Nucleocytoplasmic Recycling of the Nuclear Localization Signal Receptor α Subunit In Vivo Is Dependent on a Nuclear Export Signal, Energy, and RCC1

Nuclear protein import requires a nuclear localization signal (NLS) receptor and at least three other cytoplasmic factors. The α subunit of the NLS receptor, Rag cohort 1 (Rch1), enters the nucleus, probably in a complex with the β subunit of the receptor, as well as other import factors and the import substrate. To learn more about which factors and/or events end the import reaction and how the import factors return to the cytoplasm, we have studied nucleocytoplasmic shuttling of Rch1 in vivo. Recombinant Rch1 microinjected into Vero or tsBN2 cells was found primarily in the cytoplasm. Rch1 injected into the nucleus was rapidly exported in a temperature-dependent manner. In contrast, a mutant of Rch1 lacking the first 243 residues accumulated in the nuclei of Vero cells after cytoplasmic injection. After nuclear injection, the truncated Rch1 was retained in the nucleus, but either Rch1 residues 207–217 or a heterologous nuclear export signal, but not a mutant form of residues 207–217, restored nuclear export. Loss of the nuclear transport factor RCC1 (regulator of chromosome condensation) at the nonpermissive temperature in the thermosensitive mutant cell line tsBN2 caused nuclear accumulation of wild-type Rch1 injected into the cytoplasm. However, free Rch1 injected into nuclei of tsBN2 cells at the nonpermissive temperature was exported. These results suggested that RCC1 acts at an earlier step in Rch1 recycling, possibly the disassembly of an import complex that contains Rch1 and the import substrate. Consistent with this possibility, incubation of purified RanGTP and RCC1 with NLS receptor and import substrate prevented assembly of receptor/substrate complexes or stimulated their disassembly.     

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.     

Importin alpha/beta-mediated nuclear protein import is regulated in a cell cycle-dependent manner

Functional nuclear proteins are selectively imported into the nucleus by transport factors such as importins alpha and beta. The relationship between the efficiency of nuclear protein import and the cell cycle was measured using specific import substrates for the importin alpha/beta-mediated pathway. After the microinjection of SV40 T antigen nuclear localization signal (NLS)-containing substrates into the cytoplasm of synchronized culture cells at a certain phase of the cell cycle, the nuclear import of the substrates was measured kinetically. Cell cycle-dependent change in import efficiency, but not capacity, was found. That is, import efficiency was found low in the early S, G2/M, and M/G1 phases compared with other phases. In addition, we found that the extent of co-imunoprecipitation of importin alpha with importin beta from cell extracts was strongly associated with import efficiency. These results indicate that the importin alpha/beta-mediated nuclear import machinery is regulated in a cell cycle-dependent manner through the modulation of interaction modes between importins alpha and beta.     

The translocation of transportin–cargo complexes through nuclear pores is independent of both Ran and energy

Active transport between nucleus and cytoplasm proceeds through nuclear pore complexes (NPCs) and is mediated largely by shuttling transport receptors that use direct RanGTP binding to coordinate loading and unloading of cargo [1], [2], [3], [4]. Import receptors such as importin β or transportin bind their substrates at low RanGTP levels in the cytoplasm and release them upon encountering RanGTP in the nucleus, where a high RanGTP concentration is predicted. This substrate release is, in the case of import by the importin α/β heterodimer, coupled directly to importin β release from the NPCs. If the importin β –RanGTP interaction is prevented, import intermediates arrest at the nuclear side of the NPCs [5], [6]. This arrest makes it difficult to probe directly the Ran and energy requirements of the actual translocation from the cytoplasmic to the nuclear side of the NPC, which immediately precedes substrate release. Here, we have shown that in the case of transportin, dissociation of transportin–substrate complexes is uncoupled from transportin release from NPCs. This allowed us to dissect the requirements of translocation through the NPC, substrate release and transportin recycling. Surprisingly, translocation of transportin–substrate complexes into the nucleus requires neither Ran nor nucleoside triphosphates (NTPs). It is only nuclear RanGTP, not GTP hydrolysis, that is needed for dissociation of transportin–substrate complexes and for re-export of transportin to the cytoplasm. GTP hydrolysis is apparently required only to restore the import competence of the re-exported transportin and, thus, for multiple rounds of transportin-dependent import. In addition, we provide evidence that at least one type of substrate can also complete NPC passage mediated by importin β independently of Ran and energy.     

Evidence for distinct substrate specificities of importin alpha family members in nuclear protein import.

Importin alpha plays a pivotal role in the classical nuclear protein import pathway. Importin alpha shuttles between nucleus and cytoplasm, binds nuclear localization signal-bearing proteins, and functions as an adapter to access the importin beta-dependent import pathway. In contrast to what is found for importin beta, several isoforms of importin alpha, which can be grouped into three subfamilies, exist in higher eucaryotes. We describe here a novel member of the human family, importin alpha7. To analyze specific functions of the distinct importin alpha proteins, we recombinantly expressed and purified five human importin alpha's along with importin alpha from Xenopus and Saccharomyces cerevisiae. Binding affinity studies showed that all importin alpha proteins from humans or Xenopus bind their import receptor (importin beta) and their export receptor (CAS) with only marginal differences. Using an in vitro import assay based on permeabilized HeLa cells, we compared the import substrate specificities of the various importin alpha proteins. When the substrates were tested singly, only the import of RCC1 showed a strong preference for one family member, importin alpha3, whereas most of the other substrates were imported by all importin alpha proteins with similar efficiencies. However, strikingly different substrate preferences of the various importin alpha proteins were revealed when two substrates were offered simultaneously.     

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.     

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.     

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.     

beta-catenin can be transported into the nucleus in a Ran-unassisted manner

The nuclear accumulation of beta-catenin plays an important role in the Wingless/Wnt signaling pathway. This study describes an examination of the nuclear import of beta-catenin in living mammalian cells and in vitro semi-intact cells. When injected into the cell cytoplasm, beta-catenin rapidly migrated into the nucleus in a temperature-dependent and wheat germ agglutinin-sensitive manner. In the cell-free import assay, beta-catenin rapidly migrates into the nucleus without the exogenous addition of cytosol, Ran, or ATP/GTP. Cytoplasmic injection of mutant Ran defective in its GTP hydrolysis did not prevent beta-catenin import. Studies using tsBN2, a temperature-sensitive mutant cell line that possesses a point mutation in the RCC1 gene, showed that the import of beta-catenin is insensitive to nuclear Ran-GTP depletion. These results show that beta-catenin possesses the ability to constitutively translocate through the nuclear pores in a manner similar to importin beta in a Ran-unassisted manner. We further showed that beta-catenin also rapidly exits the nucleus in homokaryons, suggesting that the regulation of nuclear levels of beta-catenin involves both nuclear import and export of this molecule.     

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.     

Importin-beta-like nuclear transport receptors

In recent years, our understanding of macromolecular transport processes across the nuclear envelope has grown dramatically, and a large number of soluble transport receptors mediating either nuclear import or nuclear export have been identified. Most of these receptors belong to one large family of proteins, all of which share homology with the protein import receptor importin β (also named karyopherin β). Members of this family have been classified as importins or exportins on the basis of the direction they carry their cargo. To date, the family includes 14 members in the yeast Saccharomyces cerevisiae and at least 22 members in humans. Importins and exportins are regulated by the small GTPase Ran, which is thought to be highly enriched in the nucleus in its GTP-bound form. Importins recognize their substrates in the cytoplasm and transport them through nuclear pores into the nucleus. In the nucleoplasm, RanGTP binds to importins, inducing the release of import cargoes. In contrast, exportins interact with their substrates only in the nucleus in the presence of RanGTP and release them after GTP hydrolysis in the cytoplasm, causing disassembly of the export complex. Thus, common features of all importin-β-like transport factors are their ability to shuttle between the nucleus and the cytoplasm, their interaction with RanGTP as well as their ability to recognize specific transport substrates.     

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.     

Importin 7 and importin alpha/importin beta are nuclear import receptors for the glucocorticoid receptor

The vertebrate glucocorticoid receptor (GR) is cytoplasmic without hormone and localizes to the nucleus after hormone binding. GR has two nuclear localization signals (NLS): NL1 is similar in sequence to the SV40 NLS; NL2 is poorly defined, residing in the ligand-binding domain. We found that GR displayed similar hormone-regulated compartmentalization in Saccharomyces cerevisiae and required the Sxm1 nuclear import receptor for NL2-mediated import. Two metazoan homologues of Sxm1, importin 7 and importin 8, bound both NL1 and NL2, whereas importin alpha selectively bound NL1. In an in vitro nuclear import assay, both importin 7 and the importin alpha-importin beta heterodimer could import a GR NL1 fragment. Under these conditions, full-length GR localized to nuclei in the presence but not absence of an unidentified component in cell extracts. Interestingly, importin 7, importin 8, and importin alpha bound GR even in the absence of hormone; thus, hormonal control of localization is exerted at a step downstream of import receptor binding.     

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.     

Loss of RCC1, a nuclear DNA-binding protein, uncouples the completion of DNA replication from the activation of cdc2 protein kinase and mitosis.

The temperature-sensitive mutant cell line tsBN2, was derived from the BHK21 cell line and has a point mutation in the RCC1 gene. In tsBN2 cells, the RCC1 protein disappeared after a shift to the non-permissive temperature at any time in the cell cycle. From S phase onwards, once RCC1 function was lost at the non-permissive temperature, p34cdc2 was dephosphorylated and M-phase specific histone H1 kinase was activated. However, in G1 phase, shifting to the non-permissive temperature did not activate p34cdc2 histone H1 kinase. The activation of p34cdc2 histone H1 kinase required protein synthesis in addition to the presence of a complex between p34cdc2 and cyclin B. Upon the loss of RCC1 in S phase of tsBN2 cells and the consequent p34cdc2 histone H1 kinase activation, a normal mitotic cycle is induced, including the formation of a mitotic spindle and subsequent reformation of the interphase-microtubule network. Exit from mitosis was accompanied by the disappearance of cyclin B, and a decrease in p34cdc2 histone H1 kinase activity. The kinetics of p34cdc2 histone H1 kinase activation correlated well with the appearance of premature mitotic cells and was not affected by the presence of a DNA synthesis inhibitor. Thus the normal inhibition of p34cdc2 activation by incompletely replicated DNA is abrogated by the loss of RCC1.     

The asymmetric distribution of the constituents of the Ran system is essential for transport into and out of the nucleus.

The GTPase Ran is essential for nuclear import of proteins with a classical nuclear localization signal (NLS). Ran''s nucleotide-bound state is determined by the chromatin-bound exchange factor RCC1 generating RanGTP in the nucleus and the cytoplasmic GTPase activating protein RanGAP1 depleting RanGTP from the cytoplasm. This predicts a steep RanGTP concentration gradient across the nuclear envelope. RanGTP binding to importin-beta has previously been shown to release importin-alpha from -beta during NLS import. We show that RanGTP also induces release of the M9 signal from the second identified import receptor, transportin. The role of RanGTP distribution is further studied using three methods to collapse the RanGTP gradient. Nuclear injection of either RanGAP1, the RanGTP binding protein RanBP1 or a Ran mutant that cannot stably bind GTP. These treatments block major export and import pathways across the nuclear envelope. Different export pathways exhibit distinct sensitivities to RanGTP depletion, but all are more readily inhibited than is import of either NLS or M9 proteins, indicating that the block of export is direct rather than a secondary consequence of import inhibition. Surprisingly, nuclear export of several substrates including importin-alpha and -beta, transportin, HIV Rev and tRNA appears to require nuclear RanGTP but may not require GTP hydrolysis by Ran, suggesting that the energy for their nuclear export is supplied by another source.     

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.     

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.     

HURP is a Ran-importin beta-regulated protein that stabilizes kinetochore microtubules in the vicinity of chromosomes

BACKGROUND: Formation of a bipolar mitotic spindle in somatic cells requires the cooperation of two assembly pathways, one based on kinetochore capture by centrosomal microtubules, the other on RanGTP-mediated microtubule organization in the vicinity of chromosomes. How RanGTP regulates kinetochore-microtubule (K-fiber) formation is not presently understood. RESULTS: Here we identify the mitotic spindle protein HURP as a novel target of RanGTP. We show that HURP is a direct cargo of importin beta and that in interphase cells, it shuttles between cytoplasm and nucleus. During mitosis, HURP localizes predominantly to kinetochore microtubules in the vicinity of chromosomes. Overexpression of importin beta or RanT24N (resulting in low RanGTP) negatively regulates its spindle localization, whereas overexpression of RanQ69L (mimicking high RanGTP) enhances HURP association with the spindle. Thus, RanGTP levels control HURP localization to the mitotic spindle in vivo, a conclusion supported by the analysis of tsBN2 cells (mutant in RCC1). Upon depletion of HURP, K-fiber stabilization is impaired and chromosome congression is delayed. Nevertheless, cells eventually align their chromosomes, progress into anaphase, and exit mitosis. HURP is able to bundle microtubules and, in vitro, this function is abolished upon complex formation with importin beta and regulated by Ran. These data indicate that HURP stabilizes K-fibers by virtue of its ability to bind and bundle microtubules. CONCLUSIONS: Our study identifies HURP as a novel component of the Ran-importin beta-regulated spindle assembly pathway, supporting the conclusion that K-fiber formation and stabilization involves both the centrosome-dependent microtubule search and capture mechanism and the RanGTP pathway.     

Importin alpha can migrate into the nucleus in an importin beta- and Ran-independent manner

A classical nuclear localization signal (NLS)-containing protein is transported into the nucleus via the formation of a NLS-substrate/importin alpha/beta complex. In this study, we found that importin alpha migrated into the nucleus without the addition of importin beta, Ran or any other soluble factors in an in vitro transport assay. A mutant importin alpha lacking the importin beta-binding domain efficiently entered the nucleus. Competition experiments showed that this import pathway for importin alpha is distinct from that of importin beta. These results indicate that importin alpha alone can enter the nucleus via a novel pathway in an importin beta- and Ran-independent manner. Furthermore, this process is evolutionarily conserved as similar results were obtained in Saccharomyces cerevisiae. Moreover, the import rate of importin alpha differed among individual nuclei of permeabilized cells, as demonstrated by time-lapse experiments. This heterogeneous nuclear accumulation of importin alpha was affected by the addition of ATP, but not ATPgammaS. These results suggest that the nuclear import machinery for importin alpha at individual nuclear pore complexes may be regulated by reaction(s) that require ATP hydrolysis.