An N-ethylmaleimide-sensitive cytosolic factor necessary for nuclear protein import: requirement in signal-mediated binding to the nuclear pore

We described previously an assay for authentic nuclear protein import in vitro. In this assay, exogenous nuclei are placed in an extract of Xenopus eggs; a rhodamine-labeled protein possessing a nuclear localization signal is added, and fluorescence microscopy is used to measure nuclear uptake. The requirement in this system for a cytosolic extract suggests that nuclear import is dependent on at least one cytosolic factor. We now confirm this hypothesis. Treatment of the cytosol with N-ethylmaleimide (NEM) abolishes nuclear protein import; readdition of a cytosolic fraction to the NEM-inactivated extract rescues transport. Thus, at least one NEM-sensitive factor required for transport is supplied by the cytosol. This activity, called nuclear import factor-1, or NIF-1, is ammonium-sulfate-precipitable, protease-sensitive, and heat-labile; it is therefore at least partly proteinaceous. NIF-1 stimulates, in a concentration-dependent manner, the rate at which individual nuclei accumulate protein. The effect of NIF-1 is enhanced by a second cytosolic NEM-sensitive factor, NIF-2. Earlier we identified two steps in the nuclear import reaction: (a) ATP-independent binding of a signal-sequence-bearing protein to the nuclear pore; and (b) ATP-dependent translocation of that protein through the pore. We now show that NEM inhibits signal-mediated binding, and that readdition of NIF-1 restores binding. Thus, NIF-1 is required for at least the binding step and does not require ATP for its activity. NIF-1 may act as a cytoplasmic signal receptor that escorts signal-bearing proteins to the pore, or may instead promote signal-mediated binding to the pore in another manner, as discussed.     

Identification of four nuclear transport signal-binding proteins that interact with diverse transport signals.

The transport of proteins into the nucleus requires not only the presence of a nuclear transport signal on the targeted protein but also the signal recognition proteins and the nuclear pore translocation apparatus. Complicating the search for the signal recognition proteins is the fact that the nuclear transport signals identified share little obvious homology. In this study, synthetic peptides homologous to the nuclear transport signals from the simian virus 40 large T antigen, Xenopus oocyte nucleoplasmin, adenovirus E1A, and Saccharomyces cerevisiae MAT alpha 2 proteins were coupled to a UV-photoactivable cross-linker and iodinated for use in an in vitro cross-linking reaction with cellular lysates. Four proteins, p140, p100, p70, and p55, which specifically interacted with the nuclear transport signal peptides were identified. Unique patterns of reactivity were observed with closely related pairs of nuclear transport signal peptides. Competition experiments with labeled and unlabeled peptides demonstrated that heterologous signals were able to bind the same protein and suggested that diverse signals use a common transport pathway. The subcellular distribution of the four nuclear transport signal-binding proteins suggested that nuclear transport involves both cytoplasmic and nuclear receptors. The four proteins were not bound by wheat germ agglutinin and were not associated tightly with the nuclear pore complex.     

Inhibition of in vitro nuclear transport by a lectin that binds to nuclear pores

Selective transport of proteins is a major mechanism by which biochemical differences are maintained between the cytoplasm and nucleus. To begin to investigate the molecular mechanism of nuclear transport, we used an in vitro transport system composed of a Xenopus egg extract, rat liver nuclei, and a fluorescently labeled nuclear protein, nucleoplasmin. With this system, we screened for inhibitors of transport. We found that the lectin, wheat germ agglutinin (WGA), completely inhibits the nuclear transport of fluorescently labeled nucleoplasmin. No other lectin tested affected nuclear transport. The inhibition by WGA was not seen when N-acetylglucosamine was present and was reversible by subsequent addition of sugar. When rat liver nuclei that had been incubated with ferritin-labeled WGA were examined by electron microscopy, multiple molecules of WGA were found bound to the cytoplasmic face of each nuclear pore. Gel electrophoresis and nitrocellulose transfer identified one major and several minor nuclear protein bands as binding 125I-labeled WGA. The most abundant protein of these, a 63-65-kD glycoprotein, is a candidate for the inhibitory site of action of WGA on nuclear protein transport. WGA is the first identified inhibitor of nuclear protein transport and interacts directly with the nuclear pore.     

Characterization of the nuclear protein import mechanism using Ran mutants with altered nucleotide binding specificities.

The small nuclear GTP binding protein Ran is required for transport of nuclear proteins through the nuclear pore complex (NPC). Although it is known that GTP hydrolysis by Ran is essential for this reaction, it has been unclear whether additional energy-consuming steps are also required. To uncouple the energy requirements for Ran from other nucleoside triphosphatases, we constructed a mutant derivative of Ran that has an altered nucleotide specificity from GTP to xanthosine 5' triphosphate. Using this Ran mutant, we demonstrate that nucleotide hydrolysis by Ran is sufficient to promote efficient nuclear protein import in vitro. Under these conditions, protein import could no longer be inhibited with non-hydrolysable nucleotide analogues, indicating that no Ran-independent energy-requiring steps are essential for the protein translocation reaction through the NPC. We further provide evidence that nuclear protein import requires Ran in the GDP form in the cytoplasm. This suggests that a coordinated exchange reaction from Ran-GDP to Ran-GTP at the pore is necessary for translocation into the nucleus.     

Identification of cytosolic factors required for nuclear location sequence-mediated binding to the nuclear envelope

Nuclear protein import can be separated into two distinct steps: binding to the nuclear pore complex followed by translocation to the nuclear interior. A previously identified nuclear location sequence (NLS) receptor and a 97-kD protein purified from bovine erythrocytes reconstitute the binding step in a permeabilized cell assay. Binding to the envelope is specific for a functional SV-40 large T antigen NLS and is not ATP or temperature dependent. Modification of p97 with N- ethylmaleimide (NEM) decreases binding to the pore, but interestingly, NEM treatment of the NLS receptor does not. Nuclear envelope binding is inhibited by wheat germ agglutinin suggesting a possible mechanism for the inhibition of transport by the lectin.     

Structure and function of the nuclear pore complex: new perspectives.

The double membrane of the nuclear envelope is a formidable barrier separating the nucleus and cytoplasm of eukaryotic cells. However, movement of specific macromolecules across the nuclear envelope is critical for embryonic development, cell growth and differentiation. Transfer of molecules between the nucleus and cytoplasm occurs through the aqueous channel formed by the nuclear pore complex (NPC)

1 Abbreviations: NPC, nuclear pore complex; GlcNac, N-acetylglucosamine; WGA, wheat germ agglutinin

. Although small molecules may simply diffuse across the NPC, transport of large proteins and RNA requires specific transport signals and is energy dependent. A family of pore glycoproteins modified by O-linked N-acetylglucosamine moieties are essential for transport through the NPC. Recent evidence suggests that the regulation of nuclear transport may also involve the inteaction of RNA and nuclear proteins with specific binding proteins that recognize these transport signals. Are these nuclear pore glycoproteins and signal binding proteins the ‘gatekeepers’ that control access to the genetic material? Recent evidence obtained from a combination of biochemical and genetic approaches suggests – perhaps.     

Functional Tat transport of unstructured, small, hydrophilic proteins

The twin-arginine translocation (Tat) system is a protein translocation system that is adapted to the translocation of folded proteins across biological membranes. An understanding of the folding requirements for Tat substrates is of fundamental importance for the elucidation of the transport mechanism. We now demonstrate for the first time Tat transport for fully unstructured proteins, using signal sequence fusions to naturally unfolded FG repeats from the yeast Nsp1p nuclear pore protein. The transport of unfolded proteins becomes less efficient with increasing size, consistent with only a single interaction between the system and the substrate. Strikingly, the introduction of six residues from the hydrophobic core of a globular protein completely blocked translocation. Physiological data suggest that hydrophobic surface patches abort transport at a late stage, most likely by membrane interactions during transport. This study thus explains the observed restriction of the Tat system to folded globular proteins on a molecular level.     

Translocation of beta-catenin into the nucleus independent of interactions with FG-rich nucleoporins

beta-Catenin nuclear import has been found to be independent of classical nuclear localization signal (NLS) nuclear import factors. Here, we test the hypothesis that beta-catenin interacts directly with nuclear pore proteins to mediate its own transport. We show that beta-catenin, unlike importin-beta, does not interact detectably with Phe/Gly(FG)-repeat-rich nuclear pore proteins or nucleoporins (Nups). Moreover, unlike NLS-containing proteins, beta-catenin nuclear import is not inhibited by wheat germ agglutinin (WGA) or excess importin-beta. These results suggest beta-catenin nuclear translocation does not involve direct interactions with FG-Nups. However, beta-catenin has two regions that can target it to the nucleus, and its import is cold sensitive, indicating that beta-catenin nuclear import is still an active process. Transport is blocked by a soluble form of the C-cadherin cytoplasmic domain, suggesting that masking of the nuclear targeting signal may be a mechanism of regulating beta-catenin subcellular localization.     

A protein recognized by antibodies to Asp-Asp-Asp-Glu-Asp shows specific binding activity to heterogeneous nuclear transport signals

Nuclear proteins contain a signal, termed the nuclear transport signal, that specifies their selective transport into the nucleus. Previously we reported that antibodies to Asp-Asp-Asp-Glu-Asp (DDDED) inhibited nuclear transport of nuclear proteins in vivo. We therefore tried to detect a cellular receptor of nuclear transport signals as a protein that reacted with both anti-DDDED antibody and nuclear transport signal sequences. Using two steps of affinity chromatography, anti-DDDED-Sepharose and nucleoplasmin-Sepharose, we obtained a protein of 69 kDa (p69) from the nuclear pore fraction that showed these characters. This p69 recognized by anti-DDDED antibody interacted specifically with SV40 large T antigen and nucleoplasmin transport signals.     

How proteins enter the nucleus

Nuclear protein import is a selective process. Proteins destined for the nucleus contain NLSs. These short stretches of amino acids interact with proteins located in the cytoplasm, on the nuclear envelope, and/or at the nuclear pore complex. Following binding at the pore complex, proteins are translocated through the pore into the nucleus in a manner requiring ATP. The biochemical dissection of the nuclear pore complex has begun. Alteration of protein import into the nucleus is emerging as a new and complex form of regulation. However, we are left with the following problems: How do proteins move through the cytoplasm to reach the nuclear pore? How does the nuclear pore complex open and close in a selective manner? How is ATP utilized during import? And finally, how is bi-directional traffic of both proteins and RNA through the pore regulated?     

An essential 45 kDa yeast transmembrane protein reacts with anti-nuclear pore antibodies: purification of the protein, immunolocalization and cloning of the gene.

A yeast membrane protein was isolated by its binding to tRNA Sepharose column. The 45 kDa protein shares characteristics with rat liver nuclear pore proteins in having reactivity with a monoclonal antibody (RL1) raised against rat liver nuclear pore proteins and by the binding of wheat germ agglutinin (WGA), indicating the presence of N-acetylglucosamine (GlcNAc) moieties. Immunofluorescence microscopy and cell fractionation experiments indicate that the protein is located in the nuclear envelope and the endoplasmic reticulum of the cell. The gene for the 45 kDa protein was cloned using degenerate oligonucleotides derived from the N-terminal protein sequence and confirmed by internal peptide sequences. The gene was named WBP1. The protein coding sequence of the WBP1 gene reveals an ER entry signal peptide and a C-terminal membrane spanning domain. Topological studies indicate that the C-terminus of the protein is located in the cytoplasm. The cytoplasmic tail of the protein contains the K-K-X-X signal known to be sufficient for retention of transmembrane proteins in higher eukaryotic cells. Gene disruption experiments show that the 45 kDa protein is essential for the vegetative life cycle of the yeast cell.     

RNA-mediated interaction between the peptide-binding and GTPase domains of the signal recognition particle

The signal recognition particle (SRP) targets nascent proteins to cellular membranes for insertion or secretion by recognizing polypeptides containing an N-terminal signal sequence as they emerge from the ribosome. GTP-dependent binding of SRP to its receptor protein leads to controlled release of the nascent chain into a membrane-spanning translocon pore. Here we show that the association of the SRP with its receptor triggers a marked conformational change in the complex, localizing the SRP RNA and the adjacent signal peptide-binding site at the SRP-receptor heterodimer interface. The orientation of the RNA suggests how peptide binding and GTP hydrolysis can be coupled through direct structural contact during cycles of SRP-directed protein translocation.     

GLFG and FxFG nucleoporins bind to overlapping sites on importin-beta

The interaction between nuclear pore proteins (nucleoporins) and transport factors is crucial for the translocation of macromolecules through nuclear pores. Many nucleoporins contain FG sequence repeats, and previous studies have demonstrated interactions between repeats containing FxFG or GLFG cores and transport factors. The crystal structure of residues 1-442 of importin-beta bound to a GLFG peptide indicates that this repeat core binds to the same primary site as FxFG cores. Importin-beta-I178D shows reduced binding to both FxFG and GLFG repeats, consistent with both binding to an overlapping site in the hydrophobic groove between the A-helices of HEAT repeats 5 and 6. Moreover, FxFG repeats can displace importin-beta or its S. cerevisiae homologue, Kap95, bound to GLFG repeats. Addition of soluble GLFG repeats decreases the rate of nuclear protein import in digitonin-permeabilized HeLa cells, indicating that this interaction has a role in the translocation of carrier-cargo complexes through nuclear pores. The binding of GLFG and FxFG repeats to overlapping sites on importin-beta indicates that functional differences between different repeats probably arise from differences in their spatial organization.     

Two signals mediate nuclear localization of influenza virus (A/WSN/33) polymerase basic protein 2.

Polymerase basic protein 2 (PB2), a component of the influenza virus polymerase complex, when expressed alone from cloned cDNA in the absence of other influenza virus proteins, is transported into the nucleus. In this study, we have examined the nuclear translocation signal of PB2 by making deletions and mutations in the PB2 sequence. Our studies showed that two distant regions in the polypeptide sequence were involved in the nuclear translocation of PB2. In one region, four basic residues (K-736 R K R) played a critical role in the nuclear translocation of PB2, since the deletion or mutation of these residues rendered the protein totally cytoplasmic. However, seven residues (M K R K R N S) of this region, including the four basic residues, failed to translocate a cytoplasmic reporter protein into the nucleus, suggesting that these sequences were necessary but not sufficient for nuclear translocation. Deletion of another region (amino acids 449 to 495) resulted in a mutant protein which was cytoplasmic with a perinuclear distribution. This novel phenotype suggests that a perinuclear binding step was involved prior to translocation of PB2 across the nuclear pore and that a signal might be involved in perinuclear binding. Possible involvement of these two signal sequences in the nuclear localization of PB2 is discussed.     

Bacterial Sec Protein Transport Is Rate-limited by Precursor Length: A Single Turnover Study

An in vitro real-time single turnover assay for the Escherichia coli Sec transport system was developed based on fluorescence dequenching. This assay corrects for the fluorescence quenching that occurs when fluorescent precursor proteins are transported into the lumen of inverted membrane vesicles. We found that 1) the kinetics were well fit by a single exponential, even when the ATP concentration was rate-limiting; 2) ATP hydrolysis occurred during most of the observable reaction period; and 3) longer precursor proteins transported more slowly than shorter precursor proteins. If protein transport through the SecYEG pore is the rate-limiting step of transport, which seems likely, these conclusions argue against a model in which precursor movement through the SecYEG translocon is mechanically driven by a series of rate-limiting, discrete translocation steps that result from conformational cycling of the SecA ATPase. Instead, we propose that precursor movement results predominantly from Brownian motion and that the SecA ATPase regulates pore accessibility.     

NOA1, a Novel ClpXP Substrate,Takes an Unexpected Nuclear Detour Prior to Mitochondrial Import

The mitochondrial matrix GTPase NOA1 is a nuclear encoded protein, essential for mitochondrial protein synthesis, oxidative phosphorylation and ATP production. Here, we demonstrate that newly translated NOA1 protein is imported into the nucleus, where it localizes to the nucleolus and interacts with UBF1 before nuclear export and import into mitochondria. Mutation of the nuclear localization signal (NLS) prevented both nuclear and mitochondrial import while deletion of the N-terminal mitochondrial targeting sequence (MTS) or the C-terminal RNA binding domain of NOA1 impaired mitochondrial import. Absence of the MTS resulted in accumulation of NOA1 in the nucleus and increased caspase-dependent apoptosis. We also found that export of NOA1 from the nucleus requires a leptomycin-B sensitive, Crm1-dependent nuclear export signal (NES). Finally, we show that NOA1 is a new substrate of the mitochondrial matrix protease complex ClpXP. Our results uncovered an unexpected, mandatory detour of NOA1 through the nucleolus before uptake into mitochondria. We propose that nucleo-mitochondrial translocation of proteins is more widespread than previously anticipated providing additional means to control protein bioavailability as well as cellular communication between both compartments.     

Reconstitution of nuclear protein transport with semi-intact yeast cells

We have developed an in vitro nuclear protein import reaction from semi- intact yeast cells. The reaction uses cells that have been permeabilized by freeze-thaw after spheroplast formation. Electron microscopic analysis and antibody-binding experiments show that the nuclear envelope remains intact but the plasma membrane is perforated. In the presence of ATP and cytosol derived from yeast or mammalian cells, a protein containing the nuclear localization sequence (NLS) of SV40 large T-antigen is transported into the nucleus. Proteins with mutant NLSs are not imported. In the absence of cytosol, binding of NLS- containing proteins occurs at the nuclear envelope. N-ethylmaleimide treatment of the cytosol as well as antibodies to the nuclear pore protein Nsp1 inhibit import but not binding to the nuclear envelope. Yeast mutants defective in nuclear protein transport were tested in the in vitro import reaction. Semi-intact cells from temperature-sensitive nsp1 mutants failed to import but some binding to the nuclear envelope was observed. On the other hand, no binding and thus no import into nuclei was observed in semi-intact nsp49 cells which are mutated in another nuclear pore protein. Np13 mutants, which are defective for nuclear protein import in vivo, were also deficient in the binding step under the in vitro conditions. Thus, the transport defect in these mutants is at the level of the nucleus and the point at which nuclear transport is blocked can be defined.     

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.     

Nuclear mRNA export requires specific FG nucleoporins for translocation through the nuclear pore complex

Trafficking of nucleic acids and large proteins through nuclear pore complexes (NPCs) requires interactions with NPC proteins that harbor FG (phenylalanine-glycine) repeat domains. Specialized transport receptors that recognize cargo and bind FG domains facilitate these interactions. Whether different transport receptors utilize preferential FG domains in intact NPCs is not fully resolved. In this study, we use a large-scale deletion strategy in Saccharomyces cerevisiae to generate a new set of more minimal pore (mmp) mutants that lack specific FG domains. A comparison of messenger RNA (mRNA) export versus protein import reveals unique subsets of mmp mutants with functional defects in specific transport receptors. Thus, multiple functionally independent NPC translocation routes exist for different transport receptors. Our global analysis of the FG domain requirements in mRNA export also finds a requirement for two NPC substructures-one on the nuclear NPC face and one in the NPC central core. These results pinpoint distinct steps in the mRNA export mechanism that regulate NPC translocation efficiency.     

Nuclear protein accumulation by facilitated transport and intranuclear binding

Nuclear proteins are transported from the cytoplasm into the nucleus via nuclear envelope pore complexes (NPCs). At the molecular level, the mechanisms responsible for this transport remain obscure. However, it is known that, for many proteins, the process requires ATP and proceeds against formidable nucleocytoplasmic concentration gradients. Therefore, the NPC is often thought of as an active transport site. In this article, Philip Paine presents the alternative hypothesis that, on current evidence, protein translocation across the nuclear envelope and accumulation in the nucleus can equally well be explained by facilitated transport through the NPC and subsequent intranuclear binding.