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.     

Discrimination between NL1- and NL2-Mediated Nuclear Localization of the Glucocorticoid Receptor

Glucocorticoid receptor (GR) cycles between a free liganded form that is localized to the nucleus and a heat shock protein (hsp)-immunophilin-complexed, unliganded form that is usually localized to the cytoplasm but that can also be nuclear. In addition, rapid nucleocytoplasmic exchange or shuttling of the receptor underlies its localization. Nuclear import of liganded GR is mediated through a well-characterized sequence, NL1, adjacent to the receptor DNA binding domain and a second, uncharacterized motif, NL2, that overlaps with the ligand binding domain. In this study we report that rapid nuclear import (half-life [t_1/2] of 4 to 6 min) of agonist- and antagonist-treated GR and the localization of unliganded, hsp-associated GRs to the nucleus in G₀ are mediated through NL1 and correlate with the binding of GR to pendulin/importin α. By contrast, NL2-mediated nuclear transfer of GR occurred more slowly (t_1/2 = 45 min to 1 h), was agonist specific, and appeared to be independent of binding to importin α. Together, these results suggest that NL2 mediates the nuclear import of GR through an alternative nuclear import pathway. Nuclear export of GR was inhibited by leptomycin B, suggesting that the transfer of GR to the cytoplasm is mediated through the CRM1-dependent pathway. Inhibition of GR nuclear export by leptomycin B enhanced the nuclear localization of both unliganded, wild-type GR and hormone-treated NL1⁻ GR. These results highlight that the subcellular localization of both liganded and unliganded GRs is determined, at least in part, by a flexible equilibrium between the rates of nuclear import and export.     

Differential roles of heat shock protein 70 in the in vitro nuclear import of glucocorticoid receptor and simian virus 40 large tumor antigen.

Nuclear import of glucocorticoid receptors (GRs) was analyzed in vitro with digitonin-permeabilized cells (S. A. Adam, R. Sterne-Marr, and L. Gerace, J. Cell Biol. 111:807-816, 1990). Indirect immunofluorescence methods were used to monitor the transport of GRs from rat hepatoma and fibroblast cell cytosol into HeLa nuclei. In vitro nuclear import of GRs was shown to be hormone dependent and to require ATP and incubation at ambient temperatures (i.e., 30 degrees C). Hormone-dependent dissociation of GR-bound proteins, such as the 90-kDa heat shock protein, hsp90, is part of an activation process that is obligatory for the expression of the receptor's DNA-binding activity. Inhibition of in vitro GR activation by Na2MoO4 blocked hormone-dependent nuclear import, demonstrating that receptor activation is required for nuclear import. The addition to GR-containing cytosol of antiserum directed against the cytosolic 70-kDa heat shock protein, hsp70, while effective in blocking the nuclear import of simian virus 40 large tumor antigen (SV40 TAg), did not affect hormone-dependent nuclear import of endogenous, full-length GRs or an exogenously added truncated GR protein (i.e., XGR556) that lacks a hormone-binding domain but possesses a constitutively active nuclear localization signal sequence (NLS). Depletion of hsp70 from HeLa cell cytosol did not affect the nuclear import of exogenously added XGR556 but led to inhibition of SV40 TAg nuclear import. Thus, two closely related NLSs, one contained within GRs and the other contained within SV40 TAg, are distinguished by their differential requirements for hsp70 in vitro.     

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.     

The IFNAR1 subunit of the type I IFN receptor complex contains a functional nuclear localization sequence

A nuclear localization sequence (NLS) in the type II interferon (IFN) IFN gamma, which is responsible for the nuclear translocation of both the ligand and the alpha-subunit (IFNGR1) of the receptor complex, has previously been characterized and its role in signaling examined in detail. We have now identified an NLS in the type I IFN receptor (IFNAR) common subunit IFNAR1 from humans and show that the human IFNAR1 subunit can translocate to the nucleus following human IFN beta stimulation. An NLS in human IFNAR1 is located in the extracellular domain of IFNAR1 within the sequence (382)RKIIEKKT (numbered for the precursor form). Nuclear import by the NLS functions in a conventional fashion requiring cytosolic import factors, is energy-dependent and inhibited by the prototypical NLS of the SV40 large T-antigen. These studies provide a mechanism for nuclear import of IFNAR1, as well as for type I IFN ligands, and a starting point for studying an alternate role for IFNAR1 in nuclear signaling within the type I IFN system.     

The carboxyl terminus of RNA helicase A contains a bidirectional nuclear transport domain

Human RNA helicase A was recently identified to be a shuttle protein which interacts with the constitutive transport element (CTE) of type D retroviruses. Here we show that a domain of 110 amino acids at the carboxyl terminus of helicase A is both necessary and sufficient for nuclear localization as well as rapid nuclear export of glutathione S-transferase fusion proteins. The import and export activities of this domain overlap but are separable by point mutations. This bidirectional nuclear transport domain (NTD) has no obvious sequence homology to previously identified nuclear import or export signals. However, the Ran-dependent nuclear import of NTD was efficiently competed by excess amounts of the nuclear localization signal (NLS) peptide from simian virus 40 large T antigen, suggesting that import is mediated by the classical NLS pathway. The nuclear export pathway accessed by NTD is insensitive to leptomycin B and thus is distinct from the leucine-rich nuclear export signal pathway mediated by CRM1.     

Development of a functional backbone cyclic mimetic of the HIV-1 Tat arginine-rich motif

We have used the backbone cyclic proteinomimetics approach to develop peptides that functionally mimic the arginine-rich motif (ARM) of the HIV-1 Tat protein. This consensus sequence serves both as a nuclear localization signal (NLS) and as an RNA binding domain. Based on the NMR structure of Tat, we have designed and synthesized a backbone cyclic ARM mimetic peptide library. The peptides were screened for their ability to mediate nuclear import of the corresponding BSA conjugates in permeabilized cells. One peptide, designated "Tat11," displayed active NLS properties. Nuclear import of Tat11-BSA was found to proceed by the same distinct pathway used by the Tat-NLS and not by the common importin alpha pathway, which is used by the SV40-NLS. Most of the Tat-derived backbone cyclic peptides display selective inhibitory activity as demonstrated by the inhibition of the nuclear import mediated by the Tat-NLS and not by the SV40-NLS. The Tat-ARM-derived peptides, including Tat-11, also inhibited binding of the HIV-1 Rev-ARM to its corresponding RNA element (Rev response element) with inhibition constants of 5 nm. Here we have shown for the first time (a) a functional mimetic of a protein sequence, which activates a nuclear import receptor and (b) a mimetic of a protein sequence with a dual functionality. Tat11 is a lead compound which can potentially inhibit the HIV-1 life cycle by a dual mechanism: inhibition of nuclear import and of RNA binding.     

An unconventional NLS is critical for the nuclear import of the influenza A virus nucleoprotein and ribonucleoprotein

Replication of the RNAs of influenza virus occurs in the nucleus of infected cells. The nucleoprotein (NP) has been shown to be important for the import of the viral RNA into the nucleus and has been proposed to contain at least three different nuclear localization signals (NLSs). Here, an import assay in digitonin-permeabilized cells was used to further define the contribution of these NLSs. Mutation of the unconventional NLS impaired the nuclear import of the NP. A peptide bearing the unconventional NLS could inhibit the nuclear import of the NP in this import assay and prevent the NP-karyopherin alpha interaction in a binding assay confirming the crucial role of this signal. Interestingly, a peptide containing the SV40 T antigen NLS was unable to inhibit the nuclear import of NP or the NP-karyopherin alpha interaction, suggesting that the NP and the SV40 T antigen do not share a common binding site on karyopherin alpha. We also investigated the question of which NLS(s) is/are necessary for the viral ribonucleoprotein complex to enter the nucleus. We found that the peptide containing the unconventional NLS efficiently inhibited the nuclear import of the ribonucleoprotein complexes. This finding suggests that the unconventional NLS is the major signal necessary not only for the nuclear transport of free NP but also for the import of the ribonucleoprotein complexes. Finally, viral replication could be specifically inhibited by a membrane-permeable peptide containing the unconventional NLS, confirming the crucial role of this signal during the replicative cycle of the virus.     

Protracted nuclear export of glucocorticoid receptor limits its turnover and does not require the exportin 1/CRM1-directed nuclear export pathway

Glucocorticoid receptors (GRs) are shuttling proteins, yet they preferentially accumulate within either the cytoplasmic or nuclear compartment when overall rates of nuclear import or export, respectively, are limiting. Hormone binding releases receptors from stable heteromeric complexes that restrict their interactions with soluble nuclear import factors and contribute to their cytoplasmic retention. Although hormone dissociation leads to the rapid release of GRs from chromatin, unliganded nuclear receptors are delayed in their export. We have used a chimeric GR that contains a heterologous, leucine-rich nuclear export signal sequence (NES) to assess the consequences of accelerated receptor nuclear export. Leucine-rich NESs utilize the exportin 1/CRM1-dependent nuclear export pathway, which can be blocked by leptomycin B (LMB). The fact that rapid nuclear export of the NES-GR chimera, but not the protracted export of wild-type GR, is sensitive to LMB, suggests that GR does not require the exportin 1/CRM1 pathway to exit the nucleus. Despite its more rapid export, the NES-GR chimera appears indistinguishable from wild-type GR in its transactivation activity in transiently transfected cells. However, accelerated nuclear export of the NES-GR chimera is associated with an increased rate of hormone-dependent down-regulation. The increase in NES-GR down-regulation is overcome by LMB treatment, thereby confirming the connection between receptor nuclear export and down-regulation. Given the presence of a nuclear recycling pathway for GR, the protracted rate of receptor nuclear export may increase the efficiency of biological responses to secondary hormone challenges by limiting receptor down-regulation and hormone desensitization.     

Shuttling components of nuclear import machinery involved in nuclear translocation of steroid receptors exit nucleus via exportin-1/CRM-1* independent pathway

The nucleocytoplasmic transport processes are mediated by soluble transport factors constantly navigating between nuclear and cytoplasmic compartments. Our understanding about nuclear export of general 'nuclear import factors' that deliver the cargo to the nucleus is still fragmentary. Utilizing green fluorescent protein tagged glucocorticoid receptor (GR) and relA as our working model and with judicious use of LMB, we show in living cells that all the soluble components of the nuclear import machinery exit nucleus via exportin1/CRM1 independent pathway(s).     

Modulation of nucleocytoplasmic trafficking by retention in cytoplasm or nucleus

Nuclear protein transport processes have largely been studied using in vitro semi‐intact cell systems where high concentrations of nuclear localizing substrates are used, and cytoplasmic components such as the microtubule (MT) network, are either absent or damaged. Here we use the fluorescence recovery after photobleaching (FRAP) technique to analyze the nucleocytoplasmic flux of distinct fluorescently tagged proteins over time in living cultured cells. FRAP was performed in different parts of the cell to analyze the kinetics of nucleocytoplasmic trafficking and intranuclear/cytoplasmic mobility of the tumor suppressor Rb protein and a SV40 large tumor antigen (T‐ag) derivative containing the nuclear localization sequence (NLS), both fused to green fluorescent protein (GFP). The results indicate that proteins carrying the T‐ag NLS are highly mobile in the nucleus and cytoplasm. Rb, in contrast, is largely immobile in both cellular compartments, with similar nuclear import and export kinetics. Rb nuclear export was CRM‐1‐mediated, with its reduced mobility in the cytoplasm in part due to association with MTs. Overall our results show that nuclear and cytoplasm retention modulates the rates of nuclear protein import and export in intact cells. J. Cell. Biochem. 107: 1160–1167, 2009. © 2009 Wiley‐Liss, Inc.     

Nucleocytoplasmic transport of proteins

In eukaryotic cells, the movement of macromolecules between the nucleus and cytoplasm occurs through the nuclear pore complex (NPC)--a large protein complex spanning the nuclear envelope. The nuclear transport of proteins is usually mediated by a family of transport receptors known as karyopherins. Karyopherins bind to their cargoes via recognition of nuclear localization signal (NLS) for nuclear import or nuclear export signal (NES) for export to form a transport complex. Its transport through NPC is facilitated by transient interactions between the karyopherins and NPC components. The interactions of karyopherins with their cargoes are regulated by GTPase Ran. In the current review, we describe the NPC structure, NLS, and NES, as well as the model of classic Ran-dependent transport, with special emphasis on existing alternative mechanisms; we also propose a classification of the basic mechanisms of protein transport regulation.     

Identification and characterization of a functional nuclear localization signal in the HIV-1 integrase interactor LEDGF/p75

Human lens epithelium-derived growth factor (LEDGF)/p75 protein forms a specific nuclear complex with human immunodeficiency virus type 1 (HIV-1) integrase and is essential for nuclear localization and chromosomal association of the viral protein. We now studied nuclear import of LEDGF/p75 in live and semipermeabilized cells. We show that nuclear import of LEDGF/p75 is GTP-, Ran-, importin-alpha/beta-, and energy-dependent and that the protein competes with the canonical SV40 large T antigen nuclear localization signal (NLS) for nuclear import receptors. We identified the NLS of LEDGF/p75 through deletion analysis and site-directed mutagenesis. The LEDGF/p75 NLS, 148GRKRKAEKQ156, belongs to the canonical SV40-like family. Fusion of this short peptide to the amino terminus of Escherichia coli beta-galactosidase rendered the fusion protein nuclear, confirming that the LEDGF/p75 NLS is transferable. Moreover, a single amino acid change in the NLS was sufficient to exclude the mutant LEDGF/p75 protein from the nucleus and abolish nuclear import of HIV-1 integrase.     

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.