Monopartite Nuclear Localization Signal Research Articles

The PPE2 protein of Mycobacterium tuberculosis is secreted during infection and facilitates mycobacterial survival inside the host

Mycobacterium tuberculosis secrets various effector proteins to evade host immune responses for facilitating its intracellular survival. The bacterial genome encodes several unique PE/PPE family proteins, which have been implicated to play important role in mycobacterial pathogenesis. A member of this family, PPE2 have been shown to contain a monopartite nuclear localization signal (NLS) and a DNA binding domain. In this study, we demonstrate that PPE2 protein is present in the sera of mice infected with either M. smegmatis expressing PPE2 or a clinical strain of M. tuberculosis (CDC1551). It was found that exogenously added PPE2 can permeate through the macrophage cell membrane and eventually translocate into the nucleus which requires the presence of NLS which showed considerable homology to HIV-tat like cell permeable peptides. Exogenously added PPE2 could inhibit NO production and decreased mycobacterial survival in macrophages. PPE2-null mutant of M. tuberculosis failed to inhibit NO production and had poor survival in macrophages which could be rescued by complementation with full-length PPE2. PPE2-null mutants also had poor survival in the lungs of infected mice indicating that PPE2 even when present in the bloodstream can confer a survival advantage to mycobacteria.

ZBTB34 is a hepatocellular carcinoma-associated protein with a monopartite nuclear localization signal.

ZBTB34 is a novel zinc finger protein with an unknown function. In this study, the gene expression and survival prognosis of ZBTB34 were analyzed across tumors based on the TCGA datasets. According to the bioinformatics analysis and qPCR results, liver hepatocellular carcinomas exhibit a high level of ZBTB34 expression. Additionally, the experiment supported the bioinformatics analysis findings that ZBTB34 expression was regulated by miR-125b-5p and that ZBTB34 affected ZBTB10, POLR1B, and AUH expression in HepG2 cells. Biological software analysis further revealed that ZBTB34 contains a monopartite nuclear localization signal (NLS). Arginine and lysine inside the putative NLS were substituted using the alanine-scanning mutagenesis method. The findings showed that the ability of ZBTB34 to enter the nucleus was abolished by the alanine substitution of the sequence 320RGGRARQKRA329 and the mutation of Lys327 and Arg328 residues. ZBTB34 was co-immunoprecipitated with importin α1, importin α3, importin α4, and importin β1, according to the results of the co-immunoprecipitation assay. In conclusion, ZBTB34 is a hepatocellular carcinoma-associated protein with a monopartite NLS. The nuclear import of ZBTB34 is mediated by importin α1, importin α3, importin α4, and importin β1. ZBTB34 performs its biological functions via a putative miR-125b-5p/ZBTB34/(ZBTB10, POLR1B, and AUH) signaling axis in HepG2 cells.

Henipavirus Matrix Protein Employs a Non-Classical Nuclear Localization Signal Binding Mechanism.

Nipah virus (NiV) and Hendra virus (HeV) are highly pathogenic species from the Henipavirus genus within the paramyxovirus family and are harbored by Pteropus Flying Fox species. Henipaviruses cause severe respiratory disease, neural symptoms, and encephalitis in various animals and humans, with human mortality rates exceeding 70% in some NiV outbreaks. The henipavirus matrix protein (M), which drives viral assembly and budding of the virion, also performs non-structural functions as a type I interferon antagonist. Interestingly, M also undergoes nuclear trafficking that mediates critical monoubiquitination for downstream cell sorting, membrane association, and budding processes. Based on the NiV and HeV M X-ray crystal structures and cell-based assays, M possesses a putative monopartite nuclear localization signal (NLS) (residues 82KRKKIR87; NLS1 HeV), positioned on an exposed flexible loop and typical of how many NLSs bind importin alpha (IMPα), and a putative bipartite NLS (244RR-10X-KRK258; NLS2 HeV), positioned within an α-helix that is far less typical. Here, we employed X-ray crystallography to determine the binding interface of these M NLSs and IMPα. The interaction of both NLS peptides with IMPα was established, with NLS1 binding the IMPα major binding site, and NLS2 binding as a non-classical NLS to the minor site. Co-immunoprecipitation (co-IP) and immunofluorescence assays (IFA) confirm the critical role of NLS2, and specifically K258. Additionally, localization studies demonstrated a supportive role for NLS1 in M nuclear localization. These studies provide additional insight into the critical mechanisms of M nucleocytoplasmic transport, the study of which can provide a greater understanding of viral pathogenesis and uncover a potential target for novel therapeutics for henipaviral diseases.

Structural Characterization of Porcine Adeno-Associated Virus Capsid Protein with Nuclear Trafficking Protein Importin Alpha Reveals a Bipartite Nuclear Localization Signal.

Adeno-associated viruses (AAV) are important vectors for gene therapy, and accordingly, many aspects of their cell transduction pathway have been well characterized. However, the specific mechanisms that AAV virions use to enter the host nucleus remain largely unresolved. We therefore aimed to reveal the interactions between the AAV Cap protein and the nuclear transport protein importin alpha (IMPα) at an atomic resolution. Herein we expanded upon our earlier research into the Cap nuclear localization signal (NLS) of a porcine AAV isolate, by examining the influence of upstream basic regions (BRs) towards IMPα binding. Using a high-resolution crystal structure, we identified that the IMPα binding determinants of the porcine AAV Cap comprise a bipartite NLS with an N-terminal BR binding at the minor site of IMPα, and the previously identified NLS motif binding at the major site. Quantitative assays showed a vast difference in binding affinity between the previously determined monopartite NLS, and bipartite NLS described in this study. Our results provide a detailed molecular view of the interaction between AAV capsids and the nuclear import receptor, and support the findings that AAV capsids enter the nucleus by binding the nuclear import adapter IMPα using the classical nuclear localization pathway.

Mutations in the polyhedrin NLS affect the assembly and polyhedral shape of alphabaculovirus occlusion bodies

Alphabaculoviruses produce occlusion bodies (OBs) in the nucleus of the infected cells at the late stage of infection. OBs are mainly composed of a single viral protein called polyhedrin (POLH). Autographa californica multiple nucleopolyhedrovirus (AcMNPV) POLH possesses a monopartite nuclear localization signal sequence (NLS), KRKK, from 32nd to 35th residues. However, the functions of POLH NLS of other alphabaculoviruses remain unknown. Here, POLH NLS mutants of Bombyx mori nucleopolyhedrovirus (BmNPV) were generated and NLS function as well as the relationship between NLS and OB localization or morphology was investigated. Deletion or mutation of BmNPV POLH NLS severely affected POLH and OB intracellular localization. Additionally, viruses in which the arginine residue at the 33rd position of POLH was mutated produced a lower number of OBs, which was presumably due to decreased POLH accumulation in the infected cells. Furthermore, cytoplasmic OBs were morphologically aberrant, even though nuclear OB morphology was normal in the same cell. These results indicate that NLS is required for nuclear localization and efficient accumulation of BmNPV POLH, which heavily affect the number and morphology of OBs.

Identification of Spodoptera frugiperda importin alphas that facilitate the nuclear import of Autographa californica multiple nucleopolyhedrovirus DNA polymerase.

Proteins containing nuclear localization signals (NLSs) are actively transported into the nucleus via the classic importin-α/β-mediated pathway, and NLSs are recognized by members of the importin-α family. Most studies of insect importin-αs have focused on Drosophila to date, little is known about the importin-α proteins in Lepidoptera insects. In this study, we identified four putative importin-α homologues, Spodoptera frugiperda importin-α1 (SfIMA1), SfIMA2, SfIMA4 and SfIMA7, from Sf9 cells. Immunofluorescence analysis showed that SfIMA2, SfIMA4 and SfIMA7 localized to the nucleus, while SfIMA1 distributed in cytoplasm. Additionally, SfIMA4 and SfIMA7 were also detected in the nuclear membrane of Sf9 cells. SfIMA1, SfIMA4 and SfIMA7, but not SfIMA2, were found to associate with the C terminus of AcMNPV DNA polymerase (DNApol) that harbours a typical monopartite NLS and a classic bipartite NLS. Further analysis of protein-protein interactions revealed that SfIMA1 specifically recognizes the bipartite NLS, while SfIMA4 and SfIMA7 bind to both monopartite and bipartite NLSs. Together, our results suggested that SfIMA1, SfIMA4 and SfIMA7 play important roles in the nuclear import of AcMNPV DNApol C terminus in Sf9 cells.

Genomics, molecular and evolutionary perspective of NAC transcription factors.

NAC (NAM, ATAF1,2, and CUC2) transcription factors are one of the largest transcription factor families found in the plants and are involved in diverse developmental and signalling events. Despite the availability of comprehensive genomic information from diverse plant species, the basic genomic, biochemical, and evolutionary details of NAC TFs have not been established. Therefore, NAC TFs family proteins from 160 plant species were analyzed in the current study. Study revealed, Brassica napus (410) encodes highest number and Klebsormidium flaccidum (3) encodes the lowest number of TFs. The study further revealed the presence of NAC TF in the Charophyte algae K. flaccidum. On average, the monocot plants encode higher number (141.20) of NAC TFs compared to the eudicots (125.04), gymnosperm (75), and bryophytes (22.66). Furthermore, our analysis revealed that several NAC TFs are membrane bound and contain monopartite, bipartite, and multipartite nuclear localization signals. NAC TFs were also found to encode several novel chimeric proteins and regulate a complex interactome network. In addition to the presence of NAC domain, several NAC proteins were found to encode other functional signature motifs as well. Relative expression analysis of NAC TFs in A. thaliana revealed root tissue treated with urea and ammonia showed higher level of expression and leaf tissues treated with urea showed lower level of expression. The synonymous codon usage is absent in the NAC TFs and it appears that they have evolved from orthologous ancestors and undergone vivid duplications to give rise to paralogous NAC TFs. The presence of novel chimeric NAC TFs are of particular interest and the presence of chimeric NAC domain with other functional signature motifs in the NAC TF might encode novel functional properties in the plants.

Molecular and functional characterization of the SBP-box transcription factor SPL-CNR in tomato fruit ripening and cell death.

SlSPL-CNR, an SBP-box transcription factor (TF) gene residing at the epimutant Colourless non-ripening (Cnr) locus, is involved in tomato ripening. This epimutant provides a unique model to investigate the (epi)genetic basis of fruit ripening. Here we report that SlSPL-CNR is a nucleus-localized protein with a distinct monopartite nuclear localization signal (NLS). It consists of four consecutive residues ‘ 30KRKR33’ at the N-terminus of the protein. Mutation of the NLS abolishes SlSPL-CNR’s ability to localize in the nucleus. SlSPL-CNR comprises two zinc-finger motifs (ZFMs) within the C-terminal SBP-box domain. Both ZFMs contribute to zinc-binding activity. SlSPL-CNR can induce cell death in tomato and tobacco, dependent on its nuclear localization. However, the two ZFMs have differential impacts on SlSPL-CNR’s induction of severe necrosis or mild necrotic ringspot. NLS and ZFM mutants cannot complement Cnr fruits to ripen. SlSPL-CNR interacts with SlSnRK1. Virus-induced SlSnRK1 silencing leads to reduction in expression of ripening-related genes and inhibits ripening in tomato. We conclude that SlSPL-CNR is a multifunctional protein that consists of a distinct monopartite NLS, binds to zinc, and interacts with SlSnRK1 to affect cell death and tomato fruit ripening.

Development of a novel DsRed-NLS vector with a monopartite classical nuclear localization signal.

The nuclear localization signal (NLS) marks proteins for transport to the nucleus and is used in various applications in many fields. NLSs are used to achieve efficient and stable transport of biomolecules. Previously, commercial vectors used in NLS studies contained three iterations of the NLS sequence, but these sequences can affect experimental results and alter protein function. Here, we investigated a new vector using a single classical NLS sequence with a mutation in pDsRed2-C1-wt to reduce experimental artifacts. In the newly constructed pDsRed2-C1-1NLS vector, the NLS sequence is placed near the multiple cloning sites of pDsRed2-C1-wt, and the multiple cloning site region was designed to facilitate insertion of the desired gene by site-directed mutagenesis. Fluorescent protein expression in the nucleus can be visually confirmed. The results show that the fluorescent protein was bound to the transport protein. The constructed vector had a cell survival rate of 89-95% and a transfection efficiency of 39-56% when introduced into animal cells, which are similar to those of other NLS vectors. Additionally, the constructed NLS vector can be used to demonstrate complementary binding between target proteins, and that the target protein is transported by the NLS transport system. Especially, we show that the vector can be useful for experiments involving the S100A10 gene. In addition, the constructed vector is useful for studies of genes and proteins that show potential for gene therapy or drug delivery applications.

Zika Virus NS5 Forms Supramolecular Nuclear Bodies That Sequester Importin-α and Modulate the Host Immune and Pro-Inflammatory Response in Neuronal Cells.

The Zika virus (ZIKV) epidemic in the Americas was alarming because of its link with microcephaly in neonates and Guillain-Barré syndrome in adults. The unusual pathologies induced by ZIKV infection and the knowledge that the flaviviral nonstructural protein 5 (NS5), the most conserved protein in the flavivirus proteome, can modulate the host immune response during ZIKV infection prompted us to investigate the subcellular localization of NS5 during ZIKV infection and explore its functional significance. A monopartite nuclear localization signal (NLS) sequence within ZIKV NS5 was predicted by the cNLS Mapper program, and we observed localization of ZIKV NS5 in the nucleus of infected cells by immunostaining with specific antibodies. Strikingly, ZIKV NS5 forms spherical shell-like nuclear bodies that exclude DNA. The putative monopartite NLS 390KRPR393 is necessary to direct FLAG-tagged NS5 to the nucleus as the NS5 390ARPA393 mutant protein accumulates in the cytoplasm. Furthermore, coimmunostaining experiments reveal that NS5 localizes with and sequesters importin-α, but not importin-β, in the observed nuclear bodies during virus infection. Structural and biochemical data demonstrate binding of ZIKV NS5 with importin-α and reveal important binding determinants required for their interaction and formation of complexes that give rise to the supramolecular nuclear bodies. Significantly, we demonstrate a neuronal-specific activation of the host immune response to ZIKV infection and a possible role of ZIKV NS5's nuclear localization toward this activation. This suggests that ZIKV pathogenesis may arise from a tissue-specific host response to ZIKV infection.

Nuclear import of the DSCAM ‐cytoplasmic domain drives signaling capable of inhibiting synapse formation

DSCAM and DSCAML1 are immunoglobulin and cell adhesion-type receptors serving important neurodevelopmental functions including control of axon growth, branching, neurite self-avoidance, and neuronal cell death. The signal transduction mechanisms or effectors of DSCAM receptors, however, remain poorly characterized. We used a human ORFeome library to perform a high-throughput screen in mammalian cells and identified novel cytoplasmic signaling effector candidates including the Down syndrome kinase Dyrk1a, STAT3, USP21, and SH2D2A. Unexpectedly, we also found that the intracellular domains (ICDs) of DSCAM and DSCAML1 specifically and directly interact with IPO5, a nuclear import protein of the importin beta family, via a conserved nuclear localization signal. The DSCAM ICD is released by γ-secretase-dependent cleavage, and both the DSCAM and DSCAML1 ICDs efficiently translocate to the nucleus. Furthermore, RNA sequencing confirms that expression of the DSCAM as well as the DSCAML1 ICDs alone can profoundly alter the expression of genes associated with neuronal differentiation and apoptosis, as well as synapse formation and function. Gain-of-function experiments using primary cortical neurons show that increasing the levels of either the DSCAM or the DSCAML1 ICD leads to an impairment of neurite growth. Strikingly, increased expression of either full-length DSCAM or the DSCAM ICD, but not the DSCAML1 ICD, significantly decreases synapse numbers in primary hippocampal neurons. Taken together, we identified a novel membrane-to-nucleus signaling mechanism by which DSCAM receptors can alter the expression of regulators of neuronal differentiation and synapse formation and function. Considering that chromosomal duplications lead to increased DSCAM expression in trisomy 21, our findings may help uncover novel mechanisms contributing to intellectual disability in Down syndrome.

Cloning and molecular characterization of TaERF6, a gene encoding a bread wheat ethylene response factor.

Ethylene response factor proteins are important for regulating gene expression under different stresses. Different isoforms for ERF have previously isolated from bread wheat (Triticum aestivum L.) and related genera and called from TaERF1 to TaERF5. We isolated, cloned and molecular characterized a novel one based on TdERF1, an isoform in durum wheat (Triticum turgidum L.) and called TaERF6. Its cDNA was synthesized, sequenced and compared with genomic sequence to figure out intron and exon regions and determine coding sequence region. The length of TdERF1 gene was 1939 bp and cDNA was 1065 bp including two exons, the first one 259 bp and the second one 806 bp separated by a 874 bp intron with a 111 bp 5'-UTR (untranslated region) and 401 bp 3'-UTR. TaERF6 encodes a 353 amino acids protein with nearly 99% identity to TdERF1. Hydrophobic cluster analysis revealed an N-terminal hydrophobic domain contains a highly conserved motif with the consensus sequence of M [C/L/Y] [G/R] [G/R/P] [A/G/V/L/R] [I/L/R/S/P/Q] [L/I/R/H] and hydrophobic clusters in AP2/ERF domain of which tends to form -sheet. Three monopartite nuclear localization signals also identified in TaERF6 that play important role in getting back into the nucleus. The results showed several putative phosphorylation sites in TaERF6 that a motif from residues 246 to 266, the CMVII-4 motif, was predicted to phosphorylate by different kinase proteins and play important roles in TaERF6 function. Phylogenetic analysis showed 7 clusters (I to VII) and 10 subclusters according to their relatedness in Poaceae family.

Variations in nuclear localization strategies among pol X family enzymes.

Despite the essential roles of pol X family enzymes in DNA repair, information about the structural basis of their nuclear import is limited. Recent studies revealed the unexpected presence of a functional nuclear localization signal (NLS) in DNA polymerase β, indicating the importance of active nuclear targeting, even for enzymes likely to leak into and out of the nucleus. The current studies further explore the active nuclear transport of these enzymes by identifying and structurally characterizing the functional NLS sequences in the three remaining human pol X enzymes: terminal deoxynucleotidyl transferase (TdT), DNA polymerase mu (pol μ) and DNA polymerase lambda (pol λ). NLS identifications are based on Importin α (Impα) binding affinity determined by fluorescence polarization of fluorescein-labeled NLS peptides, X-ray crystallographic analysis of the Impα∆IBB•NLS complexes and fluorescence-based subcellular localization studies. All three polymerases use NLS sequences located near their N-terminus; TdT and pol μ utilize monopartite NLS sequences, while pol λ utilizes a bipartite sequence, unique among the pol X family members. The pol μ NLS has relatively weak measured affinity for Impα, due in part to its proximity to the N-terminus that limits non-specific interactions of flanking residues preceding the NLS. However, this effect is partially mitigated by an N-terminal sequence unsupportive of Met1 removal by methionine aminopeptidase, leading to a 3-fold increase in affinity when the N-terminal methionine is present. Nuclear targeting is unique to each pol X family enzyme with variations dependent on the structure and unique functional role of each polymerase.

Functional domains of SP110 that modulate its transcriptional regulatory function and cellular translocation

BackgroundSP110, an interferon-induced nuclear protein, belongs to the SP100/SP140 protein family. Very recently, we showed that SP110b, an SP110 isoform, controls host innate immunity to Mycobacterium tuberculosis infection by regulating nuclear factor-κB (NF-κB) activity. However, it remains unclear how the structure of SP110 relates to its cellular functions. In this study, we provide experimental data illustrating the protein domains that are responsible for its functions.MethodsWe examined the effects of SP110 isoforms and a series of deletion mutants of SP110 on transcriptional regulation by luciferase reporter assays. We also employed confocal microscopy to determine the cellular distributions of enhanced green fluorescent protein-tagged SP110 isoforms and SP110 mutants. In addition, we performed immunoprecipitation and Western blotting analyses to identify the regions of SP110 that are responsible for protein interactions.ResultsUsing reporter assays, we first demonstrated that SP110 isoforms have different regulatory effects on NF-κB-mediated transcription, supporting the notion that SP110 isoforms may have distinct cellular functions. Analysis of deletion mutants of SP110 showed that the interaction of the N-terminal fragment (amino acids 1–276) of SP110 with p50, a subunit of NF-κB, in the cytoplasm plays a crucial role in the down-regulation of the p50-driven tumor necrosis factor-α (TNFα) promoter activity in the nucleus, while the middle and C-terminal regions of SP110 localize it to various cellular compartments. Surprisingly, a nucleolar localization signal (NoLS) that contains one monopartite nuclear localization signal (NLS) and one bipartite NLS was identified in the middle region of SP110. The identification of a cryptic NoLS in the SP110 suggests that although this protein forms nuclear speckles in the nucleoplasm, it may be directed into the nucleolus to carry out distinct functions under certain cellular conditions.ConclusionsThe findings from this study elucidating the multidomain structure of the SP110 not only identify functional domains of SP110 that are required for transcriptional regulation, cellular translocation, and protein interactions but also implicate that SP110 has additional functions through its unexpected activity in the nucleolus.

Identification and characterization of a doublesex gene which regulates the expression of insulin-like androgenic gland hormone in Fenneropenaeus chinensis

The doublesex and its homologue genes are important regulators of sexual differentiation which are conserved among animal kingdom. In the present study, we reported a doublesex gene (designated as FcDsx) identified from the Chinese shrimp F. chinensis. The gene structure, nucleotide and deduced amino acid sequences of FcDsx were characterized. The results showed that the deduced amino acid sequence of FcDsx had the common features of Dsx proteins, including a doublesex/male abnormal 3 (DM) domain, an oligomerization domain and a predicted monopartite nuclear localization signal. The expression patterns of FcDsx in different tissues and developmental stages were detected. FcDsx exhibited a sex-biased expression patterns in different tissues and its expression level increased along with developmental stages. In addition, its regulation on the expression of FcIAG, a gene important for sexual differentiation of male crustacean, was also analyzed. Putative Dsx binding site was identified on the promoter region of FcIAG and knockdown of FcDsx could reduce the expression of FcIAG, which suggested that FcDsx might be the upstream regulator of FcIAG. The present data indicated that FcDsx gene might involve in shrimp sexual differentiation process.

Baculovirus LEF-11 nuclear localization signal is important for viral DNA replication

Baculovirus LEF-11 is a small nuclear protein that is involved in viral late gene transcription and DNA replication. However, the characteristics of its nuclear localization signal and its impact on viral DNA replication are unknown. In the present study, systemic bioinformatics analysis showed that the baculovirus LEF-11 contains monopartite and bipartite classical nuclear localization signal sequences (cNLSs), which were also detected in a few alphabaculovirus species. Localization of representative LEF-11 proteins of four baculovirus genera indicated that the nuclear localization characteristics of baculovirus LEF-11 coincided with the predicted results. Moreover, Bombyx mori nucleopolyhedrovirus (BmNPV) LEF-11 could be transported into the nucleus during viral infection in the absence of a cNLSs. Further investigations demonstrated that the NLS of BmNPV LEF-11 is important for viral DNA replication. The findings of the present study indicate that the characteristics of the baculovirus LEF-11 protein and the NLS is essential to virus DNA replication and nuclear transport mechanisms.

Transcription factor gene TuGTγ-3 is involved in the stripe rust resistance in Triticum urartu.

Wheat stripe rust, caused by Puccinia striiformis West. f. sp. tritici Eriks. ＆Henn. (Pst), is a serious fungal disease. Identification of new genes associate with stripe rust resistance is important for developing disease resistant wheat cultivars and studying the mechanism of disease resistance. Trihelix is a plant specific transcription factor family, which is involved in regulation of growth and development, morphogenesis, and response to stresses. So far, no study reports on the relationship between the Trihelix family and wheat stripe rust. In this study, a gene in the GTγ subfamily of Trihelix family, designated TuGTγ-3, was cloned from Triticum urartu Tum. (2n=2x=14, AA). The results of sequencing demonstrated that TuGTγ-3 gene consisted of a complete open reading frame (ORF), and its coding sequence was 1329 bp in length, which encoded a protein with 442 amino acids. The predicted molecular weight of this protein was 50.31 kDa and the theoretical isoelectric point was 6.12. Bioinformatic analysis revealed that TuGTγ-3 protein had a monopartite nuclear localization signal (GLPMQKKMRYT), and had neither transmembrane domain nor signal peptide. The conserved trihelix domain, the fourth α-helix and the CC domain were located in the regions of Q115?R187, F234?Y241 and K362?K436, respectively. Dissection of secondary structure showed that TuGTγ-3 protein comprised of 43.89% α-helix, 9.51% extended strand, 9.95% β-turn and 36.65% random coil structures. Based on the BLAST search against the genome database of common wheat from IWGSC, TuGTγ-3 was located on the long arm of chromosome 5A. Transient expression experiment using onion inner epidermal cell showed that the fusion protein TuGTγ-3-GFP distributed mainly in nuclear and slightly in cytoplasm. Expression profiles in different organs indicated that expression level of TuGTγ-3 was much higher in leaves than that in roots or leaf sheaths, and the expression in leaves was extremely up-regulated by infection of the Pst race CYR32. Furthermore, the BSMV-VIGS experiment demonstrated that the transcription factor TuGTγ-3 positively regulated resistance to stripe rust in T. urartu.

DNA polymerase β contains a functional nuclear localization signal at its N-terminus.

DNA polymerase β (pol β) requires nuclear localization to fulfil its DNA repair function. Although its small size has been interpreted to imply the absence of a need for active nuclear import, sequence and structural analysis suggests that a monopartite nuclear localization signal (NLS) may reside in the N-terminal lyase domain. Binding of this domain to Importin α1 (Impα1) was confirmed by gel filtration and NMR studies. Affinity was quantified by fluorescence polarization analysis of a fluorescein-tagged peptide corresponding to pol β residues 2–13. These studies indicate high affinity binding, characterized by a low micromolar Kd, that is selective for the murine Importin α1 (mImpα1) minor site, with the Kd strengthening to ∼140 nM for the full lyase domain (residues 2–87). A further reduction in Kd obtains in binding studies with human Importin α5 (hImpα5), which in some cases has been demonstrated to bind small domains connected to the NLS. The role of this NLS was confirmed by fluorescent imaging of wild-type and NLS-mutated pol β(R4S,K5S) in mouse embryonic fibroblasts lacking endogenous pol β. Together these data demonstrate that pol β contains a specific NLS sequence in the N-terminal lyase domain that promotes transport of the protein independent of its interaction partners. Active nuclear uptake allows development of a nuclear/cytosolic concentration gradient against a background of passive diffusion.

Nuclear import of dimerized ribosomal protein Rps3 in complex with its chaperone Yar1.

After their cytoplasmic synthesis, ribosomal proteins need to be transported into the nucleus, where they assemble with ribosomal RNA into pre-ribosomal particles. Due to their physicochemical properties, they need protection from aggregation on this path. Newly synthesized ribosomal protein Rps3 forms a dimer that is associated with one molecule of its specific chaperone Yar1. Here we report that redundant pathways contribute to the nuclear import of Rps3, with the classical importin α/β pathway (Kap60/Kap95 in yeast) constituting a main import route. The Kap60/Kap95 heterodimer mediates efficient nuclear import of Rps3 by recognition of an N-terminal monopartite nuclear localization signal (NLS). This Rps3-NLS is located directly adjacent to the Yar1-binding site and, upon binding of Kap60 to Rps3, Yar1 is displaced from the ribosomal protein in vitro. While Yar1 does not directly interact with Kap60 in vitro, affinity purifications of Yar1 and Rps3, however, revealed that Kap60 is present in the Rps3/Yar1 complex in vivo. Indeed we could reconstitute such a protein complex containing Rps3 and both Yar1 and Kap60 in vitro. Our data suggest that binding of Yar1 to one N-domain and binding of Kap60 to the second N-domain of dimerized Rps3 orchestrates import and protection of the ribosomal protein.

Tumor suppressor RYBP harbors three nuclear localization signals and its cytoplasm-located mutant exerts more potent anti-cancer activities than corresponding wild type

Ectopically expressed Ring1 and YY1 binding protein (RYBP) induces tumor cell apoptosis through promoting the formation of the death-inducing signaling complex (DISC) in the cytoplasm. However, transiently overexpressed as well as endogenous RYBP in tumor tissues were observed to be mainly located in the nucleus while that in adjacent non-tumor tissues distributed majorly in the cytoplasm. Currently, we do not know the nuclear localization signals and biological function of different subcellular location of RYBP. In this study, we employed bioinformatic analysis, deletion, point mutation, enhanced green fluorescence protein (EGFP) fusion and others, to investigate the elements responsible for RYBP nuclear import and to explore the anti-tumor activities of cytoplasm- and nuclear-located RYBP. Herein, we identified three functional monopartite nuclear localization signals (NLSs), all of which located at the N-terminus of RYBP. Through four basic amino acid replacements within the NLSs, we obtained a cytoplasm-located RYBP mutant (RYBPmut). Compared with wild-type counterpart, RYBPmut exhibited more potent abilities to bind to caspase 8, to prevent MDM2-mediated polyubiquitination and degradation of p53, thereby leading to its stabilization. Further investigation revealed that, in contrast to its wild type, RYBPmut showed more potentials to inhibit tumor cell proliferation and to induce apoptosis, in both p53-dependent and -independent manner. Collectively, our current study revealed the molecular mechanism responsible for RYBP nuclear translocation, and provided evidences to support that RYBPmut could be a more promising candidate agent for cancer treatment.