scholarly journals The distribution of different classes of nuclear localization signals (NLSs) in diverse organisms and the utilization of the minor NLS-binding site inplantnuclear import factor importin-α

2013 ◽  
Vol 8 (10) ◽  
pp. e25976 ◽  
Author(s):  
Chiung-Wen Chang ◽  
Rafael Miguez Couñago ◽  
Simon J. Williams ◽  
Mikael Boden ◽  
Bostjan Kobe
Keyword(s):  
Binding Site ◽  
Nuclear Localization ◽  
Nuclear Localization Signals ◽  
Importin Α

scholarly journals Importin alpha and nonclassical nuclear localization signal

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.

scholarly journals TNPO3-Mediated Nuclear Entry of the Rous Sarcoma Virus Gag Protein Is Independent of the Cargo-Binding Domain

2020 ◽  
Vol 94 (17) ◽  
Author(s):  
Breanna L. Rice ◽  
Matthew S. Stake ◽  
Leslie J. Parent
Keyword(s):  
Nuclear Localization ◽  
Nuclear Import ◽  
Rous Sarcoma Virus ◽  
Binding Domain ◽  
Nuclear Entry ◽  
Gag Protein ◽  
Nuclear Localization Signals ◽  
Rous Sarcoma ◽  
Importin Α ◽  
Sarcoma Virus

ABSTRACT Retroviral Gag polyproteins orchestrate the assembly and release of nascent virus particles from the plasma membranes of infected cells. Although it was traditionally thought that Gag proteins trafficked directly from the cytosol to the plasma membrane, we discovered that the oncogenic avian alpharetrovirus Rous sarcoma virus (RSV) Gag protein undergoes transient nucleocytoplasmic transport as an intrinsic step in virus assembly. Using a genetic approach in yeast, we identified three karyopherins that engage the two independent nuclear localization signals (NLSs) in Gag. The primary NLS is in the nucleocapsid (NC) domain of Gag and binds directly to importin-α, which recruits importin-β to mediate nuclear entry. The second NLS (TNPO3), which resides in the matrix (MA) domain, is dependent on importin-11 and transportin-3 (TNPO3), which are known as MTR10p and Kap120p in yeast, although it is not clear whether these import factors are independent or additive. The functions of importin-α/importin-β and importin-11 have been verified in avian cells, whereas the role of TNPO3 has not been studied. In this report, we demonstrate that TNPO3 directly binds to Gag and mediates its nuclear entry. To our surprise, this interaction did not require the cargo-binding domain (CBD) of TNPO3, which typically mediates nuclear entry for other binding partners of TNPO3, including SR domain-containing splicing factors and tRNAs that reenter the nucleus. These results suggest that RSV hijacks this host nuclear import pathway using a unique mechanism, potentially allowing other cargo to simultaneously bind TNPO3. IMPORTANCE RSV Gag nuclear entry is facilitated using three distinct host import factors that interact with nuclear localization signals in the Gag MA and NC domains. Here, we show that the MA region is required for nuclear import of Gag through the TNPO3 pathway. Gag nuclear entry does not require the CBD of TNPO3. Understanding the molecular basis for TNPO3-mediated nuclear trafficking of the RSV Gag protein may lead to a deeper appreciation for whether different import factors play distinct roles in retrovirus replication.

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

1999 ◽  
Vol 19 (2) ◽  
pp. 1210-1217 ◽  
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.

scholarly journals Phospholipid Scramblase 1 Contains a Nonclassical Nuclear Localization Signal with Unique Binding Site in Importin α

2004 ◽  
Vol 280 (11) ◽  
pp. 10599-10606 ◽  
Author(s):  
Min-Hsuan Chen ◽  
Iris Ben-Efraim ◽  
Gregory Mitrousis ◽  
Nancy Walker-Kopp ◽  
Peter J. Sims ◽  
...  
Keyword(s):  
Binding Site ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Phospholipid Scramblase ◽  
Importin Α ◽  
Localization Signal ◽  
Phospholipid Scramblase 1

scholarly journals Phosphorylation adjacent to the nuclear localization signal of human dUTPase abolishes nuclear import: structural and mechanistic insights

2013 ◽  
Vol 69 (12) ◽  
pp. 2495-2505 ◽  
Author(s):  
Gergely Róna ◽  
Mary Marfori ◽  
Máté Borsos ◽  
Ildikó Scheer ◽  
Enikő Takács ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Nucleocytoplasmic Transport ◽  
Wild Type ◽  
Post Translational Modification ◽  
Nuclear Localization Signals ◽  
M Phase ◽  
Importin Α ◽  
Localization Signal

Phosphorylation adjacent to nuclear localization signals (NLSs) is involved in the regulation of nucleocytoplasmic transport. The nuclear isoform of human dUTPase, an enzyme that is essential for genomic integrity, has been shown to be phosphorylated on a serine residue (Ser11) in the vicinity of its nuclear localization signal; however, the effect of this phosphorylation is not yet known. To investigate this issue, an integrated set of structural, molecular and cell biological methods were employed. It is shown that NLS-adjacent phosphorylation of dUTPase occurs during the M phase of the cell cycle. Comparison of the cellular distribution of wild-type dUTPase with those of hyperphosphorylation- and hypophosphorylation-mimicking mutants suggests that phosphorylation at Ser11 leads to the exclusion of dUTPase from the nucleus. Isothermal titration microcalorimetry and additional independent biophysical techniques show that the interaction between dUTPase and importin-α, the karyopherin molecule responsible for `classical' NLS binding, is weakened significantly in the case of the S11E hyperphosphorylation-mimicking mutant. The structures of the importin-α–wild-type and the importin-α–hyperphosphorylation-mimicking dUTPase NLS complexes provide structural insights into the molecular details of this regulation. The data indicate that the post-translational modification of dUTPase during the cell cycle may modulate the nuclear availability of this enzyme.

scholarly journals Human mRNA Cap Methyltransferase: Alternative Nuclear Localization Signal Motifs Ensure Nuclear Localization Required for Viability

2005 ◽  
Vol 25 (7) ◽  
pp. 2644-2649 ◽  
Author(s):  
Beth Shafer ◽  
Chun Chu ◽  
Aaron J. Shatkin
Keyword(s):  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Nuclear Localization Signals ◽  
Truncation Mutant ◽  
Capping Enzyme ◽  
Importin Α ◽  
Localization Signal ◽  
Targeting Signals ◽  
Sirna Knockdown ◽  
Interfering Rna

ABSTRACT A characteristic feature of gene expression in eukaryotes is the addition of a 5′-terminal 7-methylguanine cap (m7GpppN) to nascent pre-mRNAs in the nucleus catalyzed by capping enzyme and cap methyltransferase. Small interfering RNA (siRNA) knockdown of cap methyltransferase in HeLa cells resulted in apoptosis as measured by terminal deoxynucleotidyltransferase-mediated dUTP-tetramethylrhodamine nick end labeling assay, demonstrating the importance of mRNA 5′-end methylation for mammalian cell viability. Nuclear localization of cap methyltransferase is mediated by interaction with importin-α, which facilitates its transport and selective binding to transcripts containing 5′-terminal GpppN. The methyltransferase 96-144 region has been shown to be necessary for importin binding, and N-terminal fusion of this sequence to nonnuclear proteins proved sufficient for nuclear localization. The targeting sequence was narrowed to amino acids 120 to 129, including a required 126KRK. Although full-length methyltransferase (positions 1 to 476) contains the predicted nuclear localization signals 57RKRK, 80KKRK, 103KKRKR, and 194KKKR, mutagenesis studies confirmed functional motifs only at positions 80, 103, and the previously unrecognized 126KRK. All three motifs can act as alternative nu clear targeting signals. Expression of N-truncated cap methyltransferase (120 to 476) restored viability of methyltransferase siRNA knocked-down cells. However, an enzymatically active 144-476 truncation mutant missing the three nuclear localization signals was mostly cytoplasmic and ineffective in preventing siRNA-induced loss of viability.

scholarly journals Probing the Specificity of Binding to the Major Nuclear Localization Sequence-binding Site of Importin-α Using Oriented Peptide Library Screening

2010 ◽  
Vol 285 (26) ◽  
pp. 19935-19946 ◽  
Author(s):  
Sundy N. Y. Yang ◽  
Agnes A. S. Takeda ◽  
Marcos R. M. Fontes ◽  
Jonathan M. Harris ◽  
David A. Jans ◽  
...  
Keyword(s):  
Binding Site ◽  
Nuclear Localization ◽  
Peptide Library ◽  
Nuclear Localization Sequence ◽  
Library Screening ◽  
Localization Sequence ◽  
Importin Α ◽  
Specificity Of Binding

Nuclear transport of the Neurospora crassa NIT-2 transcription factor is mediated by importin-α

2017 ◽  
Vol 474 (24) ◽  
pp. 4091-4104 ◽  
Author(s):  
Natália E. Bernardes ◽  
Agnes A.S. Takeda ◽  
Thiago R. Dreyer ◽  
Fernanda B. Cupertino ◽  
Stela Virgilio ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Neurospora Crassa ◽  
Binding Site ◽  
Nuclear Localization ◽  
Nuclear Translocation ◽  
Protein Translocation ◽  
Nuclear Transport ◽  
Nuclear Localization Sequence ◽  
Primary Role ◽  
Importin Α

The Neurospora crassa NIT-2 transcription factor belongs to the GATA transcription factor family and plays a fundamental role in the regulation of nitrogen metabolism. Because NIT-2 acts by accessing DNA inside the nucleus, understanding the nuclear import process of NIT-2 is necessary to characterize its function. Thus, in the present study, NIT-2 nuclear transport was investigated using a combination of biochemical, cellular, and biophysical methods. A complemented strain that produced an sfGFP–NIT-2 fusion protein was constructed, and nuclear localization assessments were made under conditions that favored protein translocation to the nucleus. Nuclear translocation was also investigated using HeLa cells, which showed that the putative NIT-2 nuclear localization sequence (NLS; 915TISSKRQRRHSKS927) was recognized by importin-α and that subsequent transport occurred via the classical import pathway. The interaction between the N. crassa importin-α (NcImpα) and the NIT-2 NLS was quantified with calorimetric assays, leading to the observation that the peptide bound to two sites with different affinities, which is typical of a monopartite NLS sequence. The crystal structure of the NcImpα/NIT-2 NLS complex was solved and revealed that the NIT-2 peptide binds to NcImpα with the major NLS-binding site playing a primary role. This result contrasts other recent studies that suggested a major role for the minor NLS-binding site in importin-α from the α2 family, indicating that both sites can be used for different cargo proteins according to specific metabolic requirements.

scholarly journals A Role for Caenorhabditis elegans Importin IMA-2 in Germ Line and Embryonic Mitosis

2002 ◽  
Vol 13 (9) ◽  
pp. 3138-3147 ◽  
Author(s):  
Kenneth G. Geles ◽  
Jeffrey J. Johnson ◽  
Sena Jong ◽  
Stephen A. Adam
Keyword(s):  
Caenorhabditis Elegans ◽  
Nuclear Envelope ◽  
Nuclear Localization ◽  
Germ Line ◽  
Embryonic Cells ◽  
Nuclear Localization Signals ◽  
Cytoplasmic Transport ◽  
Nuclear Envelope Assembly ◽  
Importin Α ◽  
Nuclear Envelope Formation

The importin α family of nuclear-cytoplasmic transport factors mediates the nuclear localization of proteins containing classical nuclear localization signals. Metazoan animals express multiple importin α proteins, suggesting their possible roles in cell differentiation and development. Adult Caenorhabditis elegans hermaphrodites express three importin α proteins, IMA-1, IMA-2, and IMA-3, each with a distinct expression and localization pattern. IMA-2 was expressed exclusively in germ line cells from the early embryonic through adult stages. The protein has a dynamic pattern of localization dependent on the stage of the cell cycle. In interphase germ cells and embryonic cells, IMA-2 is cytoplasmic and nuclear envelope associated, whereas in developing oocytes, the protein is cytoplasmic and intranuclear. During mitosis in germ line cells and embryos, IMA-2 surrounded the condensed chromosomes but was not directly associated with the mitotic spindle. The timing of IMA-2 nuclear localization suggested that the protein surrounded the chromosomes after fenestration of the nuclear envelope in prometaphase. Depletion of IMA-2 by RNA-mediated gene interference (RNAi) resulted in embryonic lethality and a terminal aneuploid phenotype.ima-2(RNAi) embryos have severe defects in nuclear envelope formation, accumulating nucleoporins and lamin in the cytoplasm. We conclude that IMA-2 is required for proper chromosome dynamics in germ line and early embryonic mitosis and is involved in nuclear envelope assembly at the conclusion of mitosis.

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