The C-terminal nuclear localization signal of the sex-determining region Y (SRY) high mobility group domain mediates nuclear import through importin beta 1

The sex-determining factor SRY is a DNA-binding protein that diverts primordial gonads from the ovarian pathway toward male differentiation to form testes. It gains access to the nucleus through two distinct nuclear localization signals (NLSs) that flank the high mobility group (HMG) DNA-binding domain, but the mechanisms through which these NLSs operate have not been studied. In this study, we reconstitute the nuclear import of SRY in vitro, demonstrating a lack of requirement for exogenous factors for nuclear accumulation and a significant reduction in nuclear transport in the presence of antibodies to importin beta but not importin alpha. Using a range of quantitative binding assays including enzyme-linked immunosorbent assay, fluorescence polarization, and native gel mobility electrophoresis, we assess the binding of importins to SRY, demonstrating a high affinity recognition (in the low nm range) by Imp beta independent of Imp alpha. In assessing the contribution of each NLS, we found that the N-terminal NLS was recognized poorly by importins, whereas the C-terminal NLS was bound by importin beta with similar affinity to SRY. We also found that RanGTP, but not RanGDP, could dissociate the SRY-importin beta complex in solution using FP. We describe a novel double-fluorescent label DNA binding assay to demonstrate mutual exclusivity between importin beta recognition and DNA binding on the part of SRY, which may represent an alternative release mechanism upon nuclear entry. This study represents the first characterization of the nuclear import pathway for a HMG domain-containing protein. Importantly, it demonstrates for the first time that recognition of SRY by Imp beta is of comparable affinity to that with which Imp alpha/beta recognizes conventional NLS-containing substrates.     

The importin beta/importin 7 heterodimer is a functional nuclear import receptor for histone H1

Import of proteins into the nucleus proceeds through nuclear pore complexes and is largely mediated by nuclear transport receptors of the importin beta family that use direct RanGTP-binding to regulate the interaction with their cargoes. We investigated nuclear import of the linker histone H1 and found that two receptors, importin beta (Impbeta) and importin 7 (Imp7, RanBP7), play a critical role in this process. Individually, the two import receptors bind H1 weakly, but binding is strong for the Impbeta/Imp7 heterodimer. Consistent with this, import of H1 into nuclei of permeabilized mammalian cells requires exogenous Impbeta together with Imp7. Import by the Imp7/Impbeta heterodimer is strictly Ran dependent, the Ran-requiring step most likely being the disassembly of the cargo-receptor complex following translocation into the nucleus. Disassembly is brought about by direct binding of RanGTP to Impbeta and Imp7, whereby the two Ran-binding sites act synergistically. However, whereas an Impbeta/RanGTP interaction appears essential for H1 import, Ran-binding to Imp7 is dispensable. Thus, Imp7 can function in two modes. Its Ran-binding site is essential when operating as an autonomous import receptor, i.e. independently of Impbeta. Within the Impbeta/Imp7 heterodimer, however, Imp7 plays a more passive role than Impbeta and resembles an import adapter.     

Multiple importins function as nuclear transport receptors for the Rev protein of human immunodeficiency virus type 1

The Rev protein of human immunodeficiency virus type 1 is an RNA-binding protein that is required for nuclear export of unspliced and partially spliced viral mRNAs. Nuclear import of human immunodeficiency virus type 1 Rev has been suggested to depend on the classic nuclear transport receptor importin beta, but not on the adapter protein importin alpha. We now show that, similar to importin alpha, Rev is able to dissociate RanGTP from recycling importin beta, a reaction that leads to the formation of a novel import complex. Besides importin beta, the transport receptors transportin, importin 5, and importin 7 specifically interact with Rev and promote its nuclear import in digitonin-permeabilized cells. A single arginine-rich nuclear localization sequence of Rev is required for interaction with all importins tested so far. In contrast to the importin beta-binding domain of importin alpha, Rev interacts with an N-terminal fragment of importin beta. Transportin contains two independent binding sites for Rev. Hence, the mode of interaction of importin beta and transportin with Rev is clearly distinct from that with their classic import cargoes. Taken together, the viral protein takes advantage of multiple cellular transport pathways for its nuclear accumulation.     

Importin beta-depending nuclear import pathways: role of the adapter proteins in the docking and releasing steps

Nuclear imports of uridine-rich small nuclear ribonucleoprotein (U1 snRNP) and proteins with classical nuclear localization signal (cNLS-protein) are mediated by importin beta. However, due to the presence of different import signals, the adapter protein of the imported molecules and importin beta is different for each pathway. Although the adapter for cNLS-protein is importin alpha, the adapter for U1 snRNP is snurportin1 (SPN1). Herein, we show that the use of distinct adapters by importin beta results in differences at the docking and releasing step for these two import pathways. Nuclear pore complex (NPC) docking of U1 snRNP but not of cNLS-protein was inhibited by an anti-CAN/Nup214 antibody. Thus, the initial NPC-binding site is different for each pathway. Pull-down assays between immobilized SPN1 and two truncated forms of importin beta documented that SPN1 and importin alpha have different binding sites on importin beta. Importin beta fragment 1-618, which binds to SPN1 but not to importin alpha, was able to support the nuclear import of U1 snRNPs. After the translocation through the NPC, both import complexes associated with the nuclear side of the NPC. However, we found that the nature of the importin beta-binding domain of the adapters influences the release of the cargo into the nucleoplasm.     

NTF2 mediates nuclear import of Ran.

Importin beta family transport receptors shuttle between the nucleus and the cytoplasm and mediate transport of macromolecules through nuclear pore complexes (NPCs). The interactions between these receptors and their cargoes are regulated by binding RanGTP; all receptors probably exit the nucleus complexed with RanGTP, and so should deplete RanGTP continuously from the nucleus. We describe here the development of an in vitro system to study how nuclear Ran is replenished. Nuclear import of Ran does not rely on simple diffusion as Ran's small size would permit, but instead is stimulated by soluble transport factors. This facilitated import is specific for cytoplasmic RanGDP and employs nuclear transport factor 2 (NTF2) as the actual carrier. NTF2 binds RanGDP initially to NPCs and probably also mediates translocation of the NTF2-RanGDP complex to the nuclear side of the NPCs. A direct NTF2-RanGDP interaction is crucial for this process, since point mutations that disturb the RanGDP-NTF2 interaction also interfere with Ran import. The subsequent nuclear accumulation of Ran also requires GTP, but not GTP hydrolysis. The release of Ran from NTF2 into the nucleus, and thus the directionality of Ran import, probably involves nucleotide exchange to generate RanGTP, for which NTF2 has no detectable affinity, followed by binding of the RanGTP to an importin beta family transport receptor.     

The requirement of H1 histones for a heterodimeric nuclear import receptor

After synthesis in the cytoplasm, H1 histones are imported into the nucleus through an energy-dependent process that can be mediated by an importin beta-importin 7 (Impbeta-Imp7) heterodimer. H1 histones contain two structurally different types of nuclear localization signals (NLS). The first type of NLS resides within the unstructured C-terminal domain and is rich in basic amino acids. In contrast, the highly conserved central domain of the H1 histone contains comparatively few basic amino acids but also represents a functional NLS. The competence for the nuclear import of this globular domain seems to be based on its secondary structure. Here, we show that the Impbeta-Imp7 heterodimer is the only receptor for H1 import. Furthermore, we identified the import receptors mediating the in vitro transport of different NLS of the H1 histone. Using the digitonin-permeabilized cell import assay we show that Impbeta is the most efficient import receptor for the globular domain of H1 histones, whereas the heterodimer of Impbeta and Imp7 is the functional receptor for the entire C-terminal domain. However, short fragments of the C-terminal domain are imported in vitro by at least four different importins, which resembles the import pathway of ribosomal proteins and core histones. In addition, we show that heterodimerization of Impbeta with Imp7 is absolutely necessary for their proper function as an import receptor for H1 histones. These findings point to a chaperone-like function of the heterodimeric complex in addition to its function as an import receptor. It appears that the Impbeta-Imp7 heterodimer is specialized for NLS consisting of extended basic domains.     

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.     

A systems analysis of importin-{alpha}-{beta} mediated nuclear protein import

Importin-beta (Impbeta) is a major transport receptor for Ran-dependent import of nuclear cargo. Impbeta can bind cargo directly or through an adaptor such as Importin-alpha (Impalpha). Factors involved in nuclear transport have been well studied, but systems analysis can offer further insight into regulatory mechanisms. We used computer simulation and real-time assays in intact cells to examine Impalpha-beta-mediated import. The model reflects experimentally determined rates for cargo import and correctly predicts that import is limited principally by Impalpha and Ran, but is also sensitive to NTF2. The model predicts that CAS is not limiting for the initial rate of cargo import and, surprisingly, that increased concentrations of Impbeta and the exchange factor, RCC1, actually inhibit rather than stimulate import. These unexpected predictions were all validated experimentally. The model revealed that inhibition by RCC1 is caused by sequestration of nuclear Ran. Inhibition by Impbeta results from depletion nuclear RanGTP, and, in support of this mechanism, expression of mRFP-Ran reversed the inhibition.     

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.     

Importins fulfil a dual function as nuclear import receptors and cytoplasmic chaperones for exposed basic domains

Many nuclear transport pathways are mediated by importin beta-related transport receptors. Here, we identify human importin (Imp) 4b as well as mouse Imp4a, Imp9a and Imp9b as novel family members. Imp4a mediates import of the ribosomal protein (rp) S3a, while Imp9a and Imp9b import rpS7, rpL18a and apparently numerous other substrates. Ribosomal proteins, histones and many other nuclear import substrates are very basic proteins that aggregate easily with cytoplasmic polyanions such as RNA. Imp9 effectively prevents such precipitation of, for example, rpS7 and rpL18a by covering their basic domains. The same applies to Imp4, Imp5, Imp7 and Impbeta and their respective basic import substrates. The Impbeta-Imp7 heterodimer appears specialized for the most basic proteins, such as rpL4, rpL6 and histone H1, and is necessary and sufficient to keep them soluble in a cytoplasmic environment prior to rRNA or DNA binding, respectively. Thus, just as heat shock proteins function as chaperones for exposed hydrophobic patches, importins act as chaperones for exposed basic domains, and we suggest that this represents a major and general cellular function of importins.     

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.     

Nuclear import of the respiratory syncytial virus matrix protein is mediated by importin beta1 independent of importin alpha

The matrix (M) protein of respiratory syncytial virus (RSV) plays an important role in virus assembly through specific interactions with RSV nucleocapsids and envelope glycoproteins in the cytoplasm as well as with the host cell membrane. We have previously shown that M localizes to the nucleus of infected cells at an early stage in the RSV infection cycle, where it may be instrumental in inhibiting host cell processes. The present study uses transient expression of M as well as a truncated green fluorescent protein (GFP) fusion derivative to show for the first time that M is able to localize in the nucleus in the absence of other RSV gene products, through the action of amino acids 110-183, encompassing the nucleic acid binding regions of the protein, that are sufficient to target GFP to the nucleus. Using native PAGE, ELISA-based binding assays, a novel Alphascreen assay, and an in vitro nuclear transport assay, we show that M is recognized directly by the importin beta1 nuclear import receptor, which mediates its nuclear import in concert with the guanine nucleotide-binding protein Ran. Retention of M in the nucleus through binding to nuclear components, probably mediated by the putative zinc finger domain of M, also contributes to M nuclear accumulation. This is the first report of the importin binding and nuclear import properties of a gene product from a negative sense RNA virus, with implications for the function of RSV M and possibly other viral M proteins in the nucleus of infected cells.     

Efficiency of importin alpha/beta-mediated nuclear localization sequence recognition and nuclear import. Differential role of NTF2.

Little quantitative, kinetic information is available with respect to the process of nuclear import of conventional nuclear localization sequence (NLS)-containing proteins, which initially involves recognition and docking at the nuclear pore by importin alpha/beta. This study compares the binding and nuclear import properties of mouse (m) and yeast (y) importin (IMP) subunits with respect to the NLSs from the SV40 large tumor antigen (T-ag), and the Xenopus laevis phosphoprotein N1N2. m- and y-IMPalpha recognized both NLSs, with y-IMPalpha exhibiting higher affinity. m-IMPbeta greatly enhanced the binding of m-IMPalpha to the T-ag and N1N2 NLSs, but y-IMPbeta did not significantly affect the affinity of y-IMPalpha for the T-ag NLS. In contrast, y-IMPbeta enhanced y-IMPalpha binding to the NLS of N1N2, but to a lesser extent than the enhancement of m-IMPalpha binding by m-IMPbeta. NLS-dependent nuclear import was reconstituted in vitro using the different importin subunits together with the transport factors Ran and NTF2. Whereas T-ag NLS-mediated nuclear import did not exhibit an absolute requirement for NTF2, N1N2 NLS-mediated transport strictly required NTF2. High levels of NTF2 inhibited nuclear accumulation conferred by both NLSs. We conclude that different NLSs possess distinct nuclear import properties due to differences in recognition by importin and requirements for NTF2.     

Ribosomal Protein L12 Uses a Distinct Nuclear Import Pathway Mediated by Importin 11

Ribosome biogenesis requires the nuclear translocation of ribosomal proteins from their site of synthesis in the cytoplasm to the nucleus. Analyses of the import mechanisms have revealed that most ribosomal proteins can be delivered to the nucleus by multiple transport receptors (karyopherins or importins). We now provide evidence that ribosomal protein L12 (rpL12) is distinguished from the bulk of ribosomal proteins because it accesses the importin 11 pathway as a major route into the nucleus. rpL12 specifically and directly interacted with importin 11 in vitro and in vivo. Both rpL12 binding to and import by importin 11 were inhibited by another importin 11 substrate, UbcM2, indicating that these two cargoes may bind overlapping sites on the transport receptor. In contrast, the import of rpL23a, a ribosomal protein that uses the general ribosomal protein import system, was not competed by UbcM2, and in an in vitro binding assay, importin 11 did not bind to the nuclear localization signal of rpL23a. Furthermore, in a transient transfection assay, the nuclear accumulation of rpL12 was increased by coexpressed importin 11, but not by other importins. These data are consistent with importin 11 being a mediator of rpL12 nuclear import. Taken together, these results indicate that rpL12 uses a distinct nuclear import pathway that may contribute to a mechanism for regulating ribosome synthesis and/or maturation.     

Multiple pathways contribute to nuclear import of core histones

Nuclear import of the four core histones H2A, H2B, H3 and H4 is one of the main nuclear import activities during S-phase of the cell cycle. However, the molecular machinery facilitating nuclear import of core histones has not been elucidated. Here, we investigated the pathways by which histone import can occur. First, we show that core histone import can be competed by the BIB (β-like import receptor binding) domain of ribosomal protein L23a suggesting that histone import is an importin mediated process. Secondly, affinity chromatography on immobilized core histones revealed that several members of the importin β family of transport receptors are able to interact with core histones. Finally, we demonstrate that at least four known and one novel importin, importin 9, can mediate nuclear import of core histones into the nuclei of permeabilized cells. Our results suggest that multiple pathways of import exist to provide efficient nuclear uptake of these abundant, essential proteins.     

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.     

Thermodynamic analysis of H1 nuclear import: receptor tuning of importinbeta/importin7

The nuclear import of H1 linker histones is mediated by a heterodimer of transport receptors, known as importinbeta and importin7. Interestingly, both importins separately interact with H1, but only as a dimer they facilitate the translocation through the nuclear pore. We identified the H1 binding site of importin7, comprising two extended acidic loops near the C terminus of importin7. The analysis of the H1 import complex assembly by means of isothermal titration calorimetry revealed that the formation of a receptor heterodimer in vitro is an enthalpy-driven process, whereas subsequent binding of H1 to the heterodimer is entropy-driven. Furthermore, we show that the importinbeta binding domain of importin7 plays a key role in the activation of importin7 by importinbeta. This process is allosterically regulated by importinbeta and accounts for a specific tuning of the activity of the importinbeta.importin7 heterodimer. The results presented here provide new insights into cellular strategies to even energy balances in nuclear import and point toward a general regulation of importinbeta-related nuclear import processes.