HIV integration targeting: a pathway involving Transportin-3 and the nuclear pore protein RanBP2

Genome-wide siRNA screens have identified host cell factors important for efficient HIV infection, among which are nuclear pore proteins such as RanBP2/Nup358 and the karyopherin Transportin-3/TNPO3. Analysis of the roles of these proteins in the HIV replication cycle suggested that correct trafficking through the pore may facilitate the subsequent integration step. Here we present data for coupling between these steps by demonstrating that depletion of Transportin-3 or RanBP2 altered the terminal step in early HIV replication, the selection of chromosomal sites for integration. We found that depletion of Transportin-3 and RanBP2 altered integration targeting for HIV. These knockdowns reduced HIV integration frequency in gene-dense regions and near gene-associated features, a pattern that differed from that reported for depletion of the HIV integrase binding cofactor Psip1/Ledgf/p75. MLV integration was not affected by the Transportin-3 knockdown. Using siRNA knockdowns and integration targeting analysis, we also implicated several additional nuclear proteins in proper target site selection. To map viral determinants of integration targeting, we analyzed a chimeric HIV derivative containing MLV gag, and found that the gag replacement phenocopied the Transportin-3 and RanBP2 knockdowns. Thus, our data support a model in which Gag-dependent engagement of the proper transport and nuclear pore machinery mediate trafficking of HIV complexes to sites of integration.     

Distinct nuclear import and export pathways mediated by members of the karyopherin β family

Transport of proteins into and out of the nucleus occurs through nuclear pore complexes (NPCs) and is mediated by the interaction of transport factors with nucleoporins at the NPC. Nuclear import of proteins containing classical nuclear localization signals (NLSs) is mediated by a heterodimeric protein complex, composed of karyopherin α and β1, that docks via β1 the NLS-protein to the NPC. The GTPase Ran; the RanGDP binding protein, p10; and the RanGTP binding protein, RanBP1 are involved in translocation of the docked NLS-protein into the nucleus. Recently, new distinct nuclear import and export pathways that are mediated by members of the karyopherin β family have been discovered. Karyopherin β2 mediates import of mRNA binding proteins, whereas karyopherin β3 and β4 mediate import of a set of ribosomal proteins. Two other β karyopherin family members, CRM1 and CAS, mediate export of proteins containing leucine-rich nuclear export signals (NES) and reexport of karyopherin α, respectively. This growing family contains new members that constitute potential transport factors for cargoes yet to be identified in the future. The common features of the members of karyopherin β family are the ability to bind RanGTP and the ability to interact directly with nucleoporins at the NPC. The challenge for the future will be to identify the distinct or, perhaps, overlapping cargo(es) for each member of the karyopherin β superfamily and to characterize the molecular mechanisms of translocation of karyopherins together with their cargoes through the NPC. J. Cell. Biochem. 70:231–239, 1998.© 1998 Wiley-Liss, Inc.     

Redox-Dependent Control of FOXO/DAF-16 by Transportin-1

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Highlights? FOXO forms redox-sensitive, disulfide-dependent complexes with several proteins ? Transportin-1 binds to FOXO via a disulfide and regulates its nuclear localization ? Redox and insulin signaling govern FOXO nuclear localization via distinct pathways ? Redox control of longevity protein FOXO/DAF-16 is evolutionarily conserved     

Dissecting the roles of Cse1 and Nup2 in classical NLS‐cargo release in vivo

The importin α/β transport machinery mediates the nuclear import of cargo proteins that bear a classical nuclear localization sequence (cNLS). These cargo proteins are linked to the major nuclear protein import factor, importin‐β, by the importin‐α adapter, after which cargo/carrier complexes enter the nucleus through nuclear pores. In the nucleus, cargo is released by the action of RanGTP and the nuclear pore protein Nup2, after which the importins are recycled to the cytoplasm for further transport cycles. The nuclear export of importin‐α is mediated by Cse1/CAS. Here, we exploit structures of functionally important complexes to identify residues that are critical for these interactions and provide insight into how cycles of protein import and recycling of importin‐α occur in vivo using a Saccharomyces cerevisiae model. We examine how these molecular interactions impact protein localization, cargo import, function and complex formation. We show that reversing the charge of key residues in importin‐α (Arg44) or Cse1 (Asp220) results in loss of function of the respective proteins and impairs complex formation both in vitro and in vivo. To extend these results, we show that basic residues in the Nup2 N‐terminus are required for both Nup2 interaction with importin‐α and Nup2 function. These results provide a more comprehensive mechanistic model of how Cse1, RanGTP and Nup2 function in concert to mediate cNLS‐cargo release in the nucleus.     

Regulation of β-catenin nuclear dynamics by GSK-3β involves a LEF-1 positive feedback loop

Nuclear localization of β-catenin is integral to its role in Wnt signaling and cancer. Cellular stimulation by Wnt or lithium chloride (LiCl) inactivates glycogen synthase kinase-3β (GSK-3β), causing nuclear accumulation of β-catenin and transactivation of genes that transform cells. β-catenin is a shuttling protein; however, the mechanism by which GSK-3β regulates β-catenin nuclear dynamics is poorly understood. Here, fluorescence recovery after photobleaching assays were used to measure the β-catenin-green fluorescent protein dynamics in NIH 3T3 cells before and after GSK-3β inhibition. We show for the first time that LiCl and Wnt3a cause a specific increase in β-catenin nuclear retention in live cells and in fixed cells after detergent extraction. Moreover, LiCl reduced the rate of nuclear export but did not affect import, hence biasing β-catenin transport toward the nucleus. Interestingly, the S45A mutation, which blocks β-catenin phosphorylation by GSK-3β, did not alter nuclear retention or transport, implying that GSK-3β acts through an independent regulator. We compared five nuclear binding partners and identified LEF-1 as the key mediator of Wnt3a and LiCl-induced nuclear retention of β-catenin. Thus, Wnt stimulation triggered a LEF-1 positive feedback loop to enhance the nuclear chromatin-retained pool of β-catenin by 100-300%. These findings shed new light on regulation of β-catenin nuclear dynamics.     

Small molecule peptidomimetic inhibitors of importin α/β mediated nuclear transport

Nucleocytoplasmic transport of macromolecules is a fundamental process of eukaryotic cells. Translocation of proteins and many RNAs between the nucleus and cytoplasm is carried out by shuttling receptors of the β-karyopherin family, also called importins and exportins. Leptomycin B, a small molecule inhibitor of the exportin CRM1, has proved to be an invaluable tool for cell biologists, but up to now no small molecule inhibitors of nuclear import have been described. We devised a microtiter plate based permeabilized cell screen for small molecule inhibitors of the importin α/β pathway. By analyzing peptidomimetic libraries, we identified β-turn and α-helix peptidomimetic compounds that selectively inhibit nuclear import by importin α/β but not by transportin. Structure–activity relationship analysis showed that large aromatic residues and/or a histidine side chain are required for effective import inhibition by these compounds. Our validated inhibitors can be useful for in vitro studies of nuclear import, and can also provide a framework for synthesis of higher potency nuclear import inhibitors.     

Distinct roles for classical nuclear import receptors in the growth of multinucleated muscle cells

Proper muscle function is dependent on spatial and temporal control of gene expression in myofibers. Myofibers are multinucleated cells that are formed, repaired and maintained by the process of myogenesis in which progenitor myoblasts proliferate, differentiate and fuse. Gene expression is dependent upon proteins that require facilitated nuclear import, however little is known about the regulation of nucleocytoplasmic transport during the formation of myofibers. We analyzed the role of karyopherin alpha (KPNA), a key classical nuclear import receptor, during myogenesis. We established that five karyopherin alpha paralogs are expressed by primary mouse myoblasts in vitro and that their steady-state levels increase in multinucleated myotubes, suggesting a global increase in demand for classical nuclear import during myogenesis. We used siRNA-mediated knockdown to identify paralog-specific roles for KPNA1 and KPNA2 during myogenesis. KPNA1 knockdown increased myoblast proliferation, whereas KPNA2 knockdown decreased proliferation. In contrast, no proliferation defect was observed with KPNA4 knockdown. Only knockdown of KPNA2 decreased myotube growth. These results identify distinct pathways involved in myoblast proliferation and myotube growth that rely on specific nuclear import receptors suggesting that regulation of classical nuclear import pathways likely plays a critical role in controlling gene expression in skeletal muscle.     

Identification of a Karyopherin β1/β2 Proline-Tyrosine Nuclear Localization Signal in Huntingtin Protein

Among the known pathways of protein nuclear import, the karyopherin β2/transportin pathway is only the second to have a defined nuclear localization signal (NLS) consensus. Huntingtin, a 350-kDa protein, has defined roles in the nucleus, as well as a CRM1/exportin-dependent nuclear export signal; however, the NLS and exact pathway of import have remained elusive. Here, using a live cell assay and affinity chromatography, we show that huntingtin has a karyopherin β2-dependent proline-tyrosine (PY)-NLS in the amino terminus of the protein. This NLS comprises three consensus components: a basic charged sequence, a downstream conserved arginine, and a PY sequence. Unlike the classic PY-NLS, which has an unstructured intervening sequence between the consensus components, we show that a β sheet structured region separating the consensus elements is critical for huntingtin NLS function. The huntingtin PY-NLS is also capable of import through the importin/karyopherin β1 pathway but was not functional in all cell types tested. We propose that this huntingtin PY-NLS may comprise a new class of multiple import factor-dependent NLSs with an internal structural component that may regulate NLS activity.     

Conserved SR protein kinase functions in nuclear import and its action is counteracted by arginine methylation in Saccharomyces cerevisiae

Mammalian serine and arginine-rich (SR) proteins play important roles in both constitutive and regulated splicing, and SR protein-specific kinases (SRPKs) are conserved from humans to yeast. Here, we demonstrate a novel function of the single conserved SR protein kinase Sky1p in nuclear import in budding yeast. The yeast SR-like protein Npl3p is known to enter the nucleus through a composite nuclear localization signal (NLS) consisting of a repetitive arginine- glycine-glycine (RGG) motif and a nonrepetitive sequence. We found that the latter is the site for phosphorylation by Sky1p and that this phosphorylation regulates nuclear import of Npl3p by modulating the interaction of the RGG motif with its nuclear import receptor Mtr10p. The RGG motif is also methylated on arginine residues, but methylation does not affect the Npl3p-Mtr10p interaction in vitro. Remarkably, arginine methylation interferes with Sky1p-mediated phosphorylation, thereby indirectly influencing the Npl3p-Mtr10p interaction in vivo and negatively regulating nuclear import of Npl3p. These results suggest that nuclear import of Npl3p is coordinately influenced by methylation and phosphorylation in budding yeast, which may represent conserved components in the dynamic regulation of RNA processing in higher eukaryotic cells.     

Axo-glial interactions regulate the localization of axonal paranodal proteins

The SR proteins, a group of abundant arginine/serine (RS)-rich proteins, are essential pre-mRNA splicing factors that are localized in the nucleus. The RS domain of these proteins serves as a nuclear localization signal. We found that RS domain-bearing proteins do not utilize any of the known nuclear import receptors and identified a novel nuclear import receptor specific for SR proteins. The SR protein import receptor, termed transportin-SR (TRN-SR), binds specifically and directly to the RS domains of ASF/SF2 and SC35 as well as several other SR proteins. The nuclear transport regulator RanGTP abolishes this interaction. Recombinant TRN-SR mediates nuclear import of RS domain- bearing proteins in vitro. TRN-SR has amino acid sequence similarity to several members of the importin beta/transportin family. These findings strongly suggest that TRN-SR is a nuclear import receptor for the SR protein family.     

Subcellular translocation signals regulate Geminin activity during embryonic development

BACKGROUND INFORMATION: Geminin (Gem) is a protein with roles in regulating both the fidelity of DNA replication and cell fate during embryonic development. The distribution of Gem is predominantly nuclear in cells undergoing the cell cycle. Previous studies have demonstrated that Gem performs multiple activities in the nucleus and that regulation of Gem activation requires nuclear import in at least one context. In the present study, we defined structural and mechanistic features underlying subcellular localization of Gem and tested whether regulation of the subcellular localization of Gem has an impact on its activity in cell fate specification during embryonic development. RESULTS: We determined that nuclear localization of Gem is dependent on a bipartite NLS (nuclear localization signal) in the N-terminus of Xenopus Gem protein. This bipartite motif mapped to a Gem N-terminal region previously shown to regulate neural cell fate acquisition. Microinjection into Xenopus embryos demonstrated that import-deficient Gem was incapable of modulating ectodermal cell fate, but that this activity was rescued by fusion to a heterologous NLS. Cross-species comparison of Gem protein sequences revealed that the Xenopus bipartite signal is conserved in many non-mammalian vertebrates, but not in mammalian species assessed. Instead, we found that human Gem employs an alternative N-terminal motif to regulate the protein's nuclear localization. Finally, we found that additional mechanisms contributed to regulating the subcellular localization of Gem. These included a link to Crm1-dependent nuclear export and the observation that Cdt1, a protein in the pre-replication complex, could also mediate nuclear import of Gem. CONCLUSIONS: We have defined new structural and regulatory features of Gem, and showed that the activity of Gem in regulating cell fate, in addition to its cell-cycle-regulatory activity, requires control of its subcellular localization. Our data suggest that rather than being constitutively nuclear, Gem may undergo nucleocytoplasmic shuttling through several mechanisms involving distinct protein motifs. The use of multiple mechanisms for modulating Gem subcellular localization is congruent with observations that Gem levels and activity must be stringently controlled during cell-cycle progression and embryonic development.     

The VirE3 protein of Agrobacterium mimics a host cell function required for plant genetic transformation

To genetically transform plants, Agrobacterium exports its transferred DNA (T-DNA) and several virulence (Vir) proteins into the host cell. Among these proteins, VirE3 is the only one whose biological function is completely unknown. Here, we demonstrate that VirE3 is transferred from Agrobacterium to the plant cell and then imported into its nucleus via the karyopherin alpha-dependent pathway. In addition to binding plant karyopherin alpha, VirE3 interacts with VirE2, a major bacterial protein that directly associates with the T-DNA and facilitates its nuclear import. The VirE2 nuclear import in turn is mediated by a plant protein, VIP1. Our data indicate that VirE3 can mimic this VIP1 function, acting as an 'adapter' molecule between VirE2 and karyopherin alpha and 'piggy-backing' VirE2 into the host cell nucleus. As VIP1 is not an abundant protein, representing one of the limiting factors for transformation, Agrobacterium may have evolved to produce and export to the host cells its own virulence protein that at least partially complements the cellular VIP1 function necessary for the T-DNA nuclear import and subsequent expression within the infected cell.     

The interaction between importin-α and Nup153 promotes importin-α/β-mediated nuclear import

Nuclear transport is mediated by transport factors, including the importin β family members. The directionality of nuclear transport is governed by the asymmetrical distribution of the small GTPase Ran. Of note, importin α/β-mediated import of classical nuclear localization signal (cNLS)--containing cargo is more efficient than other Ran-dependent import pathways that do not require importin α. In this study, we characterized the role of importin α in nuclear transport by examining import efficiencies of cNLS-cargo/importin α/β complexes. We first depleted digitonin-permeabilized semi-intact cells of endogenous importin α and used the cells to show that the interaction between importin α and Nup153--a component of the nuclear pore complex (NPC)--is essential for efficient import of importin β-binding domain containing substrates, but not other cargoes that directly bind to importin β. Moreover, we found that the binding of importin α to Nup153 facilitates cNLS-mediated import, and demonstrated that importin α in import complexes and cargo-free importin α prebound to Nup153 promote efficient import of cNLS-containing proteins. This is the first in vitro study showing that in conjunction with Nup153, importin α contributes to directionally biased exit of cNLS-containing cargo to the nuclear side of NPCs.     

蛋白质的入核运送和它在基因表达中的调节作用

蛋白质进入细胞核是由蛋白质分子内部的核定位信号（nuclear localization signal, NLS）引导的.NLS蛋白首先与NLS受体结合,然后在多种胞浆因子及核孔复合物蛋白的作用下穿过核孔、转位入核.蛋白质上存在NLS并不一定总能够引导蛋白质入核.当NLS被修饰或遮掩时,它们便不能被核转运装置所识别.因而,NLS的遮掩被解除之前,蛋白质一直被扣留在胞浆中.以调节转录因子的入核运送来控制转录因子的活性是基因表达调节的一个新概念,也是细胞生长和分化的另一水平的调节.     

Mediators of Nuclear Protein Import Target Karyophilic Proteins to Pore Complexes of Cytoplasmic Annulate Lamellae

Nuclear pore complexes are constitutive structures of the nuclear envelope in eukaryotic cells and represent the sites where transport of molecules between nucleus and cytoplasm takes place. However, pore complexes of similar structure, but with largely unknown functional properties, are long known to occur also in certain cytoplasmic cisternae that have been termed annulate lamellae (AL). To analyze the capability of the AL pore complex to interact with the soluble mediators of nuclear protein import and their karyophilic protein substrates, we have performed a microinjection study in stage VI oocytes ofXenopus laevis.In these cells AL are especially abundant and can easily be identified by light and electron microscopy. Following injection into the cytoplasm, fluorochrome-labeled mediators of two different nuclear import pathways, importin β and transportin, not only associate with the nuclear envelope but also with AL. Likewise, nuclear localization signals (NLS) of the basic and M9 type, but not nuclear export signals, confer targeting and transient binding of fluorochrome-labeled proteins to cytoplasmic AL. Mutation or deletion of the NLS signals prevents these interactions. Furthermore, binding to AL is abolished by dominant negative inhibitors of nuclear protein import. Microinjections of gold-coupled NLS-bearing proteins reveal specific gold decoration at distinct sites within the AL pore complex. These include such at the peripheral pore complex-attached fibrils and at the central “transporter” and closely resemble those of “transport intermediates” found in electron microscopic studies of the nuclear pore complex (NPC). These data demonstrate that AL can represent distinct sites within the cytoplasm of transient accumulation of nuclear proteins and that the AL pore complex shares functional binding properties with the NPC.     

Phospholipase Cdelta1 associates with importin beta1 and translocates into the nucleus in a Ca2+-dependent manner

Phospholipase C (PLC)delta1 shuttles between the nucleus and the cytoplasm. Here, we demonstrate that treatment of MDCK cells and PC12 cells with ionomycin causes nuclear accumulation of ectopically expressed and endogenous PLCdelta1, respectively, suggesting that signals that increase [Ca2+]i trigger nuclear translocation. To clarify the molecular mechanisms involved in this translocation, we have examined whether PLCdelta1 binds with importins. PLCdelta1 interacted with importin beta1 in a Ca2+-dependent manner in vitro even in the absence of importin alpha. A PLCdelta1 mutant E341A, which lacks Ca2+-binding to the catalytic core, did not show this interaction at any physiological Ca2+ concentration and did not translocate into the nucleus after ionomycin treatment when expressed in MDCK cells. These results suggested that the nuclear import of PLCdelta1 is mediated by its Ca2+-dependent interaction with importin beta1.     

Active Nuclear Import and Export Is Independent of Lumenal Ca2+ Stores in Intact Mammalian Cells

The nuclear pore complex (NPC) mediates communication between the cytoplasm and nucleus in eukaryotic cells. Active transport of large polypeptides as well as passive diffusion of smaller (≈10 kD) macromolecules through the NPC can be inhibited by depletion of intracellular Ca²⁺ stores. However, the physiological relevance of this process for the regulation of nucleocytoplasmic trafficking is not yet clear. We expressed green fluorescent protein (GFP)–tagged glucocorticoid receptor (GR) and mitogen-activated protein (MAP) kinase–activated protein kinase 2 (MK2) to study the effect of Ca²⁺ store depletion on active transport in HM1 cells, a human embryonic kidney cell line stably transfected with the muscarinic M₁ receptor. Dexamethasone-induced nuclear import of GR-GFP and anisomycin-induced nuclear export of GFP-MK2 was monitored by confocal microscopy. We found that store depletion by carbachol, thapsigargin or ionomycin had no effect on GR-GFP import, whereas pretreatment with 1,2-bis-(o-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid–acetoxymethyl ester (BAPTA-AM) attenuated import significantly. Export of GFP-MK2 was not influenced by any pretreatment. Moreover, carbachol stimulated GFP-MK2 translocation to the cytoplasm in the absence of anisomycin. These results demonstrate that Ca²⁺ store depletion in intact HM1 cells is not directly linked to the inhibition of active protein transport through the NPC. The inhibition of GR-GFP import but not GFP-MK2 export by BAPTA-AM presumably involves a depletion-independent mechanism that interferes with components of the nuclear import pathway.