scholarly journals Structural and calorimetric studies reveal specific determinants for the binding of a high-affinity NLS to mammalian importin-alpha

2021 ◽  
Author(s):  
Hamine C Oliveira ◽  
Taina D da Silva ◽  
Guilherme H Salvador ◽  
Ivan R Moraes ◽  
Cintia A Fukuda ◽  
...  
Keyword(s):  
Hydrophobic Interactions ◽  
Consensus Sequence ◽  
Nuclear Localization Sequence ◽  
High Affinity ◽  
Importin Alpha ◽  
C Terminus ◽  
Cargo Protein ◽  
Localization Sequence ◽  
Additional Hydrogen Bond ◽  
Calorimetric Studies

The classical nuclear import pathway is mediated by importin (Impα and Impβ), which recognizes the cargo protein by its Nuclear Localization Sequence (NLS). NLSs have been extensively studied resulting in different proposed consensus; however, recent studies showed that exceptions may occur. This mechanism may be also dependent on specific characteristics of different Impα. Aiming to better understand the importance of specific residues from consensus and adjacent regions of NLSs, we studied different mutations of a high affinity NLS complexed to Impα by crystallography and calorimetry. We showed that although the consensus sequence allows Lys or Arg residues at the second residue of a monopartite sequence, the presence of Arg is very important to its binding in major and minor sites of Impα. Mutations in the N or C-terminus (position P1 or P6) of the NLS drastically reduces their affinity to the receptor, which is corroborated by the loss of hydrogen bonds and hydrophobic interactions. Surprisingly, a mutation in the far N-terminus of the NLS led to an increase in the affinity for both binding sites, corroborated by the structure with an additional hydrogen bond. The binding of NLSs to the human variant Impα1 revealed that these are similar to those found in structures presented here. For human variant Impα3 the bindings are only relevant for the major site. This study increases understanding of specific issues sparsely addressed in previous studies that are important to the task of predicting NLSs, which will be relevant in the eventual design of synthetic NLSs.

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 The midregion, nuclear localization sequence, and C terminus of PTHrP regulate skeletal development, hematopoiesis, and survival in mice

2010 ◽  
Vol 24 (6) ◽  
pp. 1947-1957 ◽  
Author(s):  
Ramiro E. Toribio ◽  
Holly A. Brown ◽  
Chad M. Novince ◽  
Brandlyn Marlow ◽  
Krista Hernon ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Skeletal Development ◽  
Nuclear Localization Sequence ◽  
C Terminus ◽  
Localization Sequence

scholarly journals PTHrP Nuclear Localization and Carboxyl Terminus Sequences Modulate Dental and Mandibular Development in Part via the Action of p27

Endocrinology ◽  
2016 ◽  
Vol 2016 (1) ◽  
pp. 72-84 ◽  
Author(s):  
Wen Sun ◽  
Jun Wu ◽  
Linying Huang ◽  
Hong Liu ◽  
Rong Wang ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Alveolar Bone ◽  
Bone Volume ◽  
Carboxyl Terminus ◽  
Nuclear Localization Sequence ◽  
Wild Type ◽  
Mineral Density ◽  
C Terminus ◽  
Localization Sequence ◽  
Dentin Sialoprotein

Abstract To determine whether the action of the PTHrP nuclear localization sequence and C terminus is mediated through p27 in modulating dental and mandibular development, compound mutant mice, which are homozygous for both p27 deletion and the PTHrP1–84 knock-in mutation (p27−/−PthrpKI/KI), were generated. Their teeth and mandibular phenotypes were compared with those of p27−/−, PthrpKUK\ and wild-type mice. At 2 weeks of age, the mandibular mineral density, alveolar bone volume, osteoblast numbers, and dental volume, dentin sialoprotein-immunopo-sitive areas in the first molar were increased significantly in p27−/− mice and decreased dramatically in both PthrpKI/KI and p27−/− PthrpKI/KI mice compared with wild-type mice; however, these parameters were partly rescued in p27−/− PthrpKI/KI mice compared with PthrpKI/KI mice. These data demonstrate that the deletion of p27 in PthrpKI/KI mice can partially rescue defects in dental and mandibular development. Furthermore, we found that deletion of p27 in PthrpKI/KI mice partially corrected the dental and mandibular phenotype by modulating cell cyclin-regulating molecules and antioxidant enzymes. This study therefore indicates that the p27 pathway may function downstream in the action of PTHrP nuclear localization sequence to regulate dental and mandibular development. (Endocrinology 157: 1372–1384, 2016)

Relationship between the function and the location of G1 cyclins inS. cerevisiae

2001 ◽  
Vol 114 (24) ◽  
pp. 4599-4611 ◽  
Author(s):  
Nicholas P. Edgington ◽  
Bruce Futcher
Keyword(s):  
Nuclear Localization ◽  
Cellular Localization ◽  
Subcellular Location ◽  
Nuclear Localization Sequence ◽  
Cyclin Dependent Kinase ◽  
Cyclin Dependent Kinases ◽  
G1 Cyclins ◽  
C Terminus ◽  
Localization Sequence ◽  
Nuclear Location

The Saccharomyces cerevisiae cyclin-dependent kinase Cdc28 forms complexes with nine different cyclins to promote cell division. These nine cyclin-Cdc28 complexes have different roles, but share the same catalytic subunit; thus, it is not clear how substrate specificity is achieved. One possible mechanism is specific sub-cellular localization of specific complexes. We investigated the location of two G1 cyclins using fractionation and microscopy. In addition, we developed ‘forced localization’ cassettes, which direct proteins to particular locations, to test the importance of localization. Cln2 was found in both nucleus and cytoplasm. A substrate of Cln2, Sic1, was also in both compartments. Cytoplasmic Cln2 was concentrated at sites of polarized growth. Forced localization showed that some functions of Cln2 required a cytoplasmic location, while other functions required a nuclear location. In addition, one function apparently required shuttling between the two compartments. The G1 cyclin Cln3 required nuclear localization. An autonomous, nuclear localization sequence was found near the C-terminus of Cln3. Our data supports the hypothesis that Cln2 and Cln3 have distinct functions and locations, and the specificity of cyclin-dependent kinases is mediated in part by subcellular location.

scholarly journals C-Terminal Anchoring of mid1p to Membranes Stabilizes Cytokinetic Ring Position in Early Mitosis in Fission Yeast

2004 ◽  
Vol 24 (24) ◽  
pp. 10621-10635 ◽  
Author(s):  
Séverine Celton-Morizur ◽  
Nicole Bordes ◽  
Vincent Fraisier ◽  
Phong T. Tran ◽  
Anne Paoletti
Keyword(s):  
Nuclear Localization Sequence ◽  
Amphipathic Helix ◽  
Ring Assembly ◽  
C Terminus ◽  
Medial Cortex ◽  
Key Factor ◽  
Localization Sequence ◽  
Membrane Bound ◽  
Ring Position ◽  
Early Mitosis

ABSTRACT mid1p is a key factor for the central positioning of the cytokinetic ring in Schizosaccharomyces pombe. In interphase and early mitosis, mid1p forms a medial cortical band overlying the nucleus, which may represent a landmark for cytokinetic ring assembly. It compacts before anaphase into a tight ring with other cytokinetic ring components. We show here that mid1p binds to the medial cortex by at least two independent means. First, mid1p C-terminus association with the cortex requires a putative amphipathic helix adjacent to mid1p nuclear localization sequence (NLS), which is predicted to insert directly into the lipid bilayer. This association is stabilized by the polybasic NLS. mid1p mutated within the helix and the NLS forms abnormal filaments in early mitosis that are not properly anchored to the medial cortex. Misplaced rings assemble in late mitosis, indicating that mid1p C-terminus binding to membranes stabilizes cytokinetic ring position. Second, the N terminus of mid1p has the ability to associate faintly with the medial cortex and is sufficient to form tight rings. In addition, we show that mid1p oligomerizes. We propose that membrane-bound oligomers of mid1p assemble recruitment “platforms” for cytokinetic ring components at the medial cortex and stabilize the ring position during its compaction.

scholarly journals Determination of the functional domains involved in nucleolar targeting of nucleolin.

1993 ◽  
Vol 4 (12) ◽  
pp. 1239-1250 ◽  
Author(s):  
L Créancier ◽  
H Prats ◽  
C Zanibellato ◽  
F Amalric ◽  
B Bugler
Keyword(s):  
Rna Binding ◽  
Chimeric Protein ◽  
Nuclear Localization Sequence ◽  
Binding Domains ◽  
Rich Domain ◽  
C Terminus ◽  
N Terminus ◽  
Localization Sequence ◽  
Nuclear Location

Nucleolin (713 aa), a major nucleolar protein, presents two structural domains: a N-terminus implicated in interaction with chromatin and a C-terminus containing four RNA-binding domains (RRMs) and a glycine/arginine-rich domain mainly involved in pre-rRNA packaging. Furthermore, nucleolin was shown to shuttle between cytoplasm and nucleolus. To get an insight on the nature of nuclear and nucleolar localization signals, a set of nucleolin deletion mutants in fusion with the prokaryotic chloramphenicol acetyltransferase (CAT) were constructed, and the resulting chimeric proteins were recognized by anti-CAT antibodies. First, a nuclear location signal bipartite and composed of two short basic stretches separated by eleven residues was characterized. Deletion of either motifs renders the protein cytoplasmic. Second, by deleting one or more domains implicated in nucleolin association either with DNA, RNA, or proteins, we demonstrated that nucleolar accumulation requires, in addition to the nuclear localization sequence, at least two of the five RRMs in presence or absence of N-terminus. However, in presence of only one RRM the N-terminus allowed a partial targeting of the chimeric protein to the nucleolus.

scholarly journals Extragenic suppressors of mutations in the cytoplasmic C terminus of SEC63 define five genes in Saccharomyces cerevisiae.

Genetics ◽  
1993 ◽  
Vol 134 (1) ◽  
pp. 159-173 ◽  
Author(s):  
M K Nelson ◽  
T Kurihara ◽  
P A Silver
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Protein ◽  
Protein Localization ◽  
Nuclear Localization Sequence ◽  
Temperature Sensitive ◽  
C Terminus ◽  
Nuclear Antigens ◽  
Regional Specificity ◽  
Localization Sequence ◽  
Sensitive Allele

Abstract Mutations in the SEC63 gene of Saccharomyces cerevisiae affect both nuclear protein localization and translocation of proteins into the endoplasmic reticulum. We now report the isolation of suppressors of sec63-101 (formerly npl1-1), a temperature-sensitive allele of SEC63. Five complementation groups of extragenic mutations, son1-son5 (suppressor of npl1-1), were identified among the recessive suppressors. The son mutations are specific to SEC63, are not bypass suppressors, and are not new alleles of previously identified secretory (SEC61, SEC62, KAR2) or nuclear protein localization genes (NPL3, NPL4, NPL6). son1 mutations show regional specificity of suppression of sec63 alleles. At low temperatures, son1 mutants grow slowly and show partial mislocalization of nuclear antigens. The SON1 gene maps to chromosome IV and encodes a nuclear protein of 531 amino acids that contains two acidic stretches and a putative nuclear localization sequence. We show that son1 mutations suppress sec63-101 by elimination of Son1p function.

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