Importin beta, transportin, RanBP5 and RanBP7 mediate nuclear import of ribosomal proteins in mammalian cells.

The assembly of eukaryotic ribosomal subunits takes place in the nucleolus and requires nuclear import of ribosomal proteins. We have studied this import in a mammalian system and found that the classical nuclear import pathway using the importin alpha/beta heterodimer apparently plays only a minor role. Instead, at least four importin beta-like transport receptors, namely importin beta itself, transportin, RanBP5 and RanBP7, directly bind and import ribosomal proteins. We found that the ribosomal proteins L23a, S7 and L5 can each be imported alternatively by any of the four receptors. We have studied rpL23a in detail and identified a very basic region to which each of the four import receptors bind avidly. This domain might be considered as an archetypal import signal that evolved before import receptors diverged in evolution. The presence of distinct binding sites for rpL23a and the M9 import signal in transportin, and for rpL23a and importin alpha in importin beta might explain how a single receptor can recognize very different import signals.     

Axo-glial interactions regulate the localization of axonal paranodal proteins

The SR proteins, a group of abundant arginine/serine (RS)-rich proteins, are essential pre-mRNA splicing factors that are localized in the nucleus. The RS domain of these proteins serves as a nuclear localization signal. We found that RS domain-bearing proteins do not utilize any of the known nuclear import receptors and identified a novel nuclear import receptor specific for SR proteins. The SR protein import receptor, termed transportin-SR (TRN-SR), binds specifically and directly to the RS domains of ASF/SF2 and SC35 as well as several other SR proteins. The nuclear transport regulator RanGTP abolishes this interaction. Recombinant TRN-SR mediates nuclear import of RS domain- bearing proteins in vitro. TRN-SR has amino acid sequence similarity to several members of the importin beta/transportin family. These findings strongly suggest that TRN-SR is a nuclear import receptor for the SR protein family.     

Cytosolic proteins that specifically bind nuclear location signals are receptors for nuclear import.

We have purified two major polypeptides of 54 and 56 kd from bovine erythrocytes that specifically bind the nuclear location sequence (NLS) of the SV40 large T antigen. When added to a permeabilized cell system for nuclear import, the purified proteins increase by 2- to 3-fold the nuclear accumulation of a fluorescent protein containing the large T antigen NLS. The import stimulation is saturable and dependent upon the presence of cytosol. Nuclear protein accumulation in vitro is sensitive to inactivation by N-ethylmaleimide (NEM). NEM inactivation can be overcome by addition of the purified NLS-binding proteins to the import system. NEM treatment of the purified proteins abolishes their ability to stimulate import but does not affect NLS binding. Our results indicate that the NLS-binding proteins are NEM-sensitive receptors for nuclear import. At least one other NEM-sensitive cytosolic activity and an NEM-insensitive cytosolic activity are also necessary for protein import in vitro.     

Conservation of complex nuclear localization signals utilizing classical and non-classical nuclear import pathways in LANA homologs of KSHV and RFHV

ORF73 latency-associated nuclear antigen (LANA) of the Kaposi's sarcoma-associated herpesvirus (KSHV) is targeted to the nucleus of infected cells where it binds to chromatin and mediates viral episome persistence, interacts with cellular proteins and plays a role in latency and tumorigenesis. A structurally related LANA homolog has been identified in the retroperitoneal fibromatosis herpesvirus (RFHV), the macaque homolog of KSHV. Here, we report the evolutionary and functional conservation of a novel bi-functional nuclear localization signal (NLS) in KSHV and RFHV LANA. N-terminal peptides from both proteins were fused to EGFP or double EGFP fusions to examine their ability to induce nuclear transport of a heterologous protein. In addition, GST-pull down experiments were used to analyze the ability of LANA peptides to interact with members of the karyopherin family of nuclear transport receptors. Our studies revealed that both LANA proteins contain an N-terminal arginine/glycine (RG)-rich domain spanning a conserved chromatin-binding motif, which binds directly to importin β1 in a RanGTP-sensitive manner and serves as an NLS in the importin β1-mediated non-classical nuclear import pathway. Embedded within this domain is a conserved lysine/arginine-(KR)-rich bipartite motif that binds directly to multiple members of the importin α family of nuclear import adaptors in a RanGTP-insensitive manner and serves as an NLS in the classical importin α/β-mediated nuclear import pathway. The positioning of a classical bipartite kr-NLS embedded within a non-classical rg-NLS is a unique arrangement in these viral proteins, whose nuclear localization is critical to their functionality and to the virus life cycle. The ability to interact with multiple import receptors provides alternate pathways for nuclear localization of LANA. Since different import receptors can import cargo to distinct subnuclear compartments, a multifunctional NLS may provide LANA with an increased ability to interact with different nuclear components in its multifunctional role to maintain viral latency.     

细胞因子和生长因子信号转导途径中信号蛋白分子的核转运机制

信号蛋白分子的入核及出核转运是细胞因子和生长因子信号转导途径中的重要环节.核定位序列（NLS）是信号蛋白分子上与入核转运相关的氨基酸序列.核孔复合物（NPC）、核转运蛋白importin和能量供应体Ran/TC4在入核转运过程中也发挥了重要作用.另外,很多细胞因子和生长因子或其受体上所含有的NLS序列也具有核定位功能,并可能通过“伴侣机制”参与其他信号蛋白分子的入核转运.     

Separate nuclear import pathways converge on the nucleoporin Nup153 and can be dissected with dominant-negative inhibitors

Background: Proteins generally enter or exit the nucleus as cargo of one of a small family of import and export receptors. These receptors bear distant homology to importin β, a subunit of the receptor for proteins with classical nuclear localisation sequences (NLSs). To understand the mechanism of nuclear transport, the next question involves identifying the nuclear pore proteins that interact with the different transport receptors as they dock at the pore and translocate through it.Results: Two pathways of nuclear import were found to intersect at a single nucleoporin, Nup153, localized on the intranuclear side of the nuclear pore. Nup153 contains separate binding sites for importin α/β, which mediates classical NLS import, and for transportin, which mediates import of different nuclear proteins. Strikingly, a Nup153 fragment containing the importin β binding site acted as a dominant-negative inhibitor of NLS import, with no effect on transportin-mediated import. Conversely, a Nup153 fragment containing the transportin binding site acted as a strong dominant-negative inhibitor of transportin import, with no effect on classical NLS import. The interaction of transportin with Nup153 could be disrupted by a non-hydrolyzable form of GTP or by a GTPase-deficient mutant of Ran, and was not observed if transportin carried cargo. Neither Nup153 fragment affected binding of the export receptor Crm1 at the nuclear rim.Conclusions: Two nuclear import pathways, mediated by importin β and transportin, converge on a single nucleoporin, Nup153. Dominant-negative fragments of Nup153 can now be used to distinguish different nuclear import pathways and, potentially, to dissect nuclear export.     

Nuclear transport and nuclear pores in yeast

The central features of nuclear import have been conserved during evolution. In yeast the nuclear accumulation of proteins follows the same selective and active transport mechanisms known from higher eukaryotes. Yeast nuclear proteins contain nuclear localization sequences (NLS) which are presumably recognized by receptors in the cytoplasm and the nuclear envelope. Subsequent to this recognition step, nuclear proteins are translocated into the nucleus via the nuclear pore complexes. The structure of the yeast nuclear pore complex resembles that of higher eukaryotes. Recently, the first putative components of the yeast nuclear import machinery have been cloned and sequenced. The genetically amenable yeast system allows for an efficient structural and functional analysis of these components. Due to the evolutionary conservation potential insights into the nuclear import mechanisms in yeast can be transferred to higher eukaryotes. Thus, yeast can be considered as a eukaryotic model system to study nuclear transport.     

Arabidopsis transportin1 is the nuclear import receptor for the circadian clock-regulated RNA-binding protein AtGRP7

We characterized the Arabidopsis orthologue of the human nuclear import receptor transportin1 (TRN1). Like the human receptor, Arabidopsis TRN1 recognizes nuclear import signals on proteins that are different from the classical basic nuclear localization signals. The M9 domain of human heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is the prototype of such signals. We show that AtTRN1 binds to similar domains in hnRNP-like proteins from plants. AtTRN1 also interacts with human hnRNP A1 and with yeast Nab2p, two classical import cargo proteins of transportin in these organisms. Like all nuclear transport receptors of the importin-beta family, AtTRN1 binds to the regulatory GTPase Ran from Arabidopsis. We demonstrated that the amino terminus of AtTRN1 is necessary for this interaction. Recombinant AtTRN1 conferred nuclear import of fluorescently labelled BSA-M9 peptide conjugates in permeabilized HeLa cells, functionally replacing human TRN1 in these in vitro nuclear import assays. We identified three plant substrate proteins that interact with AtTRN1 and contain M9-like domains: a novel Arabidopsis hnRNP that shows high similarity to human hnRNP A1 and two small RNA-binding proteins from Arabidopsis, AtGRP7 and AtGRP8. Nuclear import activity of the M9-like domains of these plant proteins was demonstrated in vivo by their ability to confer partial nuclear re-localisation of a GFP fusion protein containing a nuclear export signal. In addition, fluorescently labelled AtGRP7 was specifically imported into nuclei of permeabilized HeLa cells by Arabidopsis AtTRN1 and human TRN1. These results suggest that the transportin-mediated nuclear import pathway is highly conserved between man, yeast and plants.     

Core histones and linker histones are imported into the nucleus by different pathways

Histones are the major structural proteins in eukaryotic chromosomes. This group of small very basic proteins consists of the H1 linker histones and the core histones H2A, H2B, H3 and H4. Despite their small size, the nuclear import of histones occurs by an active transport mechanism and not simply by diffusion. Histones contain several nuclear localisation signals (NLS) that can be subdivided into two different types of signal structures. We have previously shown that H1 histones are transported by a heterodimeric import receptor complex consisting of importin beta and importin 7, and we now describe the receptors required for the import of the core histones. Competition experiments using the in vitro transport assay indicate that the import pathway of the core histones differs from that of the linker histones and of nuclear proteins with classical NLS. In vitro binding assays show that each of the import receptors importin beta, importin 5, importin 7 and transportin, has the capacity to bind to any of the four core histones. Reconstitution experiments with recombinant factors indicate that each of these factors can independently serve as an import receptor for each of the core histones.     

Transportin: Nuclear Transport Receptor of a Novel Nuclear Protein Import Pathway

Many nuclear proteins are imported into the cell nucleus by the “classical” nuclear localization signal (NLS)-mediated import pathway. In this pathway, a sequence rich in basic residues in the protein interacts with a heterodimeric complex termed importin and this, along with the GTPase Ran, mediates nuclear import of the NLS-bearing protein. The heterogeneous nuclear ribonucleoprotein (hnRNP) A1 protein contains a novel nuclear localization sequence, termed M9, that does not contain any clusters of basic residues. Very recently, we showed that M9 directs import into the nucleus by a novel protein import pathway distinct from the classical NLS pathway. A 90-kilodalton protein termed transportin was identified as a protein that specifically interacts with wild-type M9 but not transport-defective M9 mutants. Transportin and an ATP-regenerating system were found to be necessary and sufficient for import of M9-containing proteins in anin vitroimport assay. In this report, we provide additional evidence that transportin can interact directly with M9-containing proteins and also show that it can mediate import of full-length hnRNP A1. In addition, Ran, or a Ran-binding protein, is identified as a second protein component of this novel nuclear import pathway. Transportin relatives fromSaccharomyces cerevisiaewhich likely serve as additional nuclear transport receptors are described.     

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.     

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

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

The conserved amino-terminal domain of hSRP1 alpha is essential for nuclear protein import.

Nuclear proteins are targeted through the nuclear pore complex (NPC) in an energy-dependent reaction. The import reaction is mediated by nuclear localization sequences (NLS) in the substrate which are recognized by heterodimeric cytoplasmic receptors. hSRP1 alpha is an NLS-binding subunit of the human NLS receptor complex and is complexed in vivo with a second subunit of 97 kDa (p97). We show here that a short amino-terminal domain in hSRP1 alpha is necessary and sufficient for its interaction with p97. This domain is conserved in other SRP1-like proteins and its fusion to a cytoplasmic reporter protein is sufficient to promote complete nuclear import, circumventing the usual requirement for an NLS receptor interaction. The same amino-terminal domain inhibits import of NLS-containing proteins when added to an in vitro nuclear transport assay. While full-length hSRP alpha is able to leave the nucleus, the amino-terminal domain alone is not sufficient to promote exit. We conclude that hSRP1 alpha functions as an adaptor to tether NLS-containing substrates to the protein import machinery.     

Identification of an unconventional nuclear localization signal in human ribosomal protein S2

Ribosomal proteins must be imported into the nucleus after being synthesized in the cytoplasm. Since the rpS2 amino acid sequence does not contain a typical nuclear localization signal, we used deletion mutant analysis and rpS2-beta-galactosidase chimeric proteins to identify the nuclear targeting domains in rpS2. Nuclear rpS2 is strictly localized in the nucleoplasm and is not targeted to the nucleoli. Subcellular localization analysis of deletion mutants of rpS2-beta-galactosidase chimeras identified a central domain comprising 72 amino acids which is necessary and sufficient to target the chimeric beta-galactosidase to the nucleus. The nuclear targeting domain shares no significant similarity to already characterized nuclear localization signals in ribosomal proteins or other nuclear proteins. Although a Nup153 fragment containing the importinbeta binding site fused to VP22 blocks nuclear import of rpS2-beta-galactosidase fusion proteins, nuclear uptake of rpS2 could be mediated by several import receptors since it binds to importinalpha/beta and transportin.     

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.