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 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

scholarly journals Rac1 and a GTPase-activating protein, MgcRacGAP, are required for nuclear translocation of STAT transcription factors

2006 ◽  
Vol 175 (6) ◽  
pp. 937-946 ◽  
Author(s):  
Toshiyuki Kawashima ◽  
Ying Chun Bao ◽  
Yasushi Nomura ◽  
Yuseok Moon ◽  
Yukio Tonozuka ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Binding Site ◽  
Target Genes ◽  
Nuclear Translocation ◽  
Nuclear Transport ◽  
Gtpase Activating Protein ◽  
Permeabilized Cells ◽  
Importin Α ◽  
Cytokine Stimulation

STAT transcription factors are tyrosine phosphorylated upon cytokine stimulation and enter the nucleus to activate target genes. We show that Rac1 and a GTPase-activating protein, MgcRacGAP, bind directly to p-STAT5A and are required to promote its nuclear translocation. Using permeabilized cells, we find that nuclear translocation of purified p-STAT5A is dependent on the addition of GTP-bound Rac1, MgcRacGAP, importin α, and importin β. p-STAT3 also enters the nucleus via this transport machinery, and mutant STATs lacking the MgcRacGAP binding site do not enter the nucleus even after phosphorylation. We conclude that GTP-bound Rac1 and MgcRacGAP function as a nuclear transport chaperone for activated STATs.

scholarly journals SUBCELLULAR LOCALIZATION OF FoxP3 TRANSCRIPTION FACTOR IN PATIENTS WITH ACUTE CORONARY SYNDROME: COMPARATIVE ANALYSIS AND PROSPECTIVE OBSERVATION

2021 ◽  
Vol 23 (4) ◽  
pp. 731-736
Author(s):  
I. V. Kologrivova ◽  
Tatiana E. Suslova ◽  
V. V. Ryabov ◽  
M. A. Shtatolkina ◽  
O. A. Koshelskaya ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Acute Myocardial Infarction ◽  
Transcription Factor ◽  
T Lymphocytes ◽  
Subcellular Localization ◽  
Nuclear Localization ◽  
Mononuclear Cells ◽  
Nuclear Translocation ◽  
Coronary Syndrome ◽  
Treg Lymphocytes

The key cellular and molecular factors being involved in the resolution of inflammation following acute myocardial infarction remain poorly understood. T-regulatory (Treg) lymphocytes are characterized by the extreme potential to regulate the strength and direction of immune responses during the myocardial injury. The functional activity of Treg-lymphocytes depends upon the transcription factor forkhead box protein P3 (FoxP3). It may be also expressed in conventional T-lymphocytes at the stage of their activation. Nuclear localization of FoxP3 is a prerequisite factor determining its ability to impact the suppressive functions of Treglymphocytes.The aim of the present study was comparative evaluation of FoxP3+T-lymphocytes frequency and counts, combined with estimation of FoxP3 subcellular localization, in patients with acute myocardial infarction and chronic coronary syndrome and examination of changes of these parameters in the short-term follow-up of patients with myocardial infarction. The study included 14 patients with chronic coronary syndrome (8 males; 6 females; 63.2±9.0 y.o.) and 5 patients with acute anterior ST-segment elevation myocardial infarction (4 males; 1 female; 61.4±11.2 y.o.) at days 1, 3 and 7 after the event. The frequency of FoxP3+ conventional and regulatory T-lymphocytes was evaluated in peripheral blood mononuclear cells together with estimation of the level of FoxP3 nuclear localization by imaging flow cytometry.Patients with infarction were characterized by the decreased counts of FoxP3+Treg-lymphocytes compared to patients with chronic coronary syndrome, and exhibited even further decrease in the counts of FoxP3+Tregcells at day 7 after infarction, while frequency of Treg and conventional T-lymphocytes did not differ significantly. The level of FoxP3 nuclear translocation was lower both in Treg and conventional T-lymphocytes in patients at day 1 post-infarction compared to patients with chronic coronary syndrome. Absolute counts of FoxP3+Tregs with nuclear FoxP3 localization remained significantly lower both at days 1 and 7 post-infarction compared to patients with chronic coronary syndrome.Thus, here we demonstrated that FoxP3 nuclear localization experiences decrease in the course of acute myocardial infarction and may serve as a more sensitive marker of changes in Treg-lymphocyte functioning than simple evaluation of their frequency. 

The COOH-terminal nuclear localization sequence of interferon gamma regulates STAT1 alpha nuclear translocation at an intracellular site

2000 ◽  
Vol 113 (15) ◽  
pp. 2771-2781
Author(s):  
P.S. Subramaniam ◽  
J. Larkin ◽  
M.G. Mujtaba ◽  
M.R. Walter ◽  
H.M. Johnson
Keyword(s):  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Translocation ◽  
Biological Activities ◽  
Nuclear Localization Sequence ◽  
Receptor Complex ◽  
Ifn Gamma ◽  
High Affinity ◽  
Gamma Receptor ◽  
Localization Sequence

We have recently shown that the nuclear localization of IFN gamma is mediated by a polybasic nuclear localization sequence (NLS) in its C terminus. This NLS is required for the full expression of biological activity of IFN gamma, both extracellularly and intracellularly. We now show that this NLS plays an integral intracellular role in the nuclear translocation of the transcription factor STAT1 alpha activated by IFN gamma. Treatment of IFN gamma with antibodies to the C-terminal region (95–133) containing the NLS blocked the induction of STAT1 alpha nuclear translocation. The antibodies had no effect on nuclear translocation of STAT1 alpha in IFN gamma treated cells. A deletion mutant of human IFN gamma, IFN gamma (1–123), which is devoid of the C-terminal NLS region was found to be biologically inactive, but was still able to bind to the IFN gamma receptor complex on cells with a K(d) similar to that of the wild-type protein. Deletion of the NLS specifically abolished the ability of IFN gamma(1–123) to initiate the nuclear translocation of STAT1 alpha, which is required for the biological activities of IFN gamma following binding to the IFN gamma receptor complex. Thus, the NLS region appears to contribute minimally to extracellular high-affinity receptor-ligand binding, yet exerts a strong functional role in STAT1 alpha nuclear localization. A high-affinity site for the interaction of the C-terminal NLS domain of IFN gamma with a K(d) approx. 3 × 10(−8) M(−1) has been described by previous studies on the intracellular cytoplasmic domain of the IFN gamma receptor alpha-chain. To examine the role of the NLS at the intracellular level, we microinjected neutralizing antibodies raised against the C-terminal NLS domain of IFN gamma into the cytoplasm of cells before treatment of cells with IFN gamma. These intracellular antibodies specifically blocked the nuclear translocation of STAT1 alpha following the subsequent treatment of these cells extracellularly with IFN gamma. These data show that the NLS domain of IFN gamma interacts at an intracellular site to regulate STAT1 alpha nuclear import. A C-terminal peptide of murine IFN gamma, IFN gamma(95–133), that contains the NLS motif, induced nuclear translocation of STAT1 alpha when taken up intracellularly by a murine macrophage cell line. Deletion of the NLS motif specifically abrogated the ability of this intracellular peptide to cause STAT1 alpha nuclear translocation. In cells activated with IFN gamma, IFN gamma was found to as part of a complex that contained STAT1 alpha and the importin-alpha analog Npi-1, which mediates STAT1 alpha nuclear import. The tyrosine phosphorylation of STAT1 alpha, the formation of the complex IFN gamma/Npi-1/STAT1 alpha complex and the subsequent nuclear translocation of STAT1 alpha were all found to be dependent on the presence of the IFN gamma NLS. Thus, the NLS of IFN gamma functions intracellularly to directly regulate the activation and ultimate nuclear translocation STAT1 alpha.

scholarly journals Nuclear import of isoforms of the cytomegalovirus kinase pUL97 is mediated by differential activity of NLS1 and NLS2 both acting through classical importin-α binding

2012 ◽  
Vol 93 (8) ◽  
pp. 1756-1768 ◽  
Author(s):  
Rike Webel ◽  
Sara M. Ø. Solbak ◽  
Christian Held ◽  
Jens Milbradt ◽  
Andrea Groß ◽  
...  
Keyword(s):  
Virus Replication ◽  
Nuclear Localization ◽  
Evaluation System ◽  
Nuclear Translocation ◽  
Helical Structure ◽  
Nucleocytoplasmic Transport ◽  
Resonance Spectroscopy ◽  
Translational Initiation ◽  
Importin Α ◽  
Arginine Residues

The multifunctional protein kinase pUL97 of human cytomegalovirus (HCMV) strongly determines the efficiency of virus replication. Previously, the existence of two pUL97 isoforms that arise from alternative translational initiation and show a predominant nuclear localization was described. Two bipartite nuclear localization sequences, NLS1 and NLS2, were identified in the N terminus of the large isoform, whilst the small isoform exclusively contained NLS2. The current study found the following: (i) pUL97 nuclear localization in HCMV-infected primary fibroblasts showed accumulations in virus replication centres and other nuclear sections; (ii) in a quantitative evaluation system for NLS activity, the large isoform showed higher efficiency of nuclear translocation than the small isoform; (iii) NLS1 was mapped to aa 6–35 and NLS2 to aa 190–213; (iv) using surface plasmon resonance spectroscopy, the binding of both NLS1 and NLS2 to human importin-α was demonstrated, stressing the importance of individual arginine residues in the bipartite consensus motifs; (v) nuclear magnetic resonance spectroscopy of pUL97 peptides confirmed an earlier statement about the functional requirement of NLS1 embedding into an intact α-helical structure; and (vi) a bioinformatics investigation of the solvent-accessible surface suggested a high accessibility of NLS1 and an isoform-specific, variable accessibility of NLS2 for interaction with importin-α. Thus, the nucleocytoplasmic transport mechanism of the isoforms appeared to be differentially regulated, and this may have consequences for isoform-dependent functions of pUL97 during virus replication.

scholarly journals Copper Induces Cytoplasmic Retention of Fission Yeast Transcription Factor Cuf1

2006 ◽  
Vol 5 (2) ◽  
pp. 277-292 ◽  
Author(s):  
Jude Beaudoin ◽  
Simon Labbé
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Nuclear Localization ◽  
Copper Transport ◽  
Nuclear Localization Sequence ◽  
Binding Motif ◽  
Physical Interaction ◽  
Dependent Manner ◽  
C Terminus ◽  
N Terminus

ABSTRACT Copper homeostasis within the cell is established and preserved by different mechanisms. Changes in gene expression constitute a way of maintaining this homeostasis. In Schizosaccharomyces pombe, the Cuf1 transcription factor is critical for the activation of copper transport gene expression under conditions of copper starvation. However, in the presence of elevated intracellular levels of copper, the mechanism of Cuf1 inactivation to turn off gene expression remains unclear. In this study, we provide evidence that inactivation of copper transport gene expression by Cuf1 is achieved through a copper-dependent, cytosolic retention of Cuf1. We identify a minimal nuclear localization sequence (NLS) between amino acids 11 to 53 within the Cuf1 N terminus. Deletion of this region and specific mutation of the Lys13, Arg16, Arg19, Lys24, Arg28, Lys45, Arg47, Arg50, and Arg53 residues to alanine within this putative NLS is sufficient to abrogate nuclear targeting of Cuf1. Under conditions of copper starvation, Cuf1 resides in the nucleus. However, in the presence of excess copper as well as silver ions, Cuf1 is sequestered in the cytoplasm, a process which requires the putative copper binding motif, 328Cys-X-Cys-X3-Cys-X-Cys-X2-Cys-X2-His342 (designated C-rich), within the C-terminal region of Cuf1. Deletion of this region and mutation of the Cys residues within the C-rich motif result in constitutive nuclear localization of Cuf1. By coexpressing the Cuf1 N terminus with its C terminus in trans and by using a two-hybrid assay, we show that these domains physically interact with each other in a copper-dependent manner. We propose a model wherein copper induces conformational changes in Cuf1 that promote a physical interaction between the Cuf1 N terminus and the C-rich motif in the C terminus that masks the NLS. Cuf1 is thereby sequestered in the cytosol under conditions of copper excess, thereby extinguishing copper transport gene expression.

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
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