Characterization of the auto-inhibitory sequence within the N-terminal domain of importin alpha

Protein cargoes that contain a classic nuclear localization signal (NLS) are transported into the nucleus through binding to a heterodimeric receptor comprised of importin/karyopherin alpha and beta. An evolutionarily conserved auto-inhibitory sequence within the N-terminal importin beta binding (IBB) domain of importin alpha regulates NLS-cargo binding to the NLS binding pocket on importin alpha. In this study, we have used site-directed mutagenesis coupled with in vitro binding assays and in vivo analyses to investigate the intramolecular interaction of the N-terminal IBB domain and the NLS binding pocket of Saccharomyces cerevisiae importin alpha, Srp1p. We find that mutations within the IBB domain that decrease the binding affinity of the auto-inhibitory sequence for the NLS binding pocket impact importin alpha function in vivo. In addition, the severity of the in vivo phenotype is directly correlated to the reduction of auto-inhibition measured in vitro, suggesting that the in vivo phenotypes are directly related to the loss of auto-inhibitory function. We exploit a conditional auto-inhibitory mutant, srp1-55, to study the in vivo functional overlap between the N-terminal IBB domain of importin alpha and other factors implicated in NLS-cargo release, Cse1p and Nup2p. We propose that the N-terminal IBB domain of importin alpha and Cse1p function together in NLS-cargo release, whereas Nup2p contributes to cargo release/importin alpha recycling through a distinct mechanism.     

Nuclear import factors importin alpha and importin beta undergo mutually induced conformational changes upon association

A heterodimer of importin alpha and importin beta accomplishes the nuclear import of proteins carrying classical nuclear localization signals (NLS). The interaction between the two import factors is mediated by the IBB domain of importin alpha and involves an extended recognition surface as shown by X-ray crystallography. Using a combination of biochemical and biophysical techniques we have investigated the formation of the importin beta:IBB domain complex in solution. Our data suggest that upon binding to the IBB domain, importin beta adopts a compact, proteolytically resistant conformation, while simultaneously the IBB domain folds into an alpha helix. We suggest a model to describe how these dual mutually induced conformational changes may orchestrate the nuclear import of NLS cargo in vivo.     

Quantitative analysis of nuclear localization signal (NLS)-importin alpha interaction through fluorescence depolarization. Evidence for auto-inhibitory regulation of NLS binding

We have developed a quantitative in vitro steady-state fluorescence depolarization assay to measure the interaction of a nuclear localization signal (NLS) substrate with its receptors. This assay relies on the change in fluorescence depolarization of an NLS fused to the green fluorescent protein upon binding to receptor. No binding is observed in the absence of a functional NLS, and binding affinities measured correlate with previous in vivo studies of NLS function. We have used this assay to test an auto-inhibitory model for the interaction of an NLS with the NLS receptor complex. This model suggests that NLS binding to importin alpha is modulated by an auto-inhibitory sequence within the N terminus of importin alpha, which is displaced by importin beta binding. Consistent with this model, NLS substrates bind tightly to an N-terminally truncated importin alpha lacking the auto-inhibitory domain (K(d) approximately 10 nm), but measurable binding to full-length importin alpha is only observed upon addition of importin beta. Our quantitative results support the auto-inhibitory model and suggest a mechanism for a switch between a cytoplasmic, high affinity and a nuclear, low affinity NLS receptor. This predicted mode of interaction would facilitate binding of substrate in the cytoplasm and its subsequent release into the nucleus.     

Molecular basis for the recognition of a nonclassical nuclear localization signal by importin beta

Nuclear import of proteins containing a classical nuclear localization signal (NLS) involves NLS recognition by importin alpha, which associates with importin beta via the IBB domain. Other proteins, including parathyroid hormone-related protein (PTHrP), are imported into the nucleus by direct interaction with importin beta. We solved the crystal structure of a fragment of importin beta-1 (1-485) bound to the nonclassical NLS of PTHrP. The structure reveals a second extended cargo binding site on importin beta distinct from the IBB domain binding site. Using a permeabilized cell import assay we demonstrate that importin beta (1-485) can import PTHrP-coupled cargo in a Ran-dependent manner. We propose that this region contains a prototypical nuclear import receptor domain, which could have evolved into the modern importin beta superfamily.     

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.     

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.     

Snurportin1, an m3G-cap-specific nuclear import receptor with a novel domain structure.

The nuclear import of the spliceosomal snRNPs U1, U2, U4 and U5, is dependent on the presence of a complex nuclear localization signal (NLS). The latter is composed of the 5'-2,2,7-terminal trimethylguanosine (m3G) cap structure of the U snRNA and the Sm core domain. Here, we describe the isolation and cDNA cloning of a 45 kDa protein, termed snurportin1, which interacts specifically with m3G-cap but not m7G-cap structures. Snurportin1 enhances the m3G-capdependent nuclear import of U snRNPs in both Xenopus laevis oocytes and digitonin-permeabilized HeLa cells, demonstrating that it functions as an snRNP-specific nuclear import receptor. Interestingly, solely the m3G-cap and not the Sm core NLS appears to be recognized by snurportin1, indicating that at least two distinct import receptors interact with the complex snRNP NLS. Snurportin1 represents a novel nuclear import receptor which contains an N-terminal importin beta binding (IBB) domain, essential for function, and a C-terminal m3G-cap-binding region with no structural similarity to the arm repeat domain of importin alpha.     

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.     

Autoinhibition by an internal nuclear localization signal revealed by the crystal structure of mammalian importin alpha

Importin alpha is the nuclear import receptor that recognizes classical monopartite and bipartite nuclear localization signals (NLSs). The structure of mouse importin alpha has been determined at 2.5 A resolution. The structure shows a large C-terminal domain containing armadillo repeats, and a less structured N-terminal importin beta-binding domain containing an internal NLS bound to the NLS-binding site. The structure explains the regulatory switch between the cytoplasmic, high-affinity form, and the nuclear, low-affinity form for NLS binding of the nuclear import receptor predicted by the current models of nuclear import. Importin beta conceivably converts the low- to high-affinity form by binding to a site overlapping the autoinhibitory sequence. The structure also has implications for understanding NLS recognition, and the structures of armadillo and HEAT repeats.     

The importin β binding domain as a master regulator of nucleocytoplasmic transport

Specific and efficient recognition of import cargoes is essential to ensure nucleocytoplasmic transport. To this end, the prototypical karyopherin importin β associates with import cargoes directly or, more commonly, through import adaptors, such as importin α and snurportin. Adaptor proteins bind the nuclear localization sequence (NLS) of import cargoes while recruiting importin β via an N-terminal importin β binding (IBB) domain. The use of adaptors greatly expands and amplifies the repertoire of cellular cargoes that importin β can efficiently import into the cell nucleus and allows for fine regulation of nuclear import. Accordingly, the IBB domain is a dedicated NLS, unique to adaptor proteins that functions as a molecular liaison between importin β and import cargoes. This review provides an overview of the molecular role played by the IBB domain in orchestrating nucleocytoplasmic transport. Recent work has determined that the IBB domain has specialized functions at every step of the import and export pathway. Unexpectedly, this stretch of ~ 40 amino acids plays an essential role in regulating processes such as formation of the import complex, docking and translocation through the nuclear pore complex (NPC), release of import cargoes into the cell nucleus and finally recycling of import adaptors and importin β into the cytoplasm. Thus, the IBB domain is a master regulator of nucleocytoplasmic transport, whose complex molecular function is only recently beginning to emerge. This article is part of a Special Issue entitled: Regulation of Signaling and Cellular Fate through Modulation of Nuclear Protein Import.     

Nuclear import of the U1A splicesome protein is mediated by importin alpha /beta and Ran in living mammalian cells

U1A is a component of the uracil-rich small nuclear ribonucleoprotein. The molecular mechanism of nuclear import of U1A was investigated in vivo and in vitro. When recombinant deletion mutants of U1A are injected into the BHK21 cell cytoplasm, the nuclear localization signal (NLS) of U1A is found in the N-terminal half of the central domain (residues 100-144 in mouse U1A). In an in vitro assay, it was found that the U1A-NLS accumulated in only a portion of the nuclei in the absence of cytosolic extract. In contrast, the addition of importin alpha/beta and Ran induced the uniform nuclear accumulation of U1A-NLS in all cells. Furthermore, U1A was found to bind the C-terminal portion of importin alpha. In addition, the in vitro nuclear migration of full-length U1A was found to be exclusively dependent on importin alpha/beta and Ran. Moreover, in living cells, the full-length U1A accumulated in the nucleus in a Ran-dependent manner, and nuclear accumulation was inhibited by the importin beta binding domain of importin alpha. These results suggest that the nuclear import of U1A is mediated by at least two distinct pathways, an importin alpha/beta and Ran-dependent and an -independent pathway in permeabilized cells, and that the latter pathway may be suppressed in intact 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.     

Synergy of silent and hot spot mutations in importin beta reveals a dynamic mechanism for recognition of a nuclear localization signal

Molecular recognition of the importin beta-binding (IBB) domain of importin alpha by importin beta is critical for the nuclear import of protein cargoes containing a classical nuclear localization signal. We have studied the function of four conserved tryptophans of importin beta (Trp-342, Trp-430, Trp-472, and Trp-864) located at the binding interface with the IBB domain by systematic alanine substitution mutagenesis. We found that Trp-864 is a mutational hot spot that significantly affects IBB-binding and import activity, whereas residues Trp-342, Trp-430, and Trp-472 are mutationally silent when analyzed individually. Interestingly, the combination of the hot spot at residue Trp-864 with mutations in the other three tryptophans gives rise to a striking synergy that diminishes IBB domain binding by up to approximately 1000-fold and, in turn, abolishes import activity. We propose that importin beta uses the tryptophans to select and stabilize a helical conformation of the IBB domain, which, in turn, conveys specific, high affinity binding.     

Nuclear import of U snRNPs requires importin beta.

Macromolecules that are imported into the nucleus can be divided into classes according to their nuclear import signals. The best characterized class consists of proteins which carry a basic nuclear localization signal (NLS), whose transport requires the importin alpha/beta heterodimer. U snRNP import depends on both the trimethylguanosine cap of the snRNA and a signal formed when the Sm core proteins bind the RNA. Here, factor requirements for U snRNP nuclear import are studied using an in vitro system. Depletion of importin alpha, the importin subunit that binds the NLS, is found to stimulate rather than inhibit U snRNP import. This stimulation is shown to be due to a common requirement for importin beta in both U snRNP and NLS protein import. Saturation of importin beta-mediated transport with the importin beta-binding domain of importin alpha blocks U snRNP import both in vitro and in vivo. Immunodepletion of importin beta inhibits both NLS-mediated and U snRNP import. While the former requires re-addition of both importin alpha and importin beta, re-addition of importin beta alone to immunodepleted extracts was sufficient to restore efficient U snRNP import. Thus importin beta is required for U snRNP import, and it functions in this process without the NLS-specific importin alpha.     

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.     

Classical nuclear localization signals: definition, function, and interaction with importin alpha

The best understood system for the transport of macromolecules between the cytoplasm and the nucleus is the classical nuclear import pathway. In this pathway, a protein containing a classical basic nuclear localization signal (NLS) is imported by a heterodimeric import receptor consisting of the beta-karyopherin importin beta, which mediates interactions with the nuclear pore complex, and the adaptor protein importin alpha, which directly binds the classical NLS. Here we review recent studies that have advanced our understanding of this pathway and also take a bioinformatics approach to analyze the likely prevalence of this system in vivo. Finally, we describe how a predicted NLS within a protein of interest can be confirmed experimentally to be functionally important.     

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.     

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.     

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.