The yeast nucleoporin Nup2p is involved in nuclear export of importin alpha/Srp1p.

The importin alpha.beta heterodimer mediates nuclear import of proteins containing classical nuclear localization signals. After carrying its cargo into the nucleus, the importin dimer dissociates, and Srp1p (the yeast importin alpha subunit) is recycled to the cytoplasm in a complex with Cse1p and RanGTP. Nup2p is a yeast FXFG nucleoporin that contains a Ran-binding domain. We find that export of Srp1p from the nucleus is impaired in Deltanup2 mutants. Also, Srp1p fusion proteins accumulate at the nuclear rim in wild-type cells but accumulate in the nuclear interior in Deltanup2 cells. A deletion of NUP2 shows genetic interactions with mutants in SRP1 and PRP20, which encodes the Ran nucleotide exchange factor. Srp1p binds directly to an N-terminal domain of Nup2p. This region of Nup2p is sufficient to allow accumulation of an Srp1p fusion protein at the nuclear rim, but the C-terminal Ran-binding domain of Nup2p is required for efficient Srp1p export. Formation of the Srp1p.Cse1p. RanGTP export complex releases Srp1p from its binding site in Nup2p. We propose that Nup2p may act as a scaffold that facilitates formation of the Srp1p export complex.     

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.     

Nup50/Npap60 function in nuclear protein import complex disassembly and importin recycling

Nuclear import of proteins containing classical nuclear localization signals (NLS) is mediated by the importin-alpha:beta complex that binds cargo in the cytoplasm and facilitates its passage through nuclear pores, after which nuclear RanGTP dissociates the import complex and the importins are recycled. In vertebrates, import is stimulated by nucleoporin Nup50, which has been proposed to accompany the import complex through nuclear pores. However, we show here that the Nup50 N-terminal domain actively displaces NLSs from importin-alpha, which would be more consistent with Nup50 functioning to coordinate import complex disassembly and importin recycling. The crystal structure of the importin-alpha:Nup50 complex shows that Nup50 binds at two sites on importin-alpha. One site overlaps the secondary NLS-binding site, whereas the second extends along the importin-alpha C-terminus. Mutagenesis indicates that interaction at both sites is required for Nup50 to displace NLSs. The Cse1p:Kap60p:RanGTP complex structure suggests how Nup50 is then displaced on formation of the importin-alpha export complex. These results provide a rationale for understanding the series of interactions that orchestrate the terminal steps of nuclear protein import.     

Classical NLS proteins from Saccharomyces cerevisiae

Proteins can enter the nucleus through various receptor-mediated import pathways. One class of import cargos carries a classical nuclear localization signal (cNLS) containing a short cluster of basic residues. This pathway involves importin α (Impα), which possesses the cNLS binding site, and importin β (Impβ), which translocates the import complex through the nuclear pore complex. The defining criteria for a cNLS protein from Saccharomyces cerevisiae are an in vivo import defect in Impα and Impβ mutants, direct binding to purified Impα, and stimulation of this binding by Impβ. We show for the first time that endogenous S. cerevisiae proteins Prp20, Cdc6, Swi5, Cdc45, and Clb2 fulfill all of these criteria identifying them as authentic yeast cNLS cargos. Furthermore, we found that the targeting signal of Prp20 is a bipartite cNLS and that of Cdc6 is a monopartite cNLS. Basic residues present within these motifs are of different significance for the interaction with Impα. We determined the binding constants for import complexes containing the five cNLS proteins by surface plasmon resonance spectrometry. The dissociation constants for cNLS/α/β complexes differ considerably, ranging from 1 nM for Cdc6 to 112 nM for Swi5, suggesting that the nuclear import kinetics is determined by the strength of cNLS/Impα binding. Impβ enhances the affinity of Impα for cNLSs approximately 100-fold. This stimulation of cNLS binding to Impα results from a faster association in the presence of Impβ, whereas the dissociation rate is unaffected by Impβ. This implies that, after entry into the nucleus, the release of Impβ by the Ran guanosine triphosphatase (Ran GTPase) from the import complex is not sufficient to dissociate the cNLS/Impα subcomplex. Our observation that the nucleoporin Nup2, which had been previously shown to release the cNLS from Impα in vitro, is required for efficient import of all the genuine cNLS cargos supports a general role of Nup2 in import termination.     

Nup2p, a yeast nucleoporin, functions in bidirectional transport of importin alpha

Import of proteins containing a classical nuclear localization signal (NLS) into the nucleus is mediated by importin alpha and importin beta. Srp1p, the Saccharomyces cerevisiae homologue of importin alpha, returns from the nucleus in a complex with its export factor Cse1p and with Gsp1p (yeast Ran) in its GTP-bound state. We studied the role of the nucleoporin Nup2p in the transport cycle of Srp1p. Cells lacking NUP2 show a specific defect in both NLS import and Srp1p export, indicating that Nup2p is required for efficient bidirectional transport of Srp1p across the nuclear pore complex (NPC). Nup2p is located at the nuclear side of the central gated channel of the NPC and provides a binding site for Srp1p via its amino-terminal domain. We show that Nup2p effectively releases the NLS protein from importin alpha-importin and beta and strongly binds to the importin heterodimer via Srp1p. Kap95p (importin beta) is released from this complex by a direct interaction with Gsp1p-GTP. These data suggest that besides Gsp1p, which disassembles the NLS-importin alpha-importin beta complex upon binding to Kap95p in the nucleus, Nup2p can also dissociate the import complex by binding to Srp1p. We also show data indicating that Nup1p, a relative of Nup2p, plays a similar role in termination of NLS import. Cse1p and Gsp1p-GTP release Srp1p from Nup2p, which suggests that the Srp1p export complex can be formed directly at the NPC. The changed distribution of Cse1p at the NPC in nup2 mutants also supports a role for Nup2p in Srp1p export from the nucleus.     

The structure of the nuclear export receptor Cse1 in its cytosolic state reveals a closed conformation incompatible with cargo binding

Cse1 mediates nuclear export of importin alpha, the nuclear localization signal (NLS) import adaptor. We report the 3.1 A resolution structure of cargo-free Cse1, representing this HEAT repeat protein in its cytosolic state. Cse1 is compact, consisting of N- and C-terminal arches that interact to form a ring. Comparison with the structure of cargo-bound Cse1 shows a major conformational change leading to opening of the structure upon cargo binding. The largest structural changes occur within a hinge region centered at HEAT repeat 8. This repeat contains a conserved insertion that connects the RanGTP and importin alpha contact sites and that is essential for binding. In the cargo-free state, the RanGTP binding sites are occluded and the importin alpha sites are distorted. Mutations that destabilize the N- to C-terminal interaction uncouple importin alpha and Ran binding, suggesting that the closed conformation prevents association with importin alpha.     

Structural basis for Nup2p function in cargo release and karyopherin recycling in nuclear import

The yeast nucleoporin Nup2p is associated primarily with the nuclear basket of nuclear pore complexes and is required for efficient importin-alpha:beta-mediated nuclear protein import as well as efficient nuclear export of Kap60p/importin-alpha. Residues 1-51 of Nup2p bind tightly to Kap60p and are required for Nup2p function in vivo. We have determined the 2.6 A resolution crystal structure of a complex between this region of Nup2p and the armadillo repeat domain of Kap60p. Nup2p binds along the inner concave groove of Kap60p, but its interaction interface is different from that employed for nuclear localization signal (NLS) recognition although there is some overlap between them. Nup2p binds Kap60p more strongly than NLSs and accelerates release of NLSs from Kap60p. Nup2p itself is released from Kap60p by Cse1p:RanGTP only in the presence of the importin-beta binding (IBB) domain of Kap60p. These data indicate that Nup2p increases the overall rate of nuclear trafficking by coordinating nuclear import termination and importin recycling as a concerted process.     

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.     

Cse1p Is Involved in Export of Yeast Importin α from the Nucleus

Proteins bearing a nuclear localization signal (NLS) are targeted to the nucleus by the heterodimeric transporter importin. Importin α binds to the NLS and to importin β, which carries it through the nuclear pore complex (NPC). Importin disassembles in the nucleus, evidently by binding of RanGTP to importin β. The importin subunits are exported separately. We investigated the role of Cse1p, the Saccharomyces cerevisiae homologue of human CAS, in nuclear export of Srp1p (yeast importin α). Cse1p is located predominantly in the nucleus but also is present in the cytoplasm and at the NPC. We analyzed the in vivo localization of the importin subunits fused to the green fluorescent protein in wild-type and cse1-1 mutant cells. Srp1p but not importin β accumulated in nuclei of cse1-1 mutants, which are defective in NLS import but not defective in NLS-independent import pathways. Purified Cse1p binds with high affinity to Srp1p only in the presence of RanGTP. The complex is dissociated by the cytoplasmic RanGTP-binding protein Yrb1p. Combined with the in vivo results, this suggests that a complex containing Srp1p, Cse1p, and RanGTP is exported from the nucleus and is subsequently disassembled in the cytoplasm by Yrb1p. The formation of the trimeric Srp1p-Cse1p-RanGTP complex is inhibited by NLS peptides, indicating that only NLS-free Srp1p will be exported to the cytoplasm.     

Influence of cargo size on Ran and energy requirements for nuclear protein import

Previous work has shown that the transport of some small protein cargoes through the nuclear pore complex (NPC) can occur in vitro in the absence of nucleoside triphosphate hydrolysis. We now demonstrate that in the importin alpha/beta and transportin import pathways, efficient in vitro transport of large proteins, in contrast to smaller proteins, requires hydrolyzable GTP and the small GTPase Ran. Morphological and biochemical analysis indicates that the presence of Ran and GTP allows large cargo to efficiently cross central regions of the NPC. We further demonstrate that this function of RanGTP at least partly involves its direct binding to importin beta and transportin. We suggest that RanGTP functions in these pathways to promote the transport of large cargo by enhancing the ability of import complexes to traverse diffusionally restricted areas of the NPC.     

Dissociation of nuclear import cargo complexes by the protein Ran: a fluorescence correlation spectroscopy study

In nucleated cells, proteins designed for nuclear import form complexes with soluble nuclear transport receptors prior to translocation across the nuclear envelope. The directionality of transport is due to the asymmetric distribution of the protein Ran, which dissociates import cargo complexes only in its nuclear RanGTP form. Using fluorescence correlation spectroscopy, we have studied the stability of cargo complexes in solution in the presence and in the absence of RanGTP. We find that RanGTP has a higher affinity for the major import receptor, the importin alpha/beta heterodimer, when importin alpha does not carry a cargo, suggesting that some nuclear transport targets might be preferentially released.     

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.     

Molecular basis for the recognition of snurportin 1 by importin beta

The nuclear import of uridine-rich ribonucleoproteins is mediated by the transport adaptor snurportin 1 (SNP1). Similar to importin alpha, SNP1 uses an N-terminal importin beta binding (sIBB) domain to recruit the receptor importin beta and gain access to the nucleus. In this study, we demonstrate that the sIBB domain has a bipartite nature, which contains two distinct binding determinants for importin beta. The first determinant spans residues 25-65 and includes the previously identified importin alpha IBB (alphaIBB) region of homology. The second binding determinant encompasses residues 1-24 and resembles region 1011-1035 of the nucleoporin 153 (Nup153). The two binding determinants synergize within the sIBB domain to confer a low nanomolar binding affinity for importin beta (K(d) approximately 2 nm) in an interaction that, in vitro, is displaced by RanGTP. We propose that in vivo the synergy of Nup153 and nuclear RanGTP promotes translocation of uridine-rich ribonucleoproteins into the nucleus.     

The Nup358-RanGAP complex is required for efficient importin alpha/beta-dependent nuclear import

In vertebrate cells, the nucleoporin Nup358/RanBP2 is a major component of the filaments that emanate from the nuclear pore complex into the cytoplasm. Nup358 forms a complex with SUMOylated RanGAP1, the GTPase activating protein for Ran. RanGAP1 plays a pivotal role in the establishment of a RanGTP gradient across the nuclear envelope and, hence, in the majority of nucleocytoplasmic transport pathways. Here, we investigate the roles of the Nup358-RanGAP1 complex and of soluble RanGAP1 in nuclear protein transport, combining in vivo and in vitro approaches. Depletion of Nup358 by RNA interference led to a clear reduction of importin alpha/beta-dependent nuclear import of various reporter proteins. In vitro, transport could be partially restored by the addition of importin beta, RanBP1, and/or RanGAP1 to the transport reaction. In intact Nup358-depleted cells, overexpression of importin beta strongly stimulated nuclear import, demonstrating that the transport receptor is the most rate-limiting factor at reduced Nup358-concentrations. As an alternative approach, we used antibody-inhibition experiments. Antibodies against RanGAP1 inhibited the enzymatic activity of soluble and nuclear pore-associated RanGAP1, as well as nuclear import and export. Although export could be fully restored by soluble RanGAP, import was only partially rescued. Together, these data suggest a dual function of the Nup358-RanGAP1 complex as a coordinator of importin beta recycling and reformation of novel import complexes.     

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.     

20 S proteasomes are imported as precursor complexes into the nucleus of yeast

The mechanism by which yeast 20 S proteasomes are imported into the nucleus is still unresolved. Here, we provide the first evidence that 20 S proteasomes are imported as precursor complexes into the nucleus. By using the srp1-49 mutant which is deficient in nuclear import of cargos with classical nuclear localization sequences (cNLS), we show that proteasome precursor complexes associate with importin/karyopherin alphabeta, the cNLS receptor, and that they accumulate inside the cytoplasm. Reconstitution assays revealed that only precursor complexes are targeted to the nuclear envelope (NE) by karyopherin alphabeta. In support, the green fluorescent protein (GFP)-labelled maturation factor Ump1, marking precursor complexes, mainly localizes to the nucleus and around the NE. Our data suggest that nuclear 20 S proteasomes are finally matured inside the nucleus.     

Molecular basis for the rapid dissociation of nuclear localization signals from karyopherin alpha in the nucleoplasm

The yeast karyopherin heterodimer Kap60p.Kap95p facilitates nuclear import of proteins bearing a classic nuclear localization signal (NLS). The alpha subunit Kap60p binds to the NLS of cargo molecules in the cytoplasm, forming stable complexes that must ultimately dissociate in the nucleoplasm. Although Kap60p can release NLSs on its own using an autoinhibitory sequence (AIS) motif that can occupy the NLS binding site, that mechanism is too slow to support rapid nuclear import. We previously showed that the nuclear basket nucleoporin Nup2p and the exportin complex Cse1p.Gsp1p.GTP function as karyopherin release factors (KaRFs) because they can accelerate the rate of dissociation of NLSs from Kap60p. Here we dissect the molecular mechanics of their KaRF activity. We show that Cse1p accelerates dissociation of Kap60p.NLS-cargo complexes and Kap60p.Nup2p complexes by increasing the affinity of Kap60p for its AIS motif. In contrast, Nup2p uses a conserved sequence motif (VMXXRKIA) coupled to an AIS-like motif to accelerate dissociation of Kap60p.NLS complexes in a vectorial reaction mechanism. Mutation of either motif in Nup2p leads to a loss of KaRF activity and to the accumulation of Kap60p.NLS-cargo complexes in the nucleoplasm of yeast. We discuss a model whereby Nup2p, Cse1p, and Gsp1p cooperate to establish directionality in the movement of Kap60p and NLS-cargos across the nuclear pore complex.     

Conformational variability of nucleo-cytoplasmic transport factors

The transport of macromolecules between the nucleus and cytoplasm of eukaryotic cells is largely mediated by a single family of transport factors, the karyopherin or importin beta-like family. Structural and biochemical evidence suggests conformational flexibility of these modular HEAT-repeat proteins is crucial for their regulation. Here we use small angle x-ray scattering to assess the extent of conformational variation within a set of nuclear import and export factors. The study reveals that importin beta, transportin, and the exportin Xpo-t share a similar S-like superhelical conformation in their unbound state. There are no obvious differences in the overall structures that might generally distinguish nuclear export from nuclear import mediators. Two other members of the family, the exportins Cse1 and Xpo1, possess a significantly more globular conformation, indicating that the extended S-like architecture is not a hallmark of all karyopherins. Binding of RanGTP/cargo to importin beta, transportin, and Xpo-t triggers distinct conformational responses, suggesting that even closely related karyopherins employ different mechanisms of conformational regulation and that cargo and nuclear pore-interacting surfaces of the different receptors may be unique.     

A novel conserved nuclear localization signal is recognized by a group of yeast importins

Nucleo-cytoplasmic transport of proteins is mostly mediated by specific interaction between transport receptors of the importin beta family and signal sequences present in their cargo. While several signal sequences, in particular the classical nuclear localization signal (NLS) recognized by the heterodimeric importin alpha/beta complex are well known, the signals recognized by other importin beta-like transport receptors remain to be characterized in detail. Here we present the systematic analysis of the nuclear import of Saccharomyces cerevisiae Asr1p, a nonessential alcohol-responsive Ring/PHD finger protein that shuttles between nucleus and cytoplasm but accumulates in the nucleus upon alcohol stress. Nuclear import of Asr1p is constitutive and mediated by its C-terminal domain. A short sequence comprising residues 243-280 is sufficient and necessary for active targeting to the nucleus. Moreover, the nuclear import signal is conserved from yeast to mammals. In vitro, the nuclear localization signal of Asr1p directly interacts with the importins Kap114p, Kap95p, Pse1p, Kap123p, or Kap104p, interactions that are sensitive to the presence of RanGTP. In vivo, these importins cooperate in nuclear import. Interestingly, the same importins mediate nuclear transport of histone H2A. Based on mutational analysis and sequence comparison with a region mediating nuclear import of histone H2A, we identified a novel type of NLS with the consensus sequence R/KxxL(x)(n)V/YxxV/IxK/RxxxK/R that is recognized by five yeast importins and connects them into a highly efficient network for nuclear import of proteins.