Showing papers on "Importin published in 2007"

Molecular mechanism of the nuclear protein import cycle

Abstract: The nuclear import of proteins through nuclear pore complexes (NPCs) illustrates how a complex biological function can be generated by a spatially and temporally organized cycle of interactions between cargoes, carriers and the Ran GTPase. Recent work has given considerable insight into this process, especially about how interactions are coordinated and the basis for the molecular recognition that underlies the process. Although considerable progress has been made in identifying and characterizing the molecular interactions in the soluble phase that drive the nuclear protein import cycle, understanding the precise mechanism of translocation through NPCs remains a major challenge.

Structural biology of nucleocytoplasmic transport.

Abstract: In eukaryotic cells, segregation of DNA replication and RNA biogenesis in the nucleus and protein synthesis in the cytoplasm poses the requirement of transporting thousands of macromolecules between the two cellular compartments. Transport between nucleus and cytoplasm is mediated by soluble receptors that recognize specific cargoes and carry them through the nuclear pore complex (NPC), the sole gateway between the two compartments at interphase. Nucleocytoplasmic transport is specific not only in terms of cargo recognition, but also in terms of directionality, with nuclear proteins imported into the nucleus and RNAs exported from it. How is directionality achieved? How can the receptors be both specific and versatile in recognizing a multitude of cargoes? And how can their interaction with NPCs allow fast translocation? We describe the molecular mechanisms underlying nucleocytoplasmic transport as they have been revealed by structural studies of the receptors and regulators in different steps of transport cycles.

Structure and Nuclear Import Function of the C- Terminal Domain of Influenza Virus Polymerase Pb2 Subunit

Abstract: The trimeric influenza virus polymerase, comprising subunits PA, PB1 and PB2, is responsible for transcription and replication of the segmented viral RNA genome. Using a novel library-based screening technique called expression of soluble proteins by random incremental truncation (ESPRIT), we identified an independently folded C-terminal domain from PB2 and determined its solution structure by NMR. Using green fluorescent protein fusions, we show that both the domain and the full-length PB2 subunit are efficiently imported into the nucleus dependent on a previously overlooked bipartite nuclear localization sequence (NLS). The crystal structure of the domain complexed with human importin alpha5 shows how the last 20 residues unfold to permit binding to the import factor. The domain contains three surface residues implicated in adaptation from avian to mammalian hosts. One of these tethers the NLS-containing peptide to the core of the domain in the unbound state.

Nucleocytoplasmic transport of proteins.

Abstract: In eukaryotic cells, the movement of macromolecules between the nucleus and cytoplasm occurs through the nuclear pore complex (NPC)--a large protein complex spanning the nuclear envelope. The nuclear transport of proteins is usually mediated by a family of transport receptors known as karyopherins. Karyopherins bind to their cargoes via recognition of nuclear localization signal (NLS) for nuclear import or nuclear export signal (NES) for export to form a transport complex. Its transport through NPC is facilitated by transient interactions between the karyopherins and NPC components. The interactions of karyopherins with their cargoes are regulated by GTPase Ran. In the current review, we describe the NPC structure, NLS, and NES, as well as the model of classic Ran-dependent transport, with special emphasis on existing alternative mechanisms; we also propose a classification of the basic mechanisms of protein transport regulation.

Nuclear and Nucleolar Targeting of Influenza A Virus NS1 Protein: Striking Differences between Different Virus Subtypes

Abstract: Influenza A virus nonstructural protein 1 (NS1A protein) is a virulence factor which is targeted into the nucleus. It is a multifunctional protein that inhibits host cell pre-mRNA processing and counteracts host cell antiviral responses. We show that the NS1A protein can interact with all six human importin α isoforms, indicating that the nuclear translocation of NS1A protein is mediated by the classical importin α/β pathway. The NS1A protein of the H1N1 (WSN/33) virus has only one N-terminal arginine- or lysine-rich nuclear localization signal (NLS1), whereas the NS1A protein of the H3N2 subtype (Udorn/72) virus also has a second C-terminal NLS (NLS2). NLS1 is mapped to residues 35 to 41, which also function in the double-stranded RNA-binding activity of the NS1A protein. NLS2 was created by a 7-amino-acid C-terminal extension (residues 231 to 237) that became prevalent among human influenza A virus types isolated between the years 1950 to 1987. NLS2 includes basic amino acids at positions 219, 220, 224, 229, 231, and 232. Surprisingly, NLS2 also forms a functional nucleolar localization signal NoLS, a function that was retained in H3N2 type virus NS1A proteins even without the C-terminal extension. It is likely that the evolutionarily well-conserved nucleolar targeting function of NS1A protein plays a role in the pathogenesis of influenza A virus.

Aspergillus nidulans VeA subcellular localization is dependent on the importin a carrier and on light

Abstract: Summary The veA gene is a light-dependent regulator govern- ing development and secondary metabolism in Aspergillus nidulans. We have identified a putative bipartite nuclear localization signal (NLS) motif in the A. nidulans VeA amino acid sequence and demon- strated its functionality when expressed in yeast. Furthermore, migration of VeA to the nucleus was dependent on the importin a. This bipartite NLS is also functional when VeA is expressed in A. nidulans. Interestingly, we found that VeA migration to the nucleus is light-dependent. While in the dark VeA is located mainly in the nuclei, under light VeA is found abundantly in the cytoplasm. The VeA1 mutant protein (lacking the first 36 amino acids at the N-terminus) was found predominantly in the cyto- plasm independent of illumination. This indicates that the truncated bipartite NLS in VeA1 is not functional and fails to respond to light. These results might explain the lack of the morphological light-dependent response in strains carrying the veA1 allele. We also evaluated the effect of light on production of the myc- otoxin sterigmatocystin in a veA wild-type and the veA1 mutant strains and found that the highest amount of toxin was produced by the veA+ strain growing in the dark, condition favouring accumula- tion of VeA in the nucleus.

SAD2, an Importin β-Like Protein, Is Required for UV-B Response in Arabidopsis by Mediating MYB4 Nuclear Trafficking

Abstract: We report that the Arabidopsis thaliana mutant sensitive to ABA and drought2 (sad2), which harbors a T-DNA insertion in an importin β-like gene, is more tolerant to UV-B radiation than the wild type. Analysis of cyclobutane pyrimidine dimer accumulation revealed that less DNA damage occurred in sad2 than in the wild type during UV-B treatment. No significant growth difference was observed between sad2 and the wild type when treated with the genotoxic drug methyl methanesulfonate, suggesting that SAD2 functions in UV-B protection rather than in DNA damage repair. Whereas the R2R3-type transcription repressor MYB4 has previously been shown to negatively regulate the transcription of cinnamate 4-hydroxylase (C4H) and thus to regulate the synthesis of sinapate esters, expression of both MYB4 and C4H and accumulation of UV-absorbing compounds were significantly higher in sad2 than in the wild type. MYB4 did not localize to the nucleus in the sad2 mutant, suggesting that SAD2 is required for MYB4 nuclear trafficking. SAD2 and MYB4 coimmunoprecipitated, indicating that these proteins localize in the same complex in vivo. MYB4 protein specifically bound to its own promoter in gel shift assays and repressed its own expression, demonstrating that MYB4 protein and mRNA are part of a negative autoregulatory loop. This feedback loop is altered in the sad2 mutant due to the absence of MYB4 protein in the nucleus, leading to the constitutive expression of MYB4 and C4H and resulting in accumulation of UV-absorbing pigments that shield the plant from UV-B radiation.

Nuclear localization of dengue virus nonstructural protein 5 through its importin alpha/beta-recognized nuclear localization sequences is integral to viral infection

Abstract: Dengue virus nonstructural protein 5 (NS5) is a large multifunctional protein with a central role in viral replication. We previously identified two nuclear localization sequences (NLSs) within the central region of dengue virus type-2 (DENV-2) NS5 ('aNLS' and 'bNLS') that are recognized by the importin alpha/beta and importin beta1 nuclear transporters, respectively. Here, we demonstrate the importance of the kinetics of NS5 nuclear localization to virus production for the first time and show that the aNLS is responsible. Site-specific mutations in the bipartite-type aNLS or bNLS region were introduced into a reporter plasmid encoding green fluorescent protein fused to the N-terminus of DENV-2 NS5, as well as into DENV-2 genomic length complementary DNA. Mutation of basic residues in the highly conserved region of the bNLS did not affect nuclear import of NS5. In contrast, mutations in either basic cluster of the aNLS decreased NS5 nuclear accumulation and reduced virus production, with the greatest reduction observed for mutation of the second cluster (K(387)K(388)K(389)); mutagenesis of both clusters abolished NS5 nuclear import and DENV-2 virus production completely. The latter appeared to relate to the impaired ability of virus lacking nuclear-localizing NS5, as compared with wild-type virus expressing nuclear-localizing NS5, to reduce interleukin-8 production as part of the antiviral response. The results overall indicate that NS5 nuclear localization through the aNLS is integral to viral infection, with significant implications for other flaviviruses of medical importance, such as yellow fever and West Nile viruses.

A novel Crumbs3 isoform regulates cell division and ciliogenesis via importin β interactions

Abstract: The Crumbs family of apical transmembrane proteins regulates apicobasal polarity via protein interactions with a conserved C-terminal sequence, ERLI. However, one of the mammalian Crumbs proteins, Crumbs3 (CRB3) has an alternate splice form with a novel C-terminal sequence ending in CLPI (CRB3-CLPI). We report that CRB3-CLPI localizes to the cilia membrane and a membrane compartment at the mitotic spindle poles. Knockdown of CRB3-CLPI leads to both a loss of cilia and a multinuclear phenotype associated with centrosomal and spindle abnormalities. Using protein purification, we find that CRB3-CLPI interacts with importin β-1 in a Ran-regulated fashion. Importin β-1 colocalizes with CRB3-CLPI during mitosis, and a dominant-negative form of importin β-1 closely phenocopies CRB3-CLPI knockdown. Knockdown of importin β-1 blocks targeting of CRB3-CLPI to the spindle poles. Our data suggest an expanded role for Crumbs proteins in polarized membrane targeting and cell division via unique interactions with importin proteins.

Novel Nuclear Import of Vpr Promoted by Importin α Is Crucial for Human Immunodeficiency Virus Type 1 Replication in Macrophages

Abstract: Monocytes/macrophages are major targets of human immunodeficiency virus type 1 (HIV-1) infection. The viral preintegration complex (PIC) of HIV-1 enters the nuclei of monocyte-derived macrophages, but very little PIC migrates into the nuclei of immature monocytes. Vpr, one of the accessory gene products of HIV-1, is essential for the nuclear import of PIC in these cells, although the role of Vpr in the entry mechanism of PIC remains to be clarified. We have shown previously that Vpr is targeted to the nuclear envelope and then transported into the nucleus by importin alpha alone, in an importin beta-independent manner. Here we demonstrate that the nuclear import of Vpr is strongly promoted by the addition of cytoplasmic extract from macrophages but not of that from monocytes and that the nuclear import activity is lost with immunodepletion of importin alpha from the cytoplasmic extract. Immunoblot analysis and real-time PCR demonstrate that immature monocytes express importin alpha at low levels, whereas the expression of three major importin alpha isoforms markedly increases upon their differentiation into macrophages, indicating that the expression of importin alpha is required for nuclear import of Vpr. Furthermore, interaction between importin alpha and the N-terminal alpha-helical domain of Vpr is indispensable, not only for the nuclear import of Vpr but also for HIV-1 replication in macrophages. This study suggests the possibility that the binding of Vpr to importin alpha, preceding a novel nuclear import process, is a potential target for therapeutic intervention.

Intracellular transport of hepatitis B virus.

Abstract: For genome multiplication hepadnaviruses use the transcriptional machinery of the cell that is found within the nucleus. Thus the viral genome has to be transported through the cytoplasm and nuclear pore. The intracytosolic translocation is facilitated by the viral capsid that surrounds the genome and that interacts with cellular microtubules. The subsequent passage through the nuclear pore complexes (NPC) is mediated by the nuclear transport receptors importin alpha and beta. Importin alpha binds to the C-terminus of the capsid protein that comprises a nuclear localization signal (NLS). The exposure of the NLS is regulated and depends upon genome maturation and/or phosphorylation of the capsid protein. As for other karyophilic cargos using this pathway importin alpha interacts with importin beta that facilitates docking of the import complex to the NPC and the passage through the pore. Being a unique strategy, the import of the viral capsid is incomplete in that it becomes arrested inside the nuclear basket, which is a cage-like structure on the karyoplasmic face of the NPC. Presumably only this compartment provides the factors that are required for capsid disassembly and genome release that is restricted to those capsids comprising a mature viral DNA genome.

A microtubule-facilitated nuclear import pathway for cancer regulatory proteins.

Abstract: Nuclear protein import is dependent on specific targeting signals within cargo proteins recognized by importins (IMPs) that mediate translocation through the nuclear pore. Recent evidence, however, implicates a role for the microtubule (MT) network in facilitating nuclear import of the cancer regulatory proteins parathyroid hormone-related protein (PTHrP) and p53 tumor suppressor. Here we assess the extent to which MT and actin integrity may be generally required for nuclear protein import for the first time. We examine 10 nuclear-localizing proteins with diverse IMP-dependent nuclear import pathways, our results indicating that the cytoskeleton does not have a general mechanistic role in nuclear localization sequence-dependent nuclear protein import. Of the proteins examined, only the p110(Rb) tumor suppressor protein Rb, together with p53 and PTHrP, was found to require MT integrity for optimal nuclear import. Fluorescence recovery after photobleaching experiments indicated that the MT-dependent nuclear transport pathway increases both the rate and extent of Rb nuclear import but does not affect Rb nuclear export. Dynamitin overexpression experiments implicate the MT motor dynein in the import process. The results indicate that, additional to IMP/diffusion-dependent processes, certain cancer regulatory proteins utilize an MT-enhanced pathway for accelerated nuclear import that is presumably required for their nuclear functions.

Lamin B receptor plays a role in stimulating nuclear envelope production and targeting membrane vesicles to chromatin during nuclear envelope assembly through direct interaction with importin β

Abstract: Lamin B receptor (LBR), a chromatin and lamin binding protein in the inner nuclear membrane, has been proposed to play a vital role in nuclear envelope (NE) assembly. But the specific role for LBR in NE assembly remains unknown. In the present study, we show that overexpression of LBR causes membrane overproduction, inducing NE invagination and membrane stack formation, and that these processes require the transmembrane domain of LBR. Biochemical analysis shows that the N-terminal domain of LBR directly interacts with importin beta in a Ran sensitive and importin alpha independent manner. Using an in vitro NE assembly assay, we also demonstrate that blocking full length LBR binding sites on importin beta, by the addition of the LBR N-terminal domain inhibits the recruitment of LBR-containing vesicles to importin beta- or Ran-coated beads to form NE structure. Our results suggest that LBR is recruited to chromatin through direct interaction with importin beta to contribute to the fusion of membrane vesicles and formation of the NE.

Msk is required for nuclear import of TGF-β/BMP-activated Smads

Abstract: Nuclear translocation of Smad proteins is a critical step in signal transduction of transforming growth factor β (TGF-β) and bone morphogenetic proteins (BMPs). Using nuclear accumulation of the Drosophila Smad Mothers against Decapentaplegic (Mad) as the readout, we carried out a whole-genome RNAi screening in Drosophila cells. The screen identified moleskin (msk) as important for the nuclear import of phosphorylated Mad. Genetic evidence in the developing eye imaginal discs also demonstrates the critical functions of msk in regulating phospho-Mad. Moreover, knockdown of importin 7 and 8 (Imp7 and 8), the mammalian orthologues of Msk, markedly impaired nuclear accumulation of Smad1 in response to BMP2 and of Smad2/3 in response to TGF-β. Biochemical studies further suggest that Smads are novel nuclear import substrates of Imp7 and 8. We have thus identified new evolutionarily conserved proteins that are important in the signal transduction of TGF-β and BMP into the nucleus.

Location, Location, Location: Altered Transcription Factor Trafficking in Neurodegeneration

Abstract: Neurons may be particularly sensitive to disruptions in transcription factor trafficking. Survival and injury signals must traverse dendrites or axons, in addition to soma, to affect nuclear transcriptional responses. Transcription factors exhibit continued nucleocytoplasmic shuttling; the predominant localization is regulated by binding to anchoring proteins that mask nuclear localization/export signals and/or target the factor for degradation. Two functional groups of karyopherins, importins and exportins, mediate RanGTPase-dependent transport through the nuclear pore. A growing number of recent studies, in Alzheimer, Parkinson, and Lewy body diseases, amyotrophic lateral sclerosis, and human immunodeficiency virus encephalitis, implicate aberrant cytoplasmic localization of transcription factors and their regulatory kinases in degenerating neurons. Potential mechanisms include impaired nuclear import, enhanced export, suppression of degradation, and sequestration in protein aggregates or organelles and may reflect unmasking of alternative cytoplasmic functions, both physiologic and pathologic. Some "nuclear" factors also function in mitochondria, and importins are also involved in axonal protein trafficking. Detrimental consequences of a decreased nuclear to cytoplasmic balance include suppression of neuroprotective transcription mediated by cAMP- and electrophile/antioxidant-response elements and gain of toxic cytoplasmic effects. Studying the pathophysiologic mechanisms regulating transcription factor localization should facilitate strategies to bypass deficits and restore adaptive neuroprotective transcriptional responses.

Reversibility in nucleocytoplasmic transport

Abstract: Nucleocytoplasmic exchange of proteins and RNAs is mediated by receptors that usher their cargo through the nuclear pores. Peptide localization signals on each cargo determine the receptors with which it will interact. Those interactions are normally regulated by the small GTPase Ran. Hydrolysis of GTP provides the chemical energy required to create a bona fide thermodynamic pump that selectively and directionally accumulates its substrates across the nuclear envelope. A common perception is that cargo delivery is irreversible, e.g., a protein imported to the nucleus does not return to the cytoplasm except perhaps via a specific export receptor. Quantitative measurements using cell-free nuclei reconstituted in Xenopus egg extract show that nuclear accumulation follows first-order kinetics and reaches steady state at a level that follows a Michaelis–Menten function of the cytoplasmic cargo concentration. This saturation suggests that receptor-mediated translocation across the nuclear pore occurs bidirectionally. The reversibility of accumulation was demonstrated directly by exchange of the cytosolic medium and by fluorescence recovery after photobleaching. Based on our results, we offer a simple biophysical model that predicts the observed behavior. A far-reaching consequence is that the nuclear localization signal dictates the fate of a protein population rather than that of the individual molecules that bear it, which remain free to shuttle back and forth. This implies an open communication between the nucleus and cytoplasm and a ubiquitous mechanism for signaling in both directions.

A Role for Transportin in the Nuclear Import of Adenovirus Core Proteins and DNA

Abstract: Adenoviruses target their double-stranded DNA genome and its associated core proteins to the interphase nucleus; this core structure then enters through the nuclear pore complex We have used digitonin permeabilized cell import assays to study the cellular import factors involved in nuclear entry of virus DNA and the core proteins, protein V and protein VII We show that inhibition of transportin results in aberrant localization of protein V and that transportin is necessary for protein V to accumulate in the nucleolus Furthermore, inhibition of transportin results in inhibition of protein VII and DNA import, whereas disruption of the classical importin α–importin β import pathway has little effect We show that mature protein VII has different import preferences from the precursor protein, preVII from which it is derived by proteolytic processing While bacterially expressed glutathione S-transferase (GST)-preVII primarily utilizes the pathway mediated by importin α–importin β, bacterially expressed GST-VII favours the transportin pathway This is significant because while preVII is important during viral replication and assembly only mature VII is available during viral DNA import to a newly infected cell Our results implicate transportin as a key import receptor for the nuclear localization of adenovirus core

Distinct functions of the Drosophila Nup153 and Nup214 FG domains in nuclear protein transport

Abstract: The phenylanine-glycine (FG)–rich regions of several nucleoporins both bind to nuclear transport receptors and collectively provide a diffusion barrier to the nuclear pores. However, the in vivo roles of FG nucleoporins in transport remain unclear. We have inactivated 30 putative nucleoporins in cultured Drosophila melanogaster S2 cells by RNA interference and analyzed the phenotypes on importin α/β−mediated import and CRM1-dependent protein export. The fly homologues of FG nucleoporins Nup358, Nup153, and Nup54 are selectively required for import. The FG repeats of Nup153 are necessary for its function in transport, whereas the remainder of the protein maintains pore integrity. Inactivation of the CRM1 cofactor RanBP3 decreased the nuclear accumulation of CRM1 and protein export. We report a surprisingly antagonistic relationship between RanBP3 and the Nup214 FG region in determining CRM1 localization and its function in protein export. Our data suggest that peripheral metazoan FG nucleoporins have distinct functions in nuclear protein transport events.

Hyperosmotic stress signaling to the nucleus disrupts the Ran gradient and the production of RanGTP.

Abstract: The RanGTP gradient depends on nucleocytoplasmic shuttling of Ran and its nucleotide exchange in the nucleus. Here we show that hyperosmotic stress signaling induced by sorbitol disrupts the Ran protein gradient and reduces the production of RanGTP. Ran gradient disruption is rapid and is followed by early (10-20 min) and late (30-60 min) phases of recovery. Results from SB203580 and siRNA experiments suggest the stress kinase p38 is important for Ran gradient recovery. NTF2 and Mog1, which are transport factors that regulate the nuclear localization of Ran, showed kinetics of delocalization and recovery similar to Ran. Microinjection of a nuclear localization signal reporter protein revealed that sorbitol stress decreases the rate of nuclear import. Sorbitol stress also slowed RCC1 mobility in the nucleus, which is predicted to reduce RCC1 dissociation from chromatin and RanGTP production. This was tested using a FRET biosensor that registers nuclear RanGTP levels, which were reduced in response to sorbitol stress. Although sorbitol alters nucleotide levels, we show that inverting the GTP/GDP ratio in cells is not sufficient to disrupt the Ran gradient. Thus, the Ran system is a target of hyperosmotic stress signaling, and cells use protein localization-based mechanisms as part of a rapid stress response.

The Karyopherin Kap95 Regulates Nuclear Pore Complex Assembly Into Intact Nuclear Envelopes in Vivo

Abstract: Nuclear pore complex (NPC) assembly in interphase cells requires that new NPCs insert into an intact nuclear envelope (NE). Our previous work identified the Ran GTPase as an essential component in this process. We proposed that Ran is required for targeting assembly factors to the cytoplasmic NE face via a novel, vesicular intermediate. Although the molecular target was not identified, Ran is known to function by modulating protein interactions for karyopherin (Kap) β family members. Here we characterize loss-of-function Saccharomyces cerevisiae mutants in KAP95 with blocks in NPC assembly. Similar to defects in Ran cycle mutants, nuclear pore proteins are no longer localized properly to the NE in kap95 mutants. Also like Ran cycle mutants, the kap95-E126K mutant displayed enhanced lethality with nic96 and nup170 mutants. Thus, Kap95 and Ran are likely functioning at the same stage in assembly. However, although Ran cycle mutants accumulate small cytoplasmic vesicles, cells depleted of Kap95 accumulated long stretches of cytoplasmic membranes and had highly distorted NEs. We conclude that Kap95 serves as a key regulator of NPC assembly into intact NEs. Furthermore, both Kap95 and Ran may provide spatial cues necessary for targeting of vesicular intermediates in de novo NPC assembly.