for protein degradation. If yeast cells do not proliferate and transit to quiescence, metabolic changes result in the dissociation of proteasomes into proteolytic core and regulatory complexes and their sequestration into motile cytosolic proteasome storage granuli. These granuli rapidly clear with the resumption of growth, releasing the stored proteasomes, which relocalize back to the nucleus to promote cell cycle progression. Here, I report on three models of how proteasomes are transported from the cytoplasm into the nucleus of yeast cells. The first model applies for dividing yeast and is based on the canonical pathway using classical nuclear localization sequences of proteasomal subcomplexes and the classical import receptor importin/karyopherin αβ. The second model applies for quiescent yeast cells, which resume growth and use Blm10, a HEAT-like repeat protein structurally related to karyopherin β, for nuclear import of proteasome core particles. In the third model, the fully-assembled proteasome is imported into the nucleus. Our still marginal knowledge about proteasome dynamics will inspire the discussion on how protein degradation by proteasomes may be regulated in different cellular compartments of dividing and quiescent eukaryotic cells.
TL;DR: OsMADS29, a seed-specific transcription factor that affects grain filling and embryo development by regulating hormone homeostasis, requires homo- or heterodimerization with eleven other MADS proteins for its localization into the nucleus.
Abstract: OsMADS29 is a seed-specific MADS-box transcription factor that affects embryo development and grain filling by maintaining hormone homeostasis and degradation of cells in the nucellus and nucellar projection. Although it has a bipartite nuclear localization signal (NLS) sequence, the transiently expressed OsMADS29 monomer does not localize specifically in the nucleus. Dimerization of the monomers alters the intracellular localization fate of the resulting OsMADS29 homodimer, which then translocates into the nucleus. By generating domain-specific deletions/mutations, we show that two conserved amino acids (lysine23 and arginine24) in the NLS are important for nuclear localization of the OsMADS29 homodimer. Furthermore, the analyses involving interaction of OsMADS29 with 30 seed-expressed rice MADS proteins revealed 19 more MADS-box proteins, including five E-class proteins, which interacted with OsMADS29. Eleven of these complexes were observed to be localized in the nucleus. Deletion analysis revealed that the KC region (K-box and C-terminal domain) plays a pivotal role in homodimerization. These data suggest that the biological function of OsMADS29 may not only be regulated at the level of transcription and translation as reported earlier, but also at the post-translational level by way of the interaction between OsMADS29 monomers, and between OsMADS29 and other MADS-box proteins.
TL;DR: The subcellular distribution of yeast β-importins inverts upon acute glucose deprivation, likely due to collapse of the RanGTP nuclear–cytoplasmic gradient.
Abstract: The importin-β family members (karyopherins) mediate the majority of nucleocytoplasmic transport. Msn5 and Los1, members of the importin-β family, function in tRNA nuclear export. tRNAs move bidirectionally between the nucleus and the cytoplasm. Nuclear tRNA accumulation occurs upon amino acid (aa) or glucose deprivation. To understand the mechanisms regulating tRNA subcellular trafficking, we investigated whether Msn5 and Los1 are regulated in response to nutrient availability. We provide evidence that tRNA subcellular trafficking is regulated by distinct aa-sensitive and glucose-sensitive mechanisms. Subcellular distributions of Msn5 and Los1 are altered upon glucose deprivation but not aa deprivation. Redistribution of tRNA exportins from the nucleus to the cytoplasm likely provides one mechanism for tRNA nuclear distribution upon glucose deprivation. We extended our studies to other members of the importin-β family and found that all tested karyopherins invert their subcellular distributions upon glucose deprivation but not aa deprivation. Glucose availability regulates the subcellular distributions of karyopherins likely due to alteration of the RanGTP gradient since glucose deprivation causes redistribution of Ran. Thus nuclear-cytoplasmic distribution of macromolecules is likely generally altered upon glucose deprivation due to collapse of the RanGTP gradient and redistribution of karyopherins between the nucleus and the cytoplasm.
TL;DR: Investigation of the nuclear localization of NS5 using immunofluorescent staining for NS5 in infected cells shows that NS5 nuclear localization is significantly inhibited by Ivermectin, a general inhibitor of nuclear transport mediated by the cellular nuclear transport proteins importin α/β (IMPα/β).
Abstract: Dengue virus (DENV) nonstructural protein 5 (NS5) plays a central role in viral replication in the cytoplasm of infected cells. Despite this, NS5 is predominantly located in the nucleus of infected cells where it is thought to play a role in suppression of the host antiviral response. We have investigated the nuclear localization of NS5 using immunofluorescent staining for NS5 in infected cells, showing that NS5 nuclear localization is significantly inhibited by Ivermectin, a general inhibitor of nuclear transport mediated by the cellular nuclear transport proteins importin α/β (IMPα/β). Experiments in living mammalian cells transfected to express green fluorescent protein (GFP)-tagged NS5 protein confirm that NS5 is predominantly nuclear and that this localization is inhibited by Ivermectin, demonstrating that NS5 contains an Ivermectin-sensitive IMPα/β-recognized nuclear localization signal [Pryor et al. Traffic 8:795-807, 2007]. Consistent with this observation, mutation of critical residues within the nuclear localization signal (the A2 mutant; [Pryor et al. Traffic 8:795-807, 2007]) results in an 80 % reduction in nuclear localization of NS5. Finally we demonstrate direct, high-affinity binding of NS5 to IMPα/β using an AlphaScreen protein-protein binding assay.
TL;DR: It is shown that the NLS-adjacent phosphorylation by Cdk1 of human dUTPase, an enzyme essential for genomic integrity, results in dynamic cell cycle-dependent distribution of the protein.
Abstract: Phosphorylation by the cyclin-dependent kinase 1 (Cdk1) adjacent to nuclear localization signals (NLSs) is an important mechanism of regulation of nucleocytoplasmic transport. However, no systematic survey has yet been performed in human cells to analyze this regulatory process, and the corresponding cell-cycle dynamics have not yet been investigated. Here, we focused on the human proteome and found that numerous proteins, previously not identified in this context, are associated with Cdk1-dependent phosphorylation sites adjacent to their NLSs. Interestingly, these proteins are involved in key regulatory events of DNA repair, epigenetics, or RNA editing and splicing. This finding indicates that cell-cycle dependent events of genome editing and gene expression profiling may be controlled by nucleocytoplasmic trafficking. For in-depth investigations, we selected a number of these proteins and analyzed how point mutations, expected to modify the phosphorylation ability of the NLS segments, perturb nucleocytoplasmic localization. In each case, we found that mutations mimicking hyper-phosphorylation abolish nuclear import processes. To understand the mechanism underlying these phenomena, we performed a video microscopy-based kinetic analysis to obtain information on cell-cycle dynamics on a model protein, dUTPase. We show that the NLS-adjacent phosphorylation by Cdk1 of human dUTPase, an enzyme essential for genomic integrity, results in dynamic cell cycle-dependent distribution of the protein. Non-phosphorylatable mutants have drastically altered protein re-import characteristics into the nucleus during the G1 phase. Our results suggest a dynamic Cdk1-driven mechanism of regulation of the nuclear proteome composition during the cell cycle.
TL;DR: Evaluation of this set of candidate binding partners from spermatogenic cells using several bioinformatics approaches provides new evidence that individual importin αs may serve unique and nonredundant roles in mediating cellular differentiation.
Abstract: Importin α proteins function as adaptors to connect a cargo protein and importin β1 in the classical nuclear import pathway. Here we measure for the first time the stoichiometry of importins α2, α3, α4, and β1 in primary cells corresponding to 2 successive stages of rat spermatogenesis: meiotic spermatocytes and haploid round spermatids. Importin α2 levels were more than 2-fold higher in spermatocytes than in spermatids, while importins α4 and β1 levels did not differ significantly. We performed a comprehensive proteomics analysis to identify binding proteins in spermatocytes and spermatids using recombinant importin α2 and α4 proteins. Among the 100 candidate partners, 42 contained a strong classical nuclear localization signal (cNLS; score of>6 by cNLS Mapper), while 8 nuclear proteins lacked any cNLS. In addition, we developed a new strategy to predict which cargoes bind to importin α through the conserved C-terminal acidic domain (ARM repeats 9-10), and provided functional validation of a predicted importin α C-terminal binding segment in Senataxin and Smarca4. Evaluation of this set of candidate binding partners from spermatogenic cells using several bioinformatics approaches provides new evidence that individual importin αs may serve unique and nonredundant roles in mediating cellular differentiation.
TL;DR: It is shown for the first time that Dp71d shuttles between the nucleus and cytoplasm mediated by the conventional nuclear transporters, importin (IMP) α/β and the exportin CRM1, and the clear implication is that D p71d's previously unrecognised nuclear transport properties likely contribute to various, important physiological roles.