Recognition Of Nuclear Localization Signal Research Articles

Structure-based nuclear import mechanism of histones H3 and H4 mediated by Kap123.

Kap123, a major karyopherin protein of budding yeast, recognizes the nuclear localization signals (NLSs) of cytoplasmic histones H3 and H4 and translocates them into the nucleus during DNA replication. Mechanistic questions include H3- and H4-NLS redundancy toward Kap123 and the role of the conserved diacetylation of cytoplasmic H4 (K5ac and K12ac) in Kap123-mediated histone nuclear translocation. Here, we report crystal structures of full-length Kluyveromyces lactis Kap123 alone and in complex with H3- and H4-NLSs. Structures reveal the unique feature of Kap123 that possesses two discrete lysine-binding pockets for NLS recognition. Structural comparison illustrates that H3- and H4-NLSs share at least one of two lysine-binding pockets, suggesting that H3- and H4-NLSs are mutually exclusive. Additionally, acetylation of key lysine residues at NLS, particularly H4-NLS diacetylation, weakens the interaction with Kap123. These data support that cytoplasmic histone H4 diacetylation weakens the Kap123-H4-NLS interaction thereby facilitating histone Kap123-H3-dependent H3:H4/Asf1 complex nuclear translocation.

Regulation of mitotic spindle assembly factor NuMA by Importin-β.

Ran-guanosine triphosphatase orchestrates mitotic spindle assembly by modulation of the interaction between Importin-α/-β and spindle assembly factors (SAFs). The inhibition of SAFs performed by importins needs to be done without much sequestration from abundant nuclear localization signal (NLS) -containing proteins. However, the molecular mechanisms that determine NLS-binding selectivity and that inhibit activity of Importin-β-regulated SAFs (e.g., nuclear mitotic apparatus protein [NuMA]) remain undefined. Here, we present a crystal structure of the Importin-α-NuMA C terminus complex showing a novel binding pattern that accounts for selective NLS recognition. We demonstrate that, in the presence of Importin-α, Importin-β inhibits the microtubule-binding function of NuMA. Further, we have identified a high-affinity microtubule-binding region that lies carboxyl-terminal to the NLS, which is sterically masked by Importin-β on being bound by Importin-α. Our study provides mechanistic evidence of how Importin-α/-β regulates the NuMA functioning required for assembly of higher-order microtubule structures, further illuminating how Ran-governed transport factors regulate diverse SAFs and accommodate various cell demands.

Nuclear import by karyopherin-βs: Recognition and inhibition

Proteins in the karyopherin-β family mediate the majority of macromolecular transport between the nucleus and the cytoplasm. Eleven of the 19 known human karyopherin-βs and 10 of the 14S. cerevisiae karyopherin-βs mediate nuclear import through recognition of nuclear localization signals or NLSs in their cargos. This receptor-mediated process is essential to cellular viability as proteins are translated in the cytoplasm but many have functional roles in the nucleus. Many known karyopherin-β-cargo interactions were discovered through studies of the individual cargos rather than the karyopherins, and this information is thus widely scattered in the literature. We consolidate information about cargos that are directly recognized by import-karyopherin-βs and review common characteristics or lack thereof among cargos of different import pathways. Knowledge of karyopherin-β-cargo interactions is also critical for the development of nuclear import inhibitors and the understanding of their mechanisms of inhibition. This article is part of a Special Issue entitled: Regulation of Signaling and Cellular Fate through Modulation of Nuclear Protein Import.

Karyopherin α7 (KPNA7), a divergent member of the importin α family of nuclear import receptors

BackgroundClassical nuclear localization signal (NLS) dependent nuclear import is carried out by a heterodimer of importin α and importin β. NLS cargo is recognized by importin α, which is bound by importin β. Importin β mediates translocation of the complex through the central channel of the nuclear pore, and upon reaching the nucleus, RanGTP binding to importin β triggers disassembly of the complex. To date, six importin α family members, encoded by separate genes, have been described in humans.ResultsWe sequenced and characterized a seventh member of the importin α family of transport factors, karyopherin α 7 (KPNA7), which is most closely related to KPNA2. The domain of KPNA7 that binds Importin β (IBB) is divergent, and shows stronger binding to importin β than the IBB domains from of other importin α family members. With regard to NLS recognition, KPNA7 binds to the retinoblastoma (RB) NLS to a similar degree as KPNA2, but it fails to bind the SV40-NLS and the human nucleoplasmin (NPM) NLS. KPNA7 shows a predominantly nuclear distribution under steady state conditions, which contrasts with KPNA2 which is primarily cytoplasmic.ConclusionKPNA7 is a novel importin α family member in humans that belongs to the importin α2 subfamily. KPNA7 shows different subcellular localization and NLS binding characteristics compared to other members of the importin α family. These properties suggest that KPNA7 could be specialized for interactions with select NLS-containing proteins, potentially impacting developmental regulation.

Binding of p110 Retinoblastoma Protein Inhibits Nuclear Import of Simian Virus SV40 Large Tumor Antigen

Nuclear import of the simian virus 40 large tumor antigen (T-ag) is dependent on its nuclear localization signal (NLS) within amino acids 126-132 that is recognized by the importin alpha/beta1 heterodimer, as well as a protein kinase CK2 site at serine 112 upstream of the NLS, which enhances the interaction approximately 50-fold. Here we show for the first time that T-ag nuclear import is negatively regulated by N-terminal sequences (amino acids 102-110), which represent the binding site (BS) for the retinoblastoma (Rb) tumor suppressor protein (p110(Rb)). Quantitative confocal laser scanning microscopic analysis of the transport properties of T-ag constructs with or without Rb binding site mutations in living transfected cells or in a reconstituted nuclear transport system indicates that the presence of the RbBS significantly reduces nuclear accumulation of T-ag. A number of approaches, including the analysis of T-ag nuclear import in an isogenic cell pair with and without functional p110(Rb) implicate p110(Rb) binding as being responsible for the reduced nuclear accumulation, with the Ser(106) phosphorylation site within the RbBS appearing to enhance the inhibitory effect. Immunoprecipitation experiments confirmed association of T-ag and p110(Rb) and dependence thereof on negative charge at Ser(106). The involvement of p110(Rb) in modulating T-ag nuclear transport has implications for the regulation of nuclear import of other proteins from viruses of medical significance that interact with p110(Rb), and how this may relate to transformation.

Probing Nuclear Localization Signal-Importin α Binding Equilibria in Living Cells

The regulated process of protein import into the nucleus of a eukaryotic cell is mediated by specific nuclear localization signals (NLSs) that are recognized by protein-import receptors. In this study, we present fluorescence-based methods to quantitatively address the physicochemical details of NLS recognition by the receptor protein importin alpha (Impalpha) in living cells. First, by combining fluorescence recovery after photobleaching measurements and protein-concentration calibration, we quantitatively define nuclear import saturability and afford an affinity value for NLS-Impalpha binding. Second, by fluorescence lifetime imaging microscopy, we directly monitor the occurrence of NLS-Impalpha interaction and measure its effective dissociation constant (K(D)) in the actual cellular environment. Our kinetic and thermodynamic analyses independently indicate that the subsaturation of Impalpha with the expressed NLS cargo regulates nuclear import rates in living cells, in contrast to what can be predicted on the basis of available in vitro data. Finally, our experiments also provide evidence for the regulation of nuclear import mediated by the intrasteric importin beta-binding domain of Impalpha and yield the first estimate of its autoinhibition energy in living cells.

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.

Molecular Basis for the Recognition of a Nonclassical Nuclear Localization Signal by Importin β

Nuclear import of proteins containing a classical nuclear localization signal (NLS) involves NLS recognition by importin α, which associates with importin β via the IBB domain. Other proteins, including parathyroid hormone-related protein (PTHrP), are imported into the nucleus by direct interaction with importin β. We solved the crystal structure of a fragment of importin β-1 (1–485) bound to the nonclassical NLS of PTHrP. The structure reveals a second extended cargo binding site on importin β distinct from the IBB domain binding site. Using a permeabilized cell import assay we demonstrate that importin β (1–485) can import PTHrP-coupled cargo in a Ran-dependent manner. We propose that this region contains a prototypical nuclear import receptor domain, which could have evolved into the modern importin β superfamily.

Dissection of a Nuclear Localization Signal

The regulated process of protein import into the nucleus of a eukaryotic cell is mediated by specific nuclear localization signals (NLSs) that are recognized by protein import receptors. This study seeks to decipher the energetic details of NLS recognition by the receptor importin alpha through quantitative analysis of variant NLSs. The relative importance of each residue in two monopartite NLS sequences was determined using an alanine scanning approach. These measurements yield an energetic definition of a monopartite NLS sequence where a required lysine residue is followed by two other basic residues in the sequence K(K/R)X(K/R). In addition, the energetic contributions of the second basic cluster in a bipartite NLS ( approximately 3 kcal/mol) as well as the energy of inhibition of the importin alpha importin beta-binding domain ( approximately 3 kcal/mol) were also measured. These data allow the generation of an energetic scale of nuclear localization sequences based on a peptide's affinity for the importin alpha-importin beta complex. On this scale, a functional NLS has a binding constant of approximately 10 nm, whereas a nonfunctional NLS has a 100-fold weaker affinity of 1 microm. Further correlation between the current in vitro data and in vivo function will provide the foundation for a comprehensive quantitative model of protein import.

Nuclear localization signal recognition causes release of importin-α from aggregates in the cytosol

Importin-α is a cytosolic receptor that recognizes classical Nuclear Localization Signals (NLSs) and mediates import into the nucleus. We have used a number of methods to investigate the aggregation state of Xenopus importin-α both as a recombinant, purified protein and in cytosolic extracts. We have found that recombinant importin-α aggregates at a protein concentration similar to that estimated to be present in the Xenopus cytoplasm, and that the importin-α aggregation is relieved by NLS peptide binding, with the importin-α then binding the NLS as a monomer. We have also found that in HeLa cytosolic extracts, importin-α is present in an aggregated form. Similarly to the purified importin-α aggregation, NLS peptides relieve the aggregation of importin-α in the cytosol. These observations indicate that aggregation of importin-α in the cytosol may be an intrinsic property of the import receptor and may be functionally related to NLS binding.Our results suggest a novel mechanism for NLS recognition, whereby NLSs mediate disassembly of importin-α aggregates in the cytosol.

Mutual exclusivity of DNA binding and nuclear localization signal recognition by the yeast transcription factor GAL4: implications for nonviral DNA delivery.

A novel approach to nonviral DNA delivery is the use of combinations of DNA-binding proteins such as the yeast transcriptional activator GAL4 and plasmid DNA containing the specific binding sequence of the DNA-binding protein inserted within it, in addition to the gene of interest to be transferred into target cells. The amino terminal 147 amino acids of GAL4 contain a DNA-binding domain that has been shown to bind specifically to a 17 bp nucleotide recognition sequence, while the amino terminal 74 amino acids have been shown to be sufficient to target large heterologous proteins to the nucleus. Although it has been previously exploited as a gene transfer vehicle, the exact relationship between GAL4's DNA binding and nuclear targeting activities has not been investigated. Using gel mobility shift assays and ELISA-based binding assays, this study examines this issue directly, establishing the mutual exclusivity of the DNA-binding and nuclear targeting activities of GAL4. We demonstrate that GAL4(1-147) can specifically enhance transfection of plasmids containing the 17 bp recognition sequence. Interestingly, we found that the nuclear localization signal (NLS) of GAL4 is distinct from conventional NLSs, such as those of the SV40 large tumor antigen and bipartite NLSs, in that it is recognized exclusively by the nuclear pore targeting beta-subunit of the NLS-receptor importin complex, rather than the alpha-subunit. Specific binding to DNA was blocked by beta-subunit binding, while the converse was also true, making the GAL4-NLS novel in being regulated by DNA binding; this may play an important role in effecting release of GAL4 from the beta-subunit following transport through the nuclear pore. This study encompasses the first direct analysis of NLS recognition/accessibility in vehicles for nonviral DNA transfer, with the results having relevance to the use of GAL4 and comparable DNA-binding proteins in such vehicles.

The cAMP-dependent Protein Kinase Site (Ser312) Enhances Dorsal Nuclear Import through Facilitating Nuclear Localization Sequence/Importin Interaction

Control over the nuclear import of transcription factors (TFs) represents a level of gene regulation integral to cellular processes such as differentiation and transformation. The Drosophila TF Dorsal shares with other rel TF family members the fact that it contains a phosphorylation site for the cAMP-dependent protein kinase (PKA) 22 amino acids N-terminal to the nuclear localization signal (NLS) at amino acids 335-340. This study examines for the first time the nuclear import kinetics of Dorsal fusion proteins in rat hepatoma cells in vivo and in vitro. Nuclear uptake was found to be not only NLS-dependent, but also strongly dependent on the PKA site, whereby substitution of Ser312 by either Ala or Glu using site-directed mutagenesis severely reduced nuclear accumulation. Exogenous cAMP or PKA catalytic subunit significantly enhanced the nuclear import of wild-type proteins both in vivo and in vitro. Using a direct binding assay, the molecular basis of PKA site enhancement of Dorsal fusion protein nuclear import was determined to be PKA site-mediated modulation of NLS recognition by the importin 58/97 complex. The physiological relevance of these results is supported by the observation that Drosophila embryos expressing PKA site Dorsal mutant variants were impaired in development. We conclude that the Dorsal NLS and PKA site constitute a phosphorylation-regulated NLS essential to Dorsal function and able to function in heterologous mammalian cell systems, where phosphorylation modulates the affinity of NLS recognition by importin.

Differential Modes of Nuclear Localization Signal (NLS) Recognition by Three Distinct Classes of NLS Receptors

The targeting of karyophilic proteins to nuclear pores is mediated via the formation of a nuclear pore-targeting complex, through the interaction of nuclear localization signal (NLS) with its NLS receptor. Recently, a novel human protein, Qip1, was identified from a yeast two-hybrid system with DNA helicase Q1. This study demonstrates that Qip1 is a novel third class of NLS receptor that efficiently recognizes the NLS of the helicase Q1. Moreover, the data obtained in this study show that the specific interaction between Qip1 and the NLS of the helicase Q1 requires its upstream sequence of the minimal essential NLS. By using purified recombinant proteins alone in the digitonin-permeabilized cell-free transport system, it was demonstrated that the two known human NLS receptors, Rch1 and NPI-1, are able to transport all the tested NLS substrates into the nucleus, while Qip1 most efficiently transports the helicase Q1-NLS substrates, which contain its upstream sequence in so far as we have examined the system. Furthermore, in HeLa cell crude cytosol, it was found that endogenous Rch1 binds to all the tested NLS substrates, while the binding of endogenous NPI-1 is restricted to only some NLSs, despite the fact that NPI-1 itself shows binding activity to a variety of NLSs. These results indicate that at least three structurally and functionally distinct NLS receptors exist in the human single cell population, and suggest that the nuclear import of karyophilic proteins may be controlled in a complex manner at the NLS recognition step by the existence of a variety of NLS receptors with various specificities to each NLS.

Regulatory Roles of the Nuclear Envelope

Roles of the nuclear envelope are considered in the regulation of nuclear protein import, ribonucleoprotein export, and coupling of DNA replication to the cell cycle. First, evidence is discussed that indicates that neutral and acidic amino acids can be important in nuclear localization signals as well as the widely acknowledged basic amino acids. Second, the recognition of nuclear localization signals by their receptor “importin” is discussed, focusing on the different roles of the two subunits of importin. Third, a role for the α subunit of importin in RNP export is considered together with the question of how the direction of traffic through nuclear pores is determined. The final part of this article considers evidence that the nuclear membrane prevents reinitiation of DNA replication in Xenopus eggs, by excluding a “licensing factor” that is essential for DNA replication. Replication licensing in Xenopus appears to involve several proteins including the MCM (minichromosome maintenance) complex and ORC, the origin recognition complex, which must bind before the MCM complex can bind to chromatin.

Evidence that the FK506-binding immunophilin heat shock protein 56 is required for trafficking of the glucocorticoid receptor from the cytoplasm to the nucleus.

The FK506-binding immunophilin hsp56 (FKBP52) is one of several chaperone proteins associated with untrasformed steroid receptors in a multiprotein heterocomplex. The function of heat shock protein 56 (hsp56) with respect to receptor action is unknown. hsp56 is not required for glucocorticoid receptor heterocomplex assembly or for proper folding of the receptor hormone-binding domain into a high affinity steroid-binding conformation. In intact cells, the majority of the hsp56 is located in the nucleus, with a minority colocalizing with microtubules in the cytoplasm. hsp56 contains a conserved negatively charged domain that we speculate might serve as a nuclear localization signal recognition sequence. Here we show that injection of an antibody raised against this negative sequence into intact L cells impedes subsequent dexamethasone-mediated shift of the glucocorticoid receptor into the nucleus. Nonimmune rabbit serum and an antibody raised against another site on hsp56 do not affect receptor movement. Inhibition of receptor movement by the 419 antibody against the negative sequence is blocked by preincubation with purified hsp56, but not by preincubation with purified hsp90, hsp70, or BSA. These observations are consistent with the possibility that hsp56 is involved in receptor trafficking to the nucleus, possibly functioning as the nuclear localization signal recognition protein. Receptor trafficking to the nucleus is not affected by FK506, indicating that the peptidylprolyl isomerase activity of hsp56 is not involved.

Distinct functions for the two importin subunits in nuclear protein import.

The import of nuclear proteins proceeds through the nuclear pore complex and requires nuclear localization signals (NLSs), energy and soluble factors, namely importin-alpha (M(r) 60K), importin-beta (90K) and Ran. Importin-alpha is primarily responsible for NLS recognition and is a member of a protein family that includes the essential yeast nuclear pore protein SRP1p (ref. 16). As the first event, the complex of importin-alpha and importin-beta binds the import substrate in the cytosol. Here we show that this nuclear pore targeting complex initially docks as a single entity to the nuclear pore via importin-beta. Then the energy-dependent, Ran-mediated translocation through the pore results in the accumulation of import substrate and importin-alpha in the nucleus. In contrast, importin-beta accumulates at the nuclear envelope, but not in the nucleoplasm. Immunoelectron microscopy detects importin-beta on both sides of the nuclear pore. This suggests that the nuclear pore targeting complex might move as a single entity from its initial docking site through the central part of the nuclear pore before it disassembles on the nucleoplasmic side.

Nuclear localization signal binding proteins in higher plant nuclei.

The import of proteins into the nucleus is a vital process that is mediated by proteins which specifically recognize nuclear localization signals (NLSs). These factors have not been identified in plants. Previously, we demonstrated that higher plants possess a low-affinity binding site at the nuclear pore that specifically binds to several classes of functional NLSs. By the use of crosslinking reagents and a radiolabeled peptide to the bipartite NLS from the endogenous plant transcription factor Opaque2, two NLS binding proteins (NBPs) of 50-60 kDa and at least two NBPs of 30-40 kDa were identified. Competition studies indicated that labeling was specific for the functional NLS but not a mutant NLS impaired in vivo or a peptide unrelated to NLSs. Also, the apparent dissociation constant (100-300 microM) for labeling was similar to that of the binding site. Proteins of similar mass were labeled with two different crosslinking reagents, and concentration and time studies indicated that these NBPs were distinct proteins and not aggregates. Treatment with salt, detergent, or urea before or during NLS binding demonstrated that the properties of the binding site and the NBPs were identical. This tight correlation strongly indicates that some or all of the NBPs constitute the nuclear pore binding site. Overall, our results indicate that some components of NLS recognition are located at the nuclear pores in higher plants.

Nuclear transport: a guide to import receptors

After synthesis in the cytoplasm, nuclear proteins traverse the nuclear envelope as a result of the specific recognition of nuclear localization signals by import. Various approaches have now uncovered a range of proteins with at least some of the characteristics expected of import receptors. This article focuses on early steps in the nuclear import of proteins and surveys the recently identified candidate import receptors.

A yeast gene important for protein assembly into the endoplasmic reticulum and the nucleus has homology to DnaJ, an Escherichia coli heat shock protein.

When nuclear localization sequences (termed NLS) are placed at the N terminus of cytochrome c1, a mitochondrial inner membrane protein, the resulting hybrid proteins do not assemble into mitochondria when synthesized in the yeast Saccharomyces cerevisiae. Cells lacking mitochondrial cytochrome c1, but expressing the hybrid NLS-cytochrome c1 proteins, are unable to grow on glycerol since the hybrid proteins are associated primarily with the nucleus. A similar hybrid protein with a mutant NLS is transported to and assembled into the mitochondria. To identify proteins that might be involved in recognition of nuclear localization signals, we isolated conditional-lethal mutants (npl, for nuclear protein localization) that missorted NLS-cytochrome c1 to the mitochondria, allowing growth on glycerol. The gene corresponding to one complementation group (NPL1) encodes a protein with homology to DnaJ, an Escherichia coli heat shock protein. npl1-1 is allelic to sec63, a gene that affects transit of nascent secretory proteins across the endoplasmic reticulum. Rothblatt, J. A., R. J. Deshaies, S. L. Sanders, G. Daum, and R. Schekman. 1989. J. Cell Biol. 109:2641-2652. The npl1 mutants reported here also weakly affect translocation of preprocarboxypeptidaseY across the ER membrane. A normally nuclear hybrid protein containing a NLS fused to invertase and a nucleolar protein are not localized to the nucleus in npl1/sec63 cells at the nonpermissive temperature. Thus, NPL1/SEC63 may act at a very early common step in localization of proteins to the nucleus and the ER. Alternatively, by affecting ER and nuclear envelope assembly, npl1 may indirectly alter assembly of proteins into the nucleus.