Permeabilized mammalian cells as an experimental system for nuclear import of geminiviral karyophilic proteins and of synthetic peptides derived from their nuclear localization signal regions

The plant-infecting geminiviruses deliver their genome and viral proteins into the host cell nucleus. Members of the family Geminiviridae possess either a bipartite genome composed of two ∼2.6 kb DNAs or a monopartite genome of ∼3.0 kb DNA. The bipartite genome of Bean dwarf mosaic virus (BDMV) encodes several karyophilic proteins, among them the capsid protein (CP) and BV1 (nuclear shuttle protein). A CP is also encoded by the monopartite genome of Tomato yellow leaf curl virus (TYLCV). Here, an in vitro assay system was used for direct demonstration of nuclear import of BDMV BV1 and TYLCV CP, as well as synthetic peptides containing their putative nuclear localization signals (NLSs). Full-length recombinant BDMV BV1 and TYLCV CP mediated import of conjugated fluorescently labelled BSA molecules into nuclei of permeabilized mammalian cells. Fluorescently labelled and biotinylated BSA conjugates bearing the synthetic peptides containing aa 3–20 of TYLCV CP (CP-NLS) or aa 84–106 of BDMV BV1 (BV1-NLS) were also imported into the nuclei of permeabilized cells. This import was blocked by the addition of unlabelled BSA–NLS peptide conjugates or excess unlabelled free NLS peptides. The CP- and BV1-NLS peptides also mediated nuclear import of fluorescently labelled BSA molecules into the nuclei of microinjected mesophyll cells of Nicotiana benthamiana leaves, demonstrating their biological function in intact plant tissue. BV1-NLS and CP-NLS were shown to mediate specific binding to importin α, both in vitro and in vivo. These results are consistent with a common nuclear-import pathway for CP and BV1, probably via importin α.

Download Full-text

005.Efficiency of nuclear import of the chromatin-remodelling factor SRY is critical for sex determination

Reproduction Fertility and Development ◽

10.1071/srb05abs005 ◽

2005 ◽

Vol 17 (9) ◽

pp. 64

Author(s):

D. A. Jans ◽

G. Kaur ◽

I. K. H. Poon ◽

A. Delluc-Clavieries ◽

K. M. Wagstaff

Keyword(s):

Sex Determination ◽

Nuclear Localization ◽

Nuclear Import ◽

Sex Reversal ◽

Nuclear Accumulation ◽

Missense Mutations ◽

Nuclear Localization Signals ◽

Testicular Cells ◽

Xy Sex Reversal

15% of cases of human XY sex reversal are due to mutations in SRY (sex determining region on the Y chromosome), many of which map to one of SRY’s two independently acting nuclear localization signals (NLSs) flanking its DNA binding domain. The C-terminal NLS (C-NLS) targets SRY to the nucleus through a ‘conventional’ pathway dependent on the nuclear import receptor importin-β (Imp-β). No importin has been shown to bind the N-terminal NLS (N-NLS), but it is known to interact with the Ca2+-binding protein calmodulin (CaM). We examined seven distinct missense mutations in the SRY NLSs from XY sex-reversed human females for effects on nuclear import and ability to interact with CaM/Imp-β1. All mutations were found to result in reduced nuclear localization in transfected testicular cells compared to wild type. The CaM antagonist, calmidazolium chloride (CDZ), was found to significantly reduce SRY nuclear accumulation, indicating a dependence of SRY nuclear import on CaM. Intriguingly, N-NLS mutants were resistant to CDZ’s effects, implying a loss of interaction with CaM; this was confirmed directly by in vitro binding experiments using recombinantly expressed protein. Either impaired CaM or Imp-β1 binding can thus be the basis of sex-reversal in human patients. Our results implicate a CaM-dependent nuclear import pathway for SRY mediated by the N-NLS that, together with the C-NLS, is required to achieve threshold levels of SRY in the nucleus for male sex determination.

Download Full-text

The Arginine-Rich Domains Present in Human Immunodeficiency Virus Type 1 Tat and Rev Function as Direct Importin β-Dependent Nuclear Localization Signals

Molecular and Cellular Biology ◽

10.1128/mcb.19.2.1210 ◽

1999 ◽

Vol 19 (2) ◽

pp. 1210-1217 ◽

Cited By ~ 274

Author(s):

Ray Truant ◽

Bryan R. Cullen

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Nuclear Localization ◽

Nuclear Import ◽

Nuclear Localization Signals ◽

Immunodeficiency Virus ◽

Importin Α

ABSTRACT Protein nuclear import is generally mediated by basic nuclear localization signals (NLSs) that serve as targets for the importin α (Imp α) NLS receptor. Imp α is in turn bound by importin β (Imp β), which targets the resultant protein complex to the nucleus. Here, we report that the arginine-rich NLS sequences present in the human immunodeficiency virus type 1 regulatory proteins Tat and Rev fail to interact with Imp α and instead bind directly to Imp β. Using in vitro nuclear import assays, we demonstrate that Imp α is entirely dispensable for Tat and Rev nuclear import. In contrast, Imp β proved both sufficient and necessary, in that other β-like import factors, such as transportin, were unable to support Tat or Rev nuclear import. Using in vitro competition assays, it was demonstrated that the target sites on Imp β for Imp α, Tat, and Rev binding either are identical or at least overlap. The interaction of Tat and Rev with Imp β is also similar to Imp α binding in that it is inhibited by RanGTP but not RanGDP, a finding that may in part explain why the interaction of the Rev nuclear RNA export factor with target RNA species is efficient in the cell nucleus yet is released in the cytoplasm. Together, these studies define a novel class of arginine-rich NLS sequences that are direct targets for Imp β and that therefore function independently of Imp α.

Download Full-text

Identification and Functional Characterization of a Novel Nuclear Localization Signal Present in the Yeast Nab2 Poly(A)+ RNA Binding Protein

Molecular and Cellular Biology ◽

10.1128/mcb.18.3.1449 ◽

1998 ◽

Vol 18 (3) ◽

pp. 1449-1458 ◽

Cited By ~ 46

Author(s):

Ray Truant ◽

Robert A. Fridell ◽

R. Edward Benson ◽

Hal Bogerd ◽

Bryan R. Cullen

Keyword(s):

Nuclear Localization ◽

Nuclear Import ◽

Nuclear Localization Signal ◽

Nuclear Export ◽

Mammalian Cells ◽

Rna Binding ◽

Yeast Cells ◽

Vitro Assay ◽

Localization Signal

ABSTRACT The nuclear import of proteins bearing a basic nuclear localization signal (NLS) is dependent on karyopherin α/importin α, which acts as the NLS receptor, and karyopherin β1/importin β, which binds karyopherin α and mediates the nuclear import of the resultant ternary complex. Recently, a second nuclear import pathway that allows the rapid reentry into the nucleus of proteins that participate in the nuclear export of mature mRNAs has been identified. In mammalian cells, a single NLS specific for this alternate pathway, the M9 NLS of heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), has been described. The M9 NLS binds a transport factor related to karyopherin β1, termed karyopherin β2 or transportin, and does not require a karyopherin α-like adapter protein. A yeast homolog of karyopherin β2, termed Kap104p, has also been described and proposed to play a role in the nuclear import of a yeast hnRNP-like protein termed Nab2p. Here, we define a Nab2p sequence that binds to Kap104p and that functions as an NLS in both human and yeast cells despite lacking any evident similarity to basic or M9 NLSs. Using an in vitro nuclear import assay, we demonstrate that Kap104p can direct the import into isolated human cell nuclei of a substrate containing a wild-type, but not a defective mutant, Nab2p NLS. In contrast, other NLSs, including the M9 NLS, could not function as substrates for Kap104p. Surprisingly, this in vitro assay also revealed that human karyopherin β1, but not the Kap104p homolog karyopherin β2, could direct the efficient nuclear import of a Nab2p NLS substrate in vitro in the absence of karyopherin α. These data therefore identify a novel NLS sequence, active in both yeast and mammalian cells, that is functionally distinct from both basic and M9 NLS sequences.

Download Full-text

Importin alpha and nonclassical nuclear localization signal

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s205327331408365x ◽

2014 ◽

Vol 70 (a1) ◽

pp. C1634-C1634

Author(s):

Chiung-Wen Chang ◽

Rafael Couñago ◽

Simon Williams ◽

Mikael Bodén ◽

Boštjan Kobe

Keyword(s):

Binding Site ◽

Nuclear Localization ◽

Nuclear Import ◽

Specific Binding ◽

Site Specific ◽

Nuclear Localization Signals ◽

Random Peptide ◽

Importin Α ◽

Minor Site ◽

Helical Turn

In the classical nuclear import pathway, the specific recognition between the nuclear receptor (importin-α) and the nuclear localization signals (NLSs) plays an essential role on facilitating the cargo import process. Importin-α has two separate NLS-binding sites (the major and the minor sites), accommodate NLSs, comprising of one (monopartite) or two clusters (bipartite) of basic residues connected by a 10 - 12 residue linker. The major NLS-binding site is the preferential binding site for most of the monopartite NLSs characterized to date. By screening random peptide libraries using importin-α variants as bait, the bound NLS sequences could be divided into six classes [1]. The class-3 minor site-specific NLSs and class-5 plant-specific NLSs feature a shorter basic cluster. The molecular basis of the specific binding between these non-classical NLSs and importin-α was not known and in particular, there was a lack of crystal structures of plant importin-α. Here, we present the first crystal structure of plant importin-α, and explain the differential binding specificity between the class-5 plant-specific NLSs and importin-α variants [2]. The binding conformation of the class-3 minor site-specific NLSs features an α-helical turn, that is distinct from the other NLSs reported structurally [3]. Comparative bioinformatic screens not only indicate both plant-specific and minor site-specific NLSs are much less prevalent than the classical NLSs, but also reveal a greater prevalence of these two classes of non-classical NLSs in rice the proteome, compared to the others from yeast, mammals, and even other plant species. Together, our data can help to characterize novel proteins containing non-classical NLSs destined for the cell nucleus by the classical nuclear import pathway.

Download Full-text

Coat proteins of Rice tungro bacilliform virus and Mungbean yellow mosaic virus contain multiple nuclear-localization signals and interact with importin α

Journal of General Virology ◽

10.1099/vir.0.80920-0 ◽

2005 ◽

Vol 86 (6) ◽

pp. 1815-1826 ◽

Cited By ~ 38

Author(s):

O. Guerra-Peraza ◽

D. Kirk ◽

V. Seltzer ◽

K. Veluthambi ◽

A. C. Schmit ◽

...

Keyword(s):

Mosaic Virus ◽

Nuclear Localization ◽

Nuclear Import ◽

Fluorescent Protein ◽

Yellow Mosaic Virus ◽

Rice Tungro Bacilliform Virus ◽

Coat Proteins ◽

Nuclear Localization Signals ◽

Mungbean Yellow Mosaic Virus

Transport of the viral genome into the nucleus is an obligatory step in the replication cycle of plant pararetro- and geminiviruses. In both these virus types, the multifunctional coat protein (CP) is thought to be involved in this process. Here, a green fluorescent protein tagging approach was used to demonstrate nuclear import of the CPs of Rice tungro bacilliform virus (RTBV) and Mungbean yellow mosaic virus - Vigna (MYMV) in Nicotiana plumbaginifolia protoplasts. In both cases, at least two nuclear localization signals (NLSs) were identified and characterized. The NLSs of RTBV CP are located within both N- and C-terminal regions (residues 479KRPK/497KRK and 744KRK/758RRK), and those of MYMV CP within the N-terminal part (residues 3KR and 41KRRR). The MYMV and RTBV CP NLSs resemble classic mono- and bipartite NLSs, respectively. However, the N-terminal MYMV CP NLS and both RTBV CP NLSs show peculiarities in the number and position of basic residues. In vitro pull-down assays revealed interaction of RTBV and MYMV CPs with the nuclear import factor importin α, suggesting that both CPs are imported into the nucleus via an importin α-dependent pathway. The possibility that this pathway could serve for docking of virions to the nucleus is discussed.

Download Full-text

Nuclear localization signals: a driving force for nuclear transport of plasmid DNA in zebrafish

Biochemistry and Cell Biology ◽

10.1139/o97-044 ◽

1997 ◽

Vol 75 (5) ◽

pp. 633-640 ◽

Cited By ~ 24

Author(s):

Philippe Collas ◽

Peter Aleström

Keyword(s):

Nuclear Localization ◽

Plasmid Dna ◽

Nuclear Import ◽

Protein Import ◽

Nuclear Transport ◽

Nuclear Targeting ◽

Nuclear Localization Signals ◽

Expression Frequency ◽

Cytoplasmic Injection

Nuclear localization signals (NLSs) are short peptides required for nuclear transport of karyophilic proteins. We review in this paper how the nuclear targeting property of NLS peptides has been taken advantage of to enhance the efficiency of nuclear uptake of transgene DNA in zebrafish and how it may improve the efficiency of transgenesis in this species. Synthetic NLS peptides can bind to plasmid DNA by ionic interactions. Cytoplasmic injection of DNA-NLS complexes in zebrafish eggs enhances the rate and the amount of plasmid DNA taken up by embryonic nuclei. Nuclear import of DNA-NLS complexes has been duplicated in vitro and exhibits energetic and cytosolic requirements similar to those for nuclear protein import. Furthermore, binding NLSs to DNA increases expression frequency of the transgene. We suggest that NLS peptides may constitute a valuable tool to improve the efficiency of transgenesis in zebrafish and other species.

Download Full-text

Nuclear localization of Ku antigen is promoted independently by basic motifs in the Ku70 and Ku80 subunits

Journal of Cell Science ◽

10.1242/jcs.114.1.89 ◽

2001 ◽

Vol 114 (1) ◽

pp. 89-99

Author(s):

J. Bertinato ◽

C. Schild-Poulter ◽

R.J. Hache

Keyword(s):

Amino Acids ◽

Nuclear Localization ◽

Nuclear Import ◽

Nuclear Localization Signal ◽

Mammalian Cells ◽

Full Length ◽

Telomere Maintenance ◽

Nuclear Localization Signals ◽

Localization Signal ◽

Ku Antigen

The Ku antigen is a heteromeric (Ku70/Ku80), mostly nuclear protein. Ku participates in multiple nuclear processes from DNA repair to V(D)J recombination to telomere maintenance to transcriptional regulation and serves as a DNA binding subunit and allosteric regulator of DNA-dependent protein kinase. While some evidence suggests that subcellular localization of Ku may be subject to regulation, how Ku gains access to the nucleus is poorly understood. In this work, using a combination of indirect immunofluorescence and direct fluorescence, we have demonstrated that transfer of the Ku heterodimer to the nucleus is determined by basic nuclear localization signals in each of the Ku subunits that function independently. A bipartite basic nuclear localization signal between amino acids 539–556 of Ku70 was observed to be required for nuclear import of full-length Ku70 monomer, while a short Ku80 motif of four amino acids from 565–568 containing three lysines was required for the nuclear import of full-length Ku80. Ku heterodimers containing only one nuclear localization signal accumulated in the nucleus as efficiently as wild-type Ku, while site directed mutagenesis inactivating the basic motifs in each subunit, resulted in a Ku heterodimer that was completely localized to the cytoplasm. Lastly, our results indicate that mutations in Ku previously proposed to abrogate Ku70/Ku80 heterodimerization, markedly reduced the accumulation of Ku70 without affecting heterodimer formation in mammalian cells.

Download Full-text

Importin α/β mediates nuclear import of individual SUMO E1 subunits and of the holo-enzyme

Molecular Biology of the Cell ◽

10.1091/mbc.e10-05-0461 ◽

2011 ◽

Vol 22 (5) ◽

pp. 652-660 ◽

Cited By ~ 16

Author(s):

Marie Christine Moutty ◽

Volkan Sakin ◽

Frauke Melchior

Keyword(s):

Amino Acids ◽

Nuclear Localization ◽

Nuclear Import ◽

Enzyme Complex ◽

Intact Cells ◽

Nuclear Localization Signals ◽

Importin Α ◽

Essential Enzyme ◽

Nuclear Targets

SUMOylation, reversible attachment of small ubiquitin-related modifier (SUMO), serves to regulate hundreds of proteins. Consistent with predominantly nuclear targets, enzymes required for attachment and removal of SUMO are highly enriched in this compartment. This is true also for the first enzyme of the SUMOylation cascade, the SUMO E1 enzyme heterodimer, Aos1/Uba2 (SAE1/SAE2). This essential enzyme serves to activate SUMO and to transfer it to the E2-conjugating enzyme Ubc9. Although the last 40 amino acids in yeast Uba2 have been implicated in its nuclear localization, little was known about the import pathways of Aos1, Uba2, and/or of the assembled E1 heterodimer. Here we show that the mammalian E1 subunits can be imported separately, identify nuclear localization signals (NLSs) in Aos1 and in Uba2, and demonstrate that their import is mediated by importin α/β in vitro and in intact cells. Once assembled into a stable heterodimer, the E1 enzyme can still be efficiently imported by importin α/β, due to the Uba2 NLS that is still accessible. These pathways may serve distinct purposes: import of nascent subunits prior to assembly and reimport of stable E1 enzyme complex after mitosis.

Download Full-text

Defective Calmodulin-Mediated Nuclear Transport of the Sex-Determining Region of the Y Chromosome (SRY) in XY Sex Reversal

Molecular Endocrinology ◽

10.1210/me.2004-0334 ◽

2005 ◽

Vol 19 (7) ◽

pp. 1884-1892 ◽

Cited By ~ 39

Author(s):

Helena Sim ◽

Kieran Rimmer ◽

Sabine Kelly ◽

Louisa M. Ludbrook ◽

Andrew H. A. Clayton ◽

...

Keyword(s):

Y Chromosome ◽

Nuclear Import ◽

High Mobility ◽

Sex Reversal ◽

High Mobility Group ◽

Missense Mutations ◽

Nuclear Localization Signals ◽

Xy Sex Reversal

Abstract The sex-determining region of the Y chromosome (SRY) plays a key role in human sex determination, as mutations in SRY can cause XY sex reversal. Although some SRY missense mutations affect DNA binding and bending activities, it is unclear how others contribute to disease. The high mobility group domain of SRY has two nuclear localization signals (NLS). Sex-reversing mutations in the NLSs affect nuclear import in some patients, associated with defective importin-β binding to the C-terminal NLS (c-NLS), whereas in others, importin-β recognition is normal, suggesting the existence of an importin-β-independent nuclear import pathway. The SRY N-terminal NLS (n-NLS) binds calmodulin (CaM) in vitro, and here we show that this protein interaction is reduced in vivo by calmidazolium, a CaM antagonist. In calmidazolium-treated cells, the dramatic reduction in nuclear entry of SRY and an SRY-c-NLS mutant was not observed for two SRY-n-NLS mutants. Fluorescence spectroscopy studies reveal an unusual conformation of SRY.CaM complexes formed by the two n-NLS mutants. Thus, CaM may be involved directly in SRY nuclear import during gonadal development, and disruption of SRY.CaM recognition could underlie XY sex reversal. Given that the CaM-binding region of SRY is well-conserved among high mobility group box proteins, CaM-dependent nuclear import may underlie additional disease states.

Download Full-text

Modulation of Nuclear Localization of the Influenza Virus Nucleoprotein through Interaction with Actin Filaments

Journal of Virology ◽

10.1128/jvi.73.3.2222-2231.1999 ◽

1999 ◽

Vol 73 (3) ◽

pp. 2222-2231 ◽

Cited By ~ 82

Author(s):

Paul Digard ◽

Debra Elton ◽

Konrad Bishop ◽

Elizabeth Medcalf ◽

Alan Weeds ◽

...

Keyword(s):

Influenza Virus ◽

Nuclear Localization ◽

Virus Genome ◽

Actin Binding ◽

Filamentous Actin ◽

Nuclear Localization Signals ◽

Infected Cells ◽

Cytoplasmic Accumulation ◽

High Level

ABSTRACT The influenza virus genome is transcribed in the nuclei of infected cells but assembled into progeny virions in the cytoplasm. This is reflected in the cellular distribution of the virus nucleoprotein (NP), a protein which encapsidates genomic RNA to form ribonucleoprotein structures. At early times postinfection NP is found in the nucleus, but at later times it is found predominantly in the cytoplasm. NP contains several sequences proposed to act as nuclear localization signals (NLSs), and it is not clear how these are overridden to allow cytoplasmic accumulation of the protein. We find that NP binds tightly to filamentous actin in vitro and have identified a cluster of residues in NP essential for the interaction. Complexes containing RNA, NP, and actin could be formed, suggesting that viral ribonucleoproteins also bind actin. In cells, exogenously expressed NP when expressed at a high level partitioned to the cytoplasm, where it associated with F-actin stress fibers. In contrast, mutants unable to bind F-actin efficiently were imported into the nucleus even under conditions of high-level expression. Similarly, nuclear import of NLS-deficient NP molecules was restored by concomitant disruption of F-actin binding. We propose that the interaction of NP with F-actin causes the cytoplasmic retention of influenza virus ribonucleoproteins.

Download Full-text