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.     

Structural basis for RanGTP independent entry of spliceosomal U snRNPs into the nucleus

The nuclear import of assembled spliceosomal subunits, the uridine-rich small nuclear ribonucleoprotein particles (U snRNPs), is mediated by a nuclear import receptor adaptor couple of importinβ (Impβ) and snurportin1 (SPN1). In contrast to any other characterized active nuclear import, the Impβ/SPN1/U snRNP complex does not require RanGTP for the terminal release from the nuclear basket of the nuclear pore complex (NPC). The crystal structure of Impβ (127-876) in complex with the Impβ-binding (IBB) domain of SPN1 (1-65) at 2.8-Å resolution reveals that Impβ adopts an open conformation, which is unique for a functional Impβ/cargo complex, and rather surprisingly, it resembles the conformation of the Impβ/RanGTP complex. As binding of RanGTP to Impβ usually triggers the release of import complexes from the NPC, we propose that by already mimicking a conformation similar to Impβ/RanGTP the independent dissociation of Impβ/SPN1 from the nuclear basket is energetically aided.     

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.     

Gradient of increasing affinity of importin beta for nucleoporins along the pathway of nuclear import

Nuclear import and export signals on macromolecules mediate directional, receptor-driven transport through the nuclear pore complex (NPC) by a process that is suggested to involve the sequential binding of transport complexes to different nucleoporins. The directionality of transport appears to be partly determined by the nucleocytoplasmic compartmentalization of components of the Ran GTPase system. We have analyzed whether the asymmetric localization of discrete nucleoporins can also contribute to transport directionality. To this end, we have used quantitative solid phase binding analysis to determine the affinity of an importin beta cargo complex for Nup358, the Nup62 complex, and Nup153, which are in the cytoplasmic, central, and nucleoplasmic regions of the NPC, respectively. These nucleoporins are proposed to provide progressively more distal binding sites for importin beta during import. Our results indicate that the importin beta transport complex binds to nucleoporins with progressively increasing affinity as the complex moves from Nup358 to the Nup62 complex and to Nup153. Antibody inhibition studies support the possibility that importin beta moves from Nup358 to Nup153 via the Nup62 complex during import. These results indicate that nucleoporins themselves, as well as the nucleocytoplasmic compartmentalization of the Ran system, are likely to play an important role in conferring directionality to nuclear protein import.     

Structural basis of importin-α-mediated nuclear transport for Ku70 and Ku80

Ku70 and Ku80 form a heterodimeric complex involved in multiple nuclear processes. This complex plays a key role in DNA repair due to its ability to bind DNA double-strand breaks and facilitate repair by the nonhomologous end-joining pathway. Ku70 and Ku80 have been proposed to contain bipartite and monopartite nuclear localization sequences (NLSs), respectively, that allow them to be translocated to the nucleus independently of each other via the classical importin-α (Impα)/importin-β-mediated nuclear import pathway. To determine the structural basis of the recognition of Ku70 and Ku80 proteins by Impα, we solved the crystal structures of the complexes of Impα with the peptides corresponding to the Ku70 and Ku80 NLSs. Our structural studies confirm the binding of the Ku80 NLS as a classical monopartite NLS but reveal an unexpected binding mode for Ku70 NLS with only one basic cluster bound to the receptor. Both Ku70 and Ku80 therefore contain monopartite NLSs, and sequences outside the basic cluster make favorable interactions with Impα, suggesting that this may be a general feature in monopartite NLSs. We show that the Ku70 NLS has a higher affinity for Impα than the Ku80 NLS, consistent with more extensive interactions in its N-terminal region. The prospect of nuclear import of Ku70 and Ku80 independently of each other provides a powerful regulatory mechanism for the function of the Ku70/Ku80 heterodimer and independent functions of the two proteins.     

Expanding the Definition of the Classical Bipartite Nuclear Localization Signal

Nuclear localization signals (NLSs) are amino acid sequences that target cargo proteins into the nucleus. Rigorous characterization of NLS motifs is essential to understanding and predicting pathways for nuclear import. The best‐characterized NLS is the classical NLS (cNLS), which is recognized by the cNLS receptor, importin‐α. cNLSs are conventionally defined as having one (monopartite) or two clusters of basic amino acids separated by a 9‐12 aa linker (bipartite). Motivated by the finding that Ty1 integrase, which contains an unconventional putative bipartite cNLS with a 29 aa linker, exploits the classical nuclear import machinery, we assessed the functional boundaries for linker length within a bipartite cNLS. We confirmed that the integrase cNLS is a bona fide bipartite cNLS, then carried out a systematic analysis of linker length in an obligate bipartite cNLS cargo, which revealed that some linkers longer than conventionally defined can function in nuclear import. Linker function is dependent on the sequence and likely the inherent flexibility of the linker. Subsequently, we interrogated the Saccharomyces cerevisiae proteome to identify cellular proteins containing putative long bipartite cNLSs. We experimentally confirmed that Rrp4 contains a bipartite cNLS with a 25 aa linker. Our studies show that the traditional definition of bipartite cNLSs is too restrictive and linker length can vary depending on amino acid composition.     

Probing formation of cargo/importin‐α transport complexes in plant cells using a pathogen effector

Importin‐αs are essential adapter proteins that recruit cytoplasmic proteins destined for active nuclear import to the nuclear transport machinery. Cargo proteins interact with the importin‐α armadillo repeat domain via nuclear localization sequences (NLSs), short amino acids motifs enriched in Lys and Arg residues. Plant genomes typically encode several importin‐α paralogs that can have both specific and partially redundant functions. Although some cargos are preferentially imported by a distinct importin‐α it remains unknown how this specificity is generated and to what extent cargos compete for binding to nuclear transport receptors. Here we report that the effector protein HaRxL106 from the oomycete pathogen Hyaloperonospora arabidopsidis co‐opts the host cell's nuclear import machinery. We use HaRxL106 as a probe to determine redundant and specific functions of importin‐α paralogs from Arabidopsis thaliana. A crystal structure of the importin‐α3/MOS6 armadillo repeat domain suggests that five of the six Arabidopsis importin‐αs expressed in rosette leaves have an almost identical NLS‐binding site. Comparison of the importin‐α binding affinities of HaRxL106 and other cargos in vitro and in plant cells suggests that relatively small affinity differences in vitro affect the rate of transport complex formation in vivo. Our results suggest that cargo affinity for importin‐α, sequence variation at the importin‐α NLS‐binding sites and tissue‐specific expression levels of importin‐αs determine formation of cargo/importin‐α transport complexes in plant cells.     

Quantitative analysis of Tat peptide binding to import carriers reveals unconventional nuclear transport properties

A detailed study of nuclear import mediated by the HIV-1 Tat peptide (47YGRKKRRQRRR57, TatRRR) is reported. Fluorescence-based measurements, calibration of protein concentrations, and binding assays are exploited to address the physicochemical mechanisms of Tat peptide recognition by the classical importin α (Impα) and importin β (Impβ) receptors both in vitro and in intact cells. We show that TatRRR is an unconventional nuclear localization sequence that binds directly to both Impα and Impβ carriers in the absence of competitors (in vitro), whereas this property is silenced in the actual cellular environment. In the latter case, Impα/β-dependent nuclear import can be successfully restored by replacing the "RRR" stretch with "GGG". We apply a recently developed method to determine quantitatively TatGGG affinity for each receptor. Based on these results, we can rationalize previous controversial reports on the Tat peptide and provide coherent guidelines for the design of novel intracellular targeting sequences.     

Cellular stresses induce the nuclear accumulation of importin alpha and cause a conventional nuclear import block

We report here that importin alpha accumulates reversibly in the nucleus in response to cellular stresses including UV irradiation, oxidative stress, and heat shock. The nuclear accumulation of importin alpha appears to be triggered by a collapse in the Ran gradient, resulting in the suppression of the nuclear export of importin alpha. In addition, nuclear retention and the importin beta/Ran-independent import of importin alpha also facilitate its rapid nuclear accumulation. The findings herein show that the classical nuclear import pathway is down-regulated via the removal of importin alpha from the cytoplasm in response to stress. Moreover, whereas the nuclear accumulation of heat shock cognate 70 is more sensitive to heat shock than the other stresses, importin alpha is able to accumulate in the nucleus at all the stress conditions tested. These findings suggest that the stress-induced nuclear accumulation of importin alpha can be involved in a common physiological response to various stress conditions.     

Kap95p binding induces the switch loops of RanGDP to adopt the GTP-bound conformation: implications for nuclear import complex assembly dynamics

The asymmetric distribution of the nucleotide-bound state of Ran across the nuclear envelope is crucial for determining the directionality of nuclear transport. In the nucleus, Ran is primarily in the guanosine 5′-triphosphate (GTP)-bound state, whereas in the cytoplasm, Ran is primarily guanosine 5′-diphosphate (GDP)-bound. Conformational changes within the Ran switch I and switch II loops are thought to modulate its affinity for importin-β. Here, we show that RanGDP and importin-β form a stable complex with a micromolar dissociation constant. This complex can be dissociated by importin-β binding partners such as importin-α. Surprisingly, the crystal structure of the Kap95p-RanGDP complex shows that Kap95p induces the switch I and II regions of RanGDP to adopt a conformation that resembles that of the GTP-bound form. The structure of the complex provides insights into the structural basis for the gradation of affinities regulating nuclear protein transport.     

Identification of Critical Motifs within HIV-1 Integrase Required for Importin α3 Interaction and Viral cDNA Nuclear Import

The viral cDNA nuclear import is an important requirement for human immunodeficiency virus type 1 (HIV-1) replication in dividing and nondividing cells. Our recent study identified a specific interaction of importin α3 (Impα3) with HIV-1 integrase (IN) and its involvement in viral cDNA nuclear import. In this study, we have performed a more detailed investigation on the molecular mechanism of how HIV-1 IN interacts with Impα3. Our results revealed a reduced interaction between the two IN mutants INKK215,9AA (IN215,9) and INRK263,4AA (IN263,4) with Impα3, while an IN double mutant, IN215,9/263,4, was severely impaired for its Impα3-binding ability, even though it was still found interacting with other cofactors, IN interactor I and Transportin3. Immunostaining and fractionation analysis have shown that YFP-IN215,9/263,4 failed to localize in the nucleus of transfected cells. Also, we found that both major and minor nuclear localization signal binding grooves of Impα3 are involved in interaction with IN. All of these results suggest a cargo protein-import receptor type of interaction. Finally, the effect of IN215,9/263,4 mutations on HIV-1 replication was evaluated, and real-time quantitative PCR analysis showed that, while mutant virus (v215,9/263,4) had a slightly lowered total viral DNA, the 2-long-terminal-repeat DNA, a marker for nuclear import, was greatly reduced during v215,9/263,4 infection in both dividing and nondividing cells. Also, by cell fractionation assay, we found that a significant proportion of viral cDNA was still retained in cytoplasmic fraction of v215,9/263,4-infected cells. Overall, our study provides strong evidence that ²¹¹KELQKQITK and ²⁶²RRKAK regions of IN C-terminal domain are required for Impα3 interaction and HIV-1 cDNA nuclear import.     

Nuclear import by karyopherin-βs: Recognition and inhibition

Proteins in the karyopherin-β family mediate the majority of macromolecular transport between the nucleus and the cytoplasm. Eleven of the 19 known human karyopherin-βs and 10 of the 14 S. cerevisiae karyopherin-βs mediate nuclear import through recognition of nuclear localization signals or NLSs in their cargos. This receptor-mediated process is essential to cellular viability as proteins are translated in the cytoplasm but many have functional roles in the nucleus. Many known karyopherin-β-cargo interactions were discovered through studies of the individual cargos rather than the karyopherins, and this information is thus widely scattered in the literature. We consolidate information about cargos that are directly recognized by import-karyopherin-βs and review common characteristics or lack thereof among cargos of different import pathways. Knowledge of karyopherin-β-cargo interactions is also critical for the development of nuclear import inhibitors and the understanding of their mechanisms of inhibition. This article is part of a Special Issue entitled: Regulation of Signaling and Cellular Fate through Modulation of Nuclear Protein Import.     

Structural Basis of High‐Affinity Nuclear Localization Signal Interactions with Importin‐α

Classical nuclear localization signals (cNLSs), comprising one (monopartite cNLSs) or two clusters of basic residues connected by a 10–12 residue linker (bipartite cNLSs), are recognized by the nuclear import factor importin‐α. The cNLSs bind along a concave groove on importin‐α; however, specificity determinants of cNLSs remain poorly understood. We present a structural and interaction analysis study of importin‐α binding to both designed and naturally occurring high‐affinity cNLS‐like sequences; the peptide inhibitors Bimax1 and Bimax2, and cNLS peptides of cap‐binding protein 80. Our data suggest that cNLSs and cNLS‐like sequences can achieve high affinity through maximizing interactions at the importin‐α minor site, and by taking advantage of multiple linker region interactions. Our study defines an extended set of binding cavities on the importin‐α surface, and also expands on recent observations that longer linker sequences are allowed, and that long‐range electrostatic complementarity can contribute to cNLS‐binding affinity. Altogether, our study explains the molecular and structural basis of the results of a number of recent studies, including systematic mutagenesis and peptide library approaches, and provides an improved level of understanding on the specificity determinants of a cNLS. Our results have implications for identifying cNLSs in novel proteins.     

Contribution of the residue at position 4 within classical nuclear localization signals to modulating interaction with importins and nuclear targeting

Nuclear import involves the recognition by importin (IMP) superfamily members of nuclear localization signals (NLSs) within protein cargoes destined for the nucleus, the best understood being recognition of classical NLSs (cNLSs) by the IMPα/β1 heterodimer. Although the cNLS consensus [K-(K/R)-X-(K/R) for positions P2–P5] is generally accepted, recent studies indicated that the contribution made by different residues at the P4 position can vary. Here, we apply a combination of microscopy, molecular dynamics, crystallography, in vitro binding, and bioinformatics approaches to show that the nature of residues at P4 indeed modulates cNLS function in the context of a prototypical Simian Virus 40 large tumor antigen-derived cNLS (KKRK, P2–5). Indeed, all hydrophobic substitutions in place of R impaired binding to IMPα and nuclear targeting, with the largest effect exerted by a G residue at P4. Substitution of R with neutral hydrophobic residues caused the loss of electrostatic and van der Waals interactions between the P4 residue side chains and IMPα. Detailed bioinformatics analysis confirmed the importance of the P4 residue for cNLS function across the human proteome, with specific residues such as G being associated with low activity. Furthermore, we validate our findings for two additional cNLSs from human cytomegalovirus (HCMV) DNA polymerase catalytic subunit UL54 and processivity factor UL44, where a G residue at P4 results in a 2–3-fold decrease in NLS activity. Our results thus showed that the P4 residue makes a hitherto poorly appreciated contribution to nuclear import efficiency, which is essential to determining the precise nuclear levels of cargoes.     

Importins and Beyond: Non-Conventional Nuclear Transport Mechanisms

The movement of proteins between the cytoplasm and the nucleus conventionally involves the recognition of nuclear targeting signals by members of the importin (Imp) superfamily of nuclear transporters, followed by translocation through the nuclear envelope-embedded nuclear pore complexes (NPCs). It is becoming increasingly apparent, however, that distinct alternative pathways for nuclear transport exist and are relatively abundant. This review examines several of these novel pathways, including facilitation of Imp-dependent transport by microtubule motors, and Imp-independent pathways involving either other transport molecules such as the calcium-binding protein calmodulin or through direct binding to the components of the NPC. The existence of these pathways and the fact that many proteins appear to possess separate Imp-dependent and -independent nuclear import mechanisms ensure that the cell can function under conditions in which Imp-dependent transport is inhibited and/or modulate the efficiency of Imp-dependent transport itself, according to the need.     

The nucleoporin Nup358/RanBP2 promotes nuclear import in a cargo- and transport receptor-specific manner

In vertebrates, the nuclear pore complex (NPC), the gate for transport of macromolecules between the nucleus and the cytoplasm, consists of approximately 30 different nucleoporins (Nups). The Nup and SUMO E3-ligase Nup358/RanBP2 are the major components of the cytoplasmic filaments of the NPC. In this study, we perform a structure-function analysis of Nup358 and describe its role in nuclear import of specific proteins. In a screen for nuclear proteins that accumulate in the cytoplasm upon Nup358 depletion, we identified proteins that were able to interact with Nup358 in a receptor-independent manner. These included the importin α/β-cargo DBC-1 (deleted in breast cancer 1) and DMAP-1 (DNA methyltransferase 1 associated protein 1). Strikingly, a short N-terminal fragment of Nup358 was sufficient to promote import of DBC-1, whereas DMAP-1 required a larger portion of Nup358 for stimulated import. Neither the interaction of RanGAP with Nup358 nor its SUMO-E3 ligase activity was required for nuclear import of all tested cargos. Together, Nup358 functions as a cargo- and receptor-specific assembly platform, increasing the efficiency of nuclear import of proteins through various mechanisms.     

A monoclonal antibody to the COOH-terminal acidic portion of Ran inhibits both the recycling of Ran and nuclear protein import in living cells

A small GTPase Ran is a key regulator for active nuclear transport. In immunoblotting analysis, a monoclonal antibody against recombinant human Ran, designated ARAN1, was found to recognize an epitope in the COOH-terminal domain of Ran. In a solution binding assay, ARAN1 recognized Ran when complexed with importin beta, transportin, and CAS, but not the Ran-GTP or the Ran-GDP alone, indicating that the COOH-terminal domain of Ran is exposed via its interaction with importin beta-related proteins. In addition, ARAN1 suppressed the binding of RanBP1 to the Ran-importin beta complex. When injected into the nucleus of BHK cells, ARAN1 was rapidly exported to the cytoplasm, indicating that the Ran-importin beta-related protein complex is exported as a complex from the nucleus to the cytoplasm in living cells. Moreover, ARAN1, when injected into the cultured cells induces the accumulation of endogenous Ran in the cytoplasm and prevents the nuclear import of SV-40 T-antigen nuclear localization signal substrates. From these findings, we propose that the binding of RanBP1 to the Ran-importin beta complex is required for the dissociation of the complex in the cytoplasm and that the released Ran is recycled to the nucleus, which is essential for the nuclear protein transport.     

Importin β-type nuclear transport receptors have distinct binding affinities for Ran-GTP

Cargos destined to enter or leave the cell nucleus are typically transported by receptors of the importin β family to pass the nuclear pore complex. The yeast Saccharomyces cerevisiae comprises 14 members of this protein family, which can be divided in importins and exportins. The Ran GTPase regulates the association and dissociation of receptors and cargos as well as the transport direction through the nuclear pore. All receptors bind to Ran exclusively in its GTP-bound state and this event is restricted to the nuclear compartment. We determined the Ran–GTP binding properties of all yeast transport receptors by biosensor measurements and observed that the affinity of importins for Ran–GTP differs significantly. The dissociation constants range from 230 pM to 270 nM, which is mostly based on a variability of the off-rate constants. The divergent affinity of importins for Ran–GTP suggests the existence of a novel mode of nucleocytoplasmic transport regulation. Furthermore, the cellular concentration of β-receptors and of other Ran-binding proteins was determined. We found that the number of β-receptors altogether about equals the amounts of yeast Ran, but Ran–GTP is not limiting in the nucleus. The implications of our results for nucleocytoplasmic transport mechanisms are discussed.