Nuclear import and DNA binding of human papillomavirus type 45 L1 capsid protein

During the life cycle of human papillomaviruses (HPVs), the L1 capsid proteins seem to enter the nucleus twice: once after the virions infect the cells, and later during the productive phase when they assemble the replicated HPV genomic DNA into infectious virions. We established for the high-risk HPV45 that when digitonin-permeabilized HeLa cells were incubated with L1 homopentameric capsomers, the HPV45 L1 protein was imported into the nucleus in a receptor-mediated manner. In contrast, intact capsids were not able to enter the nucleus. Immunoisolation assays showed that HPV45 L1 capsomers interact with cytosolic karyopherin alpha 2 beta 1 heterodimers. HPV45 L1 bound strongly to karyopherin alpha 2, and weakly to karyopherin beta 1, as did its nuclear localization signal (NLS). Nuclear import of HPV45 L1, or of a GST-NLS(HPV45L1) fusion protein was efficiently mediated by karyopherin alpha 2 beta 1 heterodimers, and only weakly by karyopherin beta 1. Nuclear import required RanGDP, but was independent of GTP hydrolysis by Ran. Together, these data suggest that the major nuclear import pathway for HPV45 L1 major capsid protein in infected host cells is mediated by karyopherin alpha 2 beta 1 heterodimers and that GTP hydrolysis by Ran is not required for import. Remarkably, HPV45 L1 capsomers can interact nonspecifically with different types of HPV-DNA, and the DNA binding region of HPV45 L1 overlaps with its NLS sequence.     

Identification of a karyopherin alpha 2 recognition site in PLAG1, which functions as a nuclear localization signal

The activation of the pleomorphic adenoma gene 1 (PLAG1) is the most frequent gain-of-function mutation found in pleomorphic adenomas of the salivary glands. To gain more insight into the regulation of PLAG1 function, we searched for PLAG1-interacting proteins. Using the yeast two-hybrid system, we identified karyopherin alpha2 as a PLAG1-interacting protein. Physical interaction between PLAG1 and karyopherin alpha2 was confirmed by an in vitro glutathione S-transferase pull-down assay. Karyopherin alpha2 escorts proteins into the nucleus via interaction with a nuclear localization sequence (NLS) composed of short stretches of basic amino acids. Two putative NLSs were identified in PLAG1. The predicted NLS1 (KRKR) was essential for physical interaction with karyopherin alpha2 in glutathione S-transferase pull-down assay, and its mutation resulted in decreased nuclear import of PLAG1. Moreover, NLS1 was able to drive the nuclear import of the cytoplasmic protein beta-galactosidase. In contrast, predicted NLS2 of PLAG1 (KPRK) was not involved in karyopherin alpha2 binding nor in its nuclear import. The residual nuclear import of PLAG1 after mutation of the NLS1 was assigned to the zinc finger domain of PLAG1. These observations indicate that the nuclear import of PLAG1 is governed by its zinc finger domain and by NLS1, a karyopherin alpha2 recognition site.     

Dissection of the karyopherin alpha nuclear localization signal (NLS)-binding groove: functional requirements for NLS binding

Classical protein import, mediated by the binding of a classical nuclear localization signal (NLS) to the NLS receptor, karyopherin/importin alpha, is the most well studied nuclear transport process. Classical NLSs are either monopartite sequences that contain a single cluster of basic amino acids (Lys/Arg) or bipartite sequences that contain two clusters of basic residues separated by an unconserved linker region. We have created mutations in conserved residues in each of the three NLS-binding sites/regions in Saccharomyces cerevisiae karyopherin alpha (SRP1). For each mutant we have analyzed binding to both a monopartite and a bipartite NLS cargo in vitro. We have also expressed each karyopherin alpha mutant in vivo as the only cellular copy of the NLS receptor and examined the impact on cell growth and import of both monopartite and bipartite NLS-containing cargoes. Our results reveal the functional significance of specific residues within karyopherin alpha for NLS cargo binding. A karyopherin alpha variant with a mutation in the major NLS-binding site exhibits decreased binding to both monopartite and bipartite NLS cargoes, and this protein is not functional in vivo. However, we also find that a karyopherin alpha variant with a mutation in the minor NLS-binding site, which shows decreased binding only to bipartite NLS-containing cargoes, is also not functional in vivo. This suggests that the cell is dependent on the function of at least one bipartite NLS cargo that is imported into the nucleus by karyopherin alpha. Our experiments also reveal functional importance for the linker-binding region. This study provides insight into how changes in binding to cellular NLS sequences could impact cellular function. In addition, this work has led to the creation of conditional alleles of karyopherin alpha with well characterized defects in NLS binding that will be useful for identifying and characterizing novel NLS cargoes.     

Karyopherins and nuclear import

Proteins of the karyopherin alpha and karyopherin beta families play a central role in nucleocytoplasmic transport. Recently, crystal structures of karyopherin alpha and its complexes with nuclear localization signal peptides, a karyopherin beta2-Ran complex and complexes of full-length and fragments of karyopherin beta1 with import substrates, Ran and nucleoporins have been solved. These karyopherin structures provide valuable insights into understanding the molecular mechanism of nuclear import, especially substrate recognition, substrate release by GTPase and interactions with the nuclear pore complex.     

The l2 minor capsid protein of human papillomavirus type 16 interacts with a network of nuclear import receptors

The L2 minor capsid proteins enter the nucleus twice during viral infection: in the initial phase after virion disassembly and in the productive phase when, together with the L1 major capsid proteins, they assemble the replicated viral DNA into virions. In this study we investigated the interactions between the L2 protein of high-risk human papillomavirus type 16 (HPV16) and nuclear import receptors. We discovered that HPV16 L2 interacts directly with both Kapbeta(2) and Kapbeta(3). Moreover, binding of Ran-GTP to either Kapbeta(2) or Kapbeta(3) inhibits its interaction with L2, suggesting that the Kapbeta/L2 complex is import competent. In addition, we found that L2 forms a complex with the Kapalpha(2)beta(1) heterodimer via interaction with the Kapalpha(2) adapter. In agreement with the binding data, nuclear import of L2 in digitonin-permeabilized cells could be mediated by either Kapalpha(2)beta(1) heterodimers, Kapbeta(2), or Kapbeta(3). Mapping studies revealed that HPV16 L2 contains two nuclear localization signals (NLSs), in the N terminus (nNLS) and C terminus (cNLS), that could mediate its nuclear import. Together the data suggest that HPV16 L2 interacts via its NLSs with a network of karyopherins and can enter the nucleus via several import pathways mediated by Kapalpha(2)beta(1) heterodimers, Kapbeta(2), and Kapbeta(3).     

Crystallographic analysis of the specific yet versatile recognition of distinct nuclear localization signals by karyopherin alpha

BACKGROUND: Karyopherin alpha (importin alpha) is an adaptor molecule that recognizes proteins containing nuclear localization signals (NLSs). The prototypical NLS that is able to bind to karyopherin alpha is that of the SV40 T antigen, and consists of a short positively charged sequence motif. Distinct classes of NLSs (monopartite and bipartite) have been identified that are only partly conserved with respect to one another but are nevertheless recognized by the same receptor. RESULTS: We report the crystal structures of two peptide complexes of yeast karyopherin alpha (Kapalpha): one with a human c-myc NLS peptide, determined at 2.1 A resolution, and one with a Xenopus nucleoplasmin NLS peptide, determined at 2.4 A resolution. Analysis of these structures reveals the determinants of specificity for the binding of a relatively hydrophobic monopartite NLS and of a bipartite NLS peptide. The peptides bind Kapalpha in its extended surface groove, which presents a modular array of tandem binding pockets for amino acid residues. CONCLUSIONS: Monopartite and bipartite NLSs bind to a different number of amino acid binding pockets and make different interactions within them. The relatively hydrophobic monopartite c-myc NLS binds extensively at a few binding pockets in a similar manner to that of the SV40 T antigen NLS. In contrast, the bipartite nucleoplasmin NLS engages the whole array of pockets with individually more limited but overall more abundant interactions, which include the NLS two basic clusters and the backbone of its non-conserved linker region. Versatility in the specific recognition of NLSs relies on the modular.     

Nuclear localization signal and protein context both mediate importin alpha specificity of nuclear import substrates

The "classical" nuclear protein import pathway depends on importin alpha and importin beta. Importin alpha binds nuclear localization signal (NLS)-bearing proteins and functions as an adapter to access the importin beta-dependent import pathway. In humans, only one importin beta is known to interact with importin alpha, while six alpha importins have been described. Various experimental approaches provided evidence that several substrates are transported specifically by particular alpha importins. Whether the NLS is sufficient to mediate importin alpha specificity is unclear. To address this question, we exchanged the NLSs of two well-characterized import substrates, the seven-bladed propeller protein RCC1, preferentially transported into the nucleus by importin alpha3, and the less specifically imported substrate nucleoplasmin. In vitro binding studies and nuclear import assays revealed that both NLS and protein context contribute to the specificity of importin alpha binding and transport.     

Identification of different roles for RanGDP and RanGTP in nuclear protein import.

The importin-alpha/beta heterodimer and the GTPase Ran play key roles in nuclear protein import. Importin binds the nuclear localization signal (NLS). Translocation of the resulting import ligand complex through the nuclear pore complex (NPC) requires Ran and is terminated at the nucleoplasmic side by its disassembly. The principal GTP exchange factor for Ran is the nuclear protein RCC1, whereas the major RanGAP is cytoplasmic, predicting that nuclear Ran is mainly in the GTP form and cytoplasmic Ran is in the GDP-bound form. Here, we show that nuclear import depends on cytoplasmic RanGDP and free GTP, and that RanGDP binds to the NPC. Therefore, import might involve nucleotide exchange and GTP hydrolysis on NPC-bound Ran. RanGDP binding to the NPC is not mediated by the Ran binding sites of importin-beta, suggesting that translocation is not driven from these sites. Consistently, a mutant importin-beta deficient in Ran binding can deliver its cargo up to the nucleoplasmic side of the NPC. However, the mutant is unable to release the import substrate into the nucleoplasm. Thus, binding of nucleoplasmic RanGTP to importin-beta probably triggers termination, i.e. the dissociation of importin-alpha from importin-beta and the subsequent release of the import substrate into the nucleoplasm.     

Importin alpha/beta and Ran-GTP regulate XCTK2 microtubule binding through a bipartite nuclear localization signal

The small GTPase Ran is essential for spindle assembly. Ran is proposed to act through its nuclear import receptors importin alpha and/or importin beta to control the sequestration of proteins necessary for spindle assembly. To date, the molecular mechanisms by which the Ran pathway functions remain unclear. Using purified proteins, we have reconstituted Ran-regulated microtubule binding of the C-terminal kinesin XCTK2, a kinesin important for spindle assembly. We show that the tail of XCTK2 binds to microtubules and that this binding is inhibited in the presence of importin alpha and beta (alpha/beta) and restored by addition of Ran-GTP. The bipartite nuclear localization signal (NLS) in the tail of XCTK2 is essential to this process, because mutation of the NLS abolishes importin alpha/beta-mediated regulation of XCTK2 microtubule binding. Our data show that importin alpha/beta directly regulates the activity of XCTK2 and that one of the molecular mechanisms of Ran-regulated spindle assembly is identical to that used in classical NLS-driven nuclear transport.     

The positively charged termini of L2 minor capsid protein required for bovine papillomavirus infection function separately in nuclear import and DNA binding

During the papillomavirus (PV) life cycle, the L2 minor capsid protein enters the nucleus twice: in the initial phase after entry of virions into cells and in the productive phase to mediate encapsidation of the newly replicated viral genome. Therefore, we investigated the interactions of the L2 protein of bovine PV type 1 (BPV1) with the nuclear import machinery and the viral DNA. We found that BPV1 L2 bound to the karyopherin alpha2 (Kap alpha2) adapter and formed a complex with Kap alpha2beta1 heterodimers. Previous data have shown that the positively charged termini of BPV1 L2 are required for BPV1 infection after the binding of the virions to the cell surface. We determined that these BPV1 L2 termini function as nuclear localization signals (NLSs). Both the N-terminal NLS (nNLS) and the C-terminal NLS (cNLS) interacted with Kap alpha2, formed a complex with Kap alpha2beta1 heterodimers, and mediated nuclear import via a Kap alpha2beta1 pathway. Interestingly, the cNLS was also the major DNA binding site of BPV1 L2. Consistent with the promiscuous DNA encapsidation by BPV1 pseudovirions, this DNA binding occurred without nucleotide sequence specificity. Moreover, an L2 mutant encoding a scrambled version of the cNLS, which supports production of virions, rescued the DNA binding but not the Kap alpha2 interaction. These data support a model in which BPV1 L2 functions as an adapter between the viral DNA via the cNLS and the Kaps via the nNLS and facilitates nuclear import of the DNA during infection.     

Nucleocytoplasmic protein transport and recycling of Ran

The active transport of proteins into and out of the nucleus is mediated by specific signals, the nuclear localization signal (NLS) and nuclear export signal (NES), respectively. The best characterized NLS is that of the SV40 large T antigen, which contains a cluster of basic amino acids. The NESs were first identified in the protein kinase inhibitor (PKI) and HIV Rev protein, which are rich in leucine residues. The SV40 T-NLS containing transport substrates are carried into the nucleus by an importin alpha/beta heterodimer. Importin alpha recognizes the NLS and acts as an adapter between the NLS and importin beta, whereas importin beta interacts with importin alpha bound to the NLS, and acts as a carrier of the NLS/importin alpha/beta trimer. It is generally thought that importin alpha and beta are part of a large protein family. The leucine rich NES-containing proteins are exported from the nucleus by one of the importin beta family molecules, CRM1/exportin 1. A Ras-like small GTPase Ran plays a crucial role in both import/export pathways and determines the directionality of nuclear transport. It has recently been demonstrated in living cells that Ran actually shuttles between the nucleus and the cytoplasm and that the recycling of Ran is essential for the nuclear transport. Furthermore, it has been shown that nuclear transport factor 2 (NTF2) mediates the nuclear import of RanGDP. This review largely focuses on the issue concerning the functional divergence of importin alpha family molecules and the role of Ran in nucleocytoplasmic protein transport.     

Ran-unassisted Nuclear Migration of a 97-kD Component of Nuclear Pore–targeting Complex

A 97-kD component of nuclear pore-targeting complex (the β-subunit of nuclear pore–targeting complex [PTAC]/importin/karyopherin) mediates the import of nuclear localization signal (NLS)-containing proteins by anchoring the NLS receptor protein (the α-subunit of PTAC/importin/karyopherin) to the nuclear pore complex (NPC). The import requires a small GTPase Ran, which interacts directly with the β-subunit. The present study describes an examination of the behavior of the β-subunit in living cells and in digitonin-permeabilized cells. In living cells, cytoplasmically injected β-subunit rapidly migrates into the nucleus. The use of deletion mutants reveals that nuclear migration of the β-subunit requires neither Ran- nor α-subunit–binding but only the NPC-binding domain of this molecule, which is also involved in NLS-mediated import. Furthermore, unlike NLS-mediated import, a dominant-negative Ran, defective in GTP-hydrolysis, did not inhibit nuclear migration of the β-subunit. In the digitonin-permeabilized cell-free import assay, the β-subunit transits rapidly through the NPC into the nucleus in a saturating manner in the absence of exogenous addition of soluble factors. These results show that the β-subunit undergoes translocation at the NPC in a Ran-unassisted manner when it does not carry α-subunit/NLS substrate. Therefore, a requirement for Ran arises only when the β-subunit undergoes a translocation reaction together with the α-subunit/NLS substrate. The results provide an insight to the yet unsolved question regarding the mechanism by which proteins are directionally transported through the NPC, and the role of Ran in this process.     

Nuclear import of Epstein-Barr virus nuclear antigen 1 mediated by NPI-1 (Importin alpha5) is up- and down-regulated by phosphorylation of the nuclear localization signal for which Lys379 and Arg380 are essential

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1) is essential for replication of episomal EBV DNAs and maintenance of latency. Multifunctional EBNA-1 is phosphorylated, but the significance of EBNA-1 phosphorylation is not known. Here, we examined the effects on nuclear translocation of Ser phosphorylation of the EBNA-1 nuclear localization signal (NLS) sequence, 379Lys-Arg-Pro-Arg-Ser-Pro-Ser-Ser386. We found that Lys379Ala and Arg380Ala substitutions greatly reduced nuclear transport and steady-state levels of green fluorescent protein (GFP)-EBNA1, whereas Pro381Ala, Arg382Ala, Pro384Ala, and Glu378Ala substitutions did not. Microinjection of modified EBNA-1 NLS peptide-inserted proteins and NLS peptides cross-linked to bovine serum albumin (BSA) showed that Ala substitution for three NLS Ser residues reduced the efficiency of nuclear import. Similar microinjection analyses demonstrated that phosphorylation of Ser385 accelerated the rate of nuclear import, but phosphorylation of Ser383 and Ser386 reduced it. However, transfection analyses of GFP-EBNA1 mutants with the Ser-to-Ala substitution causing reduced nuclear import efficiency did not result in a decrease in the nuclear accumulation level of EBNA-1. The results suggest dynamic nuclear transport control of phosphorylated EBNA-1 proteins, although the nuclear localization level of EBNA-1 that binds to cellular chromosomes and chromatin seems unchanged. The karyopherin alpha NPI-1 (importin alpha5), a nuclear import adaptor, bound more strongly to Ser385-phosphorylated NLS than to any other phosphorylated or nonphosphorylated forms. Rch1 (importin alpha1) bound only weakly and Qip1 (importin alpha3) did not bind to the Ser385-phosphorylated NLS. These findings suggest that the amino-terminal 379Lys-Arg380 is essential for the EBNA-1 NLS and that Ser385 phosphorylation up-regulates nuclear transport efficiency of EBNA-1 by increasing its binding affinity to NPI-1, while phosphorylation of Ser386 and Ser383 down-regulates it.     

Importin beta recognizes parathyroid hormone-related protein with high affinity and mediates its nuclear import in the absence of importin alpha

Parathyroid hormone-related protein (PTHrP), expressed in a range of tumors, has endocrine, autocrine/paracrine, and intracrine actions, some of which relate to its ability to localize in the nucleus. Here we show for the first time that extracellularly added human PTHrP (amino acids 1-108) can be taken up specifically by receptor-expressing UMR106.01 osteogenic sarcoma cells and accumulate to quite high levels in the nucleus and nucleolus within 40 min. Quantitation of recognition by the nuclear localization sequence (NLS)-binding importin subunits indicated that in contrast to proteins containing conventional NLSs, PTHrP is recognized exclusively by importin beta and not by importin alpha. The sequence of PTHrP responsible for binding was mapped to amino acids 66-94, which includes an SV40 large tumor-antigen NLS-like sequence, although sequence determinants amino-terminal to this region were also necessary for high affinity binding (apparent dissociation constant of approximately 2 nM for importin beta). Nuclear import of PTHrP was assessed in vitro using purified components, demonstrating that importin beta, together with the GTP-binding protein Ran, was able to mediate efficient nuclear accumulation in the absence of importin alpha, whereas the addition of nuclear transport factor NTF2 reduced transport. The polypeptide ligand PTHrP thus appears to be accumulated in the nucleus/nucleolus through a novel, NLS-dependent nuclear import pathway independent of importin alpha and perhaps also of NTF2.     

An unconventional NLS is critical for the nuclear import of the influenza A virus nucleoprotein and ribonucleoprotein

Replication of the RNAs of influenza virus occurs in the nucleus of infected cells. The nucleoprotein (NP) has been shown to be important for the import of the viral RNA into the nucleus and has been proposed to contain at least three different nuclear localization signals (NLSs). Here, an import assay in digitonin-permeabilized cells was used to further define the contribution of these NLSs. Mutation of the unconventional NLS impaired the nuclear import of the NP. A peptide bearing the unconventional NLS could inhibit the nuclear import of the NP in this import assay and prevent the NP-karyopherin alpha interaction in a binding assay confirming the crucial role of this signal. Interestingly, a peptide containing the SV40 T antigen NLS was unable to inhibit the nuclear import of NP or the NP-karyopherin alpha interaction, suggesting that the NP and the SV40 T antigen do not share a common binding site on karyopherin alpha. We also investigated the question of which NLS(s) is/are necessary for the viral ribonucleoprotein complex to enter the nucleus. We found that the peptide containing the unconventional NLS efficiently inhibited the nuclear import of the ribonucleoprotein complexes. This finding suggests that the unconventional NLS is the major signal necessary not only for the nuclear transport of free NP but also for the import of the ribonucleoprotein complexes. Finally, viral replication could be specifically inhibited by a membrane-permeable peptide containing the unconventional NLS, confirming the crucial role of this signal during the replicative cycle of the virus.     

A nonconventional nuclear localization signal within the UL84 protein of human cytomegalovirus mediates nuclear import via the importin alpha/beta pathway

The open reading frame UL84 of human cytomegalovirus encodes a multifunctional regulatory protein which is required for viral DNA replication and binds with high affinity to the immediate-early transactivator IE2-p86. Although the exact role of pUL84 in DNA replication is unknown, the nuclear localization of this protein is a prerequisite for this function. To investigate whether the activities of pUL84 are modulated by cellular proteins we used the Saccharomyces cerevisiae two-hybrid system to screen a cDNA-library for interacting proteins. Strong interactions were found between pUL84 and four members of the importin alpha protein family. These interactions could be confirmed in vitro by pull down experiments and in vivo by coimmunoprecipitation analysis from transfected cells. Using in vitro transport assays we showed that the pUL84 nuclear import required importin alpha, importin beta, and Ran, thus following the classical importin-mediated import pathway. Deletion mutagenesis of pUL84 revealed a domain of 282 amino acids which is required for binding to the importin alpha proteins. Its function as a nuclear localization signal (NLS) was confirmed by fusion to heterologous proteins. Although containing a cluster of basic amino acids similar to classical NLSs, this cluster did not contain the NLS activity. Thus, a complex structure appears to be essential for importin alpha binding and import activity.     

The auto-inhibitory function of importin alpha is essential in vivo

Proteins that contain a classical nuclear localization signal (NLS) are recognized in the cytoplasm by a heterodimeric import receptor composed of importin/karyopherin alpha and beta. The importin alpha subunit recognizes classical NLS sequences, and the importin beta subunit directs the complex to the nuclear pore. Recent work shows that the N-terminal importin beta binding (IBB) domain of importin alpha regulates NLS-cargo binding in the absence of importin beta in vitro. To analyze the in vivo functions of the IBB domain, we created a series of mutants in the Saccharomyces cerevisiae importin alpha protein. These mutants dissect the two functions of the N-terminal IBB domain, importin beta binding and auto-inhibition. One of these importin alpha mutations, A3, decreases auto-inhibitory function without impacting binding to importin beta or the importin alpha export receptor, Cse1p. We used this mutant to show that the auto-inhibitory function is essential in vivo and to provide evidence that this auto-inhibitory-defective importin alpha remains bound to NLS-cargo within the nucleus. We propose a model where the auto-inhibitory activity of importin alpha is required for NLS-cargo release and the subsequent Cse1p-dependent recycling of importin alpha to the cytoplasm.     

Importin beta mediates nuclear translocation of Smad 3

Smad proteins are intracellular mediators of transforming growth factor-beta (TGF-beta) and related cytokines. Although ligand-induced nuclear translocation of Smad proteins is clearly established, the pathway mediating this import is yet to be determined. We previously identified a nuclear localization signal (NLS) in the N-terminal region of Smad 3, the major Smad protein involved in TGF-beta signal transduction. This basic motif (Lys(40-)Lys-Leu-Lys-Lys(44)), conserved among all the pathway-specific Smad proteins, is required for Smad 3 nuclear import in response to ligand. Here we studied the nuclear import pathway of Smad 3 mediated by this NLS. We demonstrate that the isolated Smad 3 MH1 domain displays significant specific binding to importin beta, which is diminished or eliminated by mutations in the NLS. Full-size Smad 3 exhibits weak but specific binding to importin beta, which is enhanced after phosphorylation by the type I TGF-beta receptor. In contrast, no interaction was observed between importin alpha and Smad 3 or its MH1 domain, indicating that nuclear translocation of Smad proteins may occur through direct binding to importin beta. We propose that activation of all of the pathway-specific Smad proteins (Smads 1, 2, 3, 5, 8, and 9) exposes the conserved NLS motif, which then binds directly to importin beta and triggers nuclear translocation.