The yeast nucleoporin Nsp1 binds nuclear localization sequences in vitro

1996 ◽  
Vol 74 (3) ◽  
pp. 363-372 ◽  
Author(s):  
Werner Barth ◽  
Ursula Stochaj
Keyword(s):  
Nuclear Localization ◽  
Specific Binding ◽  
T Antigen ◽  
Nuclear Localization Sequence ◽  
Nuclear Pore ◽  
Sv40 T Antigen ◽  
Nuclear Targeting ◽  
Nuclear Localization Sequences

Facilitated transport of proteins into the nucleus requires nuclear localization sequences (NLSs) be present in the protein destined for the nucleus. The specific binding of NLSs by components of the nuclear transport apparatus is essential for these targeting reactions. We now report that the yeast nucleoporin Nsp1 binds specifically nuclear localization sequences in vitro. This nucleoporin recognizes several NLSs that are functional for nuclear targeting in vivo, including the NLS of SV40 T-antigen and of the yeast transcription factor Gal4. Nsp1 is organized into three domains, and we have located NLS binding sites to the N-terminal portion and the middle repetitive region of the protein. For the interaction between the NLS of SV40 T-antigen and Nsp1, we obtained association constants of 1.2 × 107 M−1 and 5 × 107 M−1. An association constant of 5 × 107 M−1 was determined for NLS binding to the repetitive domain of Nsp1. We analyzed binding of Nsp1 and its domains to a mutant version of the NLS derived from SV40 T-antigen, which poorly functions for nuclear targeting in vivo. The affinity for the mutant signal was about two orders of magnitude lower than for the wild-type NLS.Key words: Nsp1, nuclear pore complex, nucleoporin, nuclear localization sequence, protein targeting, yeast.

scholarly journals Analysis of conserved binding proteins for nuclear localization sequences

1993 ◽  
Vol 104 (1) ◽  
pp. 89-95 ◽  
Author(s):  
U. Stochaj ◽  
M.A. Bossie ◽  
K. van Zee ◽  
A.M. Whalen ◽  
P.A. Silver
Keyword(s):  
Nuclear Localization ◽  
Binding Proteins ◽  
Nuclear Protein ◽  
Protein Localization ◽  
T Antigen ◽  
Nuclear Proteins ◽  
Nuclear Localization Sequence ◽  
Nuclear Pore ◽  
Permissive Temperature ◽  
Nuclear Localization Sequences

Correct targeting of nuclear proteins is mediated by nuclear localization sequences (NLS) which permit specific binding to the nucleus and subsequent translocation across the nuclear envelope via the nuclear pore complex. It is proposed that nuclear import is facilitated by NLS-receptors which reside in the cytoplasm and at the nuclear pore. These NLS-receptors could facilitate an early step of nuclear protein import, i.e. targeting and binding of nuclear proteins at the nuclear pore. We have generated anti-idiotype antibodies against the SV40 T-antigen nuclear localization sequence that allowed us to study NLS-binding proteins in a variety of different organisms. Proteins of similar size are recognized by these antibodies in yeast, Drosophila, rat and human cells. Cytological analysis indicates that the NLS-binding proteins reside in part at nuclear pores. One of the proteins recognized by anti-idiotype antibodies is identical to a previously identified NLS-binding protein. Using isolated yeast nuclei we demonstrate that the anti-idiotype antibodies compete for binding of nuclear proteins in vitro. We show that the yeast mutant npl3, which is defective in nuclear protein localization, has an altered distribution of antigens recognized by these anti-idiotype antibodies, at the semi-permissive temperature. Our results suggest that a set of proteins common to various eukaryotes recognizes nuclear localization sequences.

In vitro and in vivo characterisation of glial cells immortalised with a temperature sensitive SV40 T antigen-containing retrovirus

1994 ◽  
Vol 37 (2) ◽  
pp. 182-196 ◽  
Author(s):  
M. Jung ◽  
A. J. Crang ◽  
W. F. Blackemore ◽  
D. Hoppe ◽  
H. Kettenmann ◽  
...  
Keyword(s):  
Glial Cells ◽  
T Antigen ◽  
Temperature Sensitive ◽  
Sv40 T Antigen

scholarly journals Yeast NPI46 encodes a novel prolyl cis-trans isomerase that is located in the nucleolus.

1994 ◽  
Vol 126 (4) ◽  
pp. 853-862 ◽  
Author(s):  
X Shan ◽  
Z Xue ◽  
T Mélèse
Keyword(s):  
Nuclear Localization ◽  
Synthetic Peptides ◽  
Spectrophotometric Assay ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Histone H2b ◽  
Nucleolar Proteins ◽  
Nuclear Localization Sequences

We have identified a gene (NPI46) encoding a new prolyl cis-trans isomerase within the nucleolus of the yeast Saccharomyces cerevisiae. The protein encoded by NPI46 was originally found by us in a search for proteins that recognize nuclear localization sequences (NLSs) in vitro. Thus, NPI46 binds to affinity columns that contain a wild-type histone H2B NLS but not a mutant H2B NLS that is incompetent for nuclear localization in vivo. NPI46 has two domains, a highly charged NH2 terminus similar to two other mammalian nucleolar proteins, nucleolin and Nopp140, and a COOH terminus with 45% homology to a family of mammalian and yeast proline isomerases. NPI46 is capable of catalyzing the prolyl cis-trans isomerization of two small synthetic peptides, succinyl-Ala-Leu-Pro-Phe-p-nitroanilide and succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, as measured by a chymotrypsin-coupled spectrophotometric assay. By indirect immunofluorescence we have shown that NPI46 is a nucleolar protein. NPI46 is not essential for cell viability.

The SV40 T antigen nuclear localization sequence enhances nuclear import of vector DNA in embryos of a crustacean (Litopenaeus schmitti)

Gene ◽  
2004 ◽  
Vol 337 ◽  
pp. 71-77 ◽  
Author(s):  
Amilcar Arenal ◽  
Rafael Pimentel ◽  
Carmen Garcı́a ◽  
Eulogio Pimentel ◽  
Peter Aleström
Keyword(s):  
Nuclear Localization ◽  
Nuclear Import ◽  
T Antigen ◽  
Nuclear Localization Sequence ◽  
Sv40 T Antigen ◽  
Localization Sequence ◽  
Vector Dna

scholarly journals Transportin acts to regulate mitotic assembly events by target binding rather than Ran sequestration

2014 ◽  
Vol 25 (7) ◽  
pp. 992-1009 ◽  
Author(s):  
Cyril Bernis ◽  
Beth Swift-Taylor ◽  
Matthew Nord ◽  
Sarah Carmona ◽  
Yuh Min Chook ◽  
...  
Keyword(s):  
Direct Action ◽  
Nuclear Localization Sequence ◽  
Nuclear Pore ◽  
Assembly Factors ◽  
Localization Sequence ◽  
Import Receptors ◽  
Target Binding ◽  
Microtubule Aster

The nuclear import receptors importin β and transportin play a different role in mitosis: both act phenotypically as spatial regulators to ensure that mitotic spindle, nuclear membrane, and nuclear pore assembly occur exclusively around chromatin. Importin β is known to act by repressing assembly factors in regions distant from chromatin, whereas RanGTP produced on chromatin frees factors from importin β for localized assembly. The mechanism of transportin regulation was unknown. Diametrically opposed models for transportin action are as follows: 1) indirect action by RanGTP sequestration, thus down-regulating release of assembly factors from importin β, and 2) direct action by transportin binding and inhibiting assembly factors. Experiments in Xenopus assembly extracts with M9M, a superaffinity nuclear localization sequence that displaces cargoes bound by transportin, or TLB, a mutant transportin that can bind cargo and RanGTP simultaneously, support direct inhibition. Consistently, simple addition of M9M to mitotic cytosol induces microtubule aster assembly. ELYS and the nucleoporin 107–160 complex, components of mitotic kinetochores and nuclear pores, are blocked from binding to kinetochores in vitro by transportin, a block reversible by M9M. In vivo, 30% of M9M-transfected cells have spindle/cytokinesis defects. We conclude that the cell contains importin β and transportin “global positioning system”or “GPS” pathways that are mechanistically parallel.

scholarly journals Nup62 is recruited to pathological condensates and promotes TDP-43 insolubility in C9orf72 and sporadic ALS/FTLD.

2021 ◽  
Author(s):  
Amanda Gleixner ◽  
Brandie Morris Verdone ◽  
Charlton Otte ◽  
Nandini Ramesh ◽  
Jenna Gale ◽  
...  
Keyword(s):  
Phase Transitions ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Localization Sequence ◽  
Nuclear Pore ◽  
Permeability Barrier ◽  
Nucleocytoplasmic Trafficking ◽  
Mechanisms Of Toxicity ◽  
Sporadic Als

Abstract Amyotrophic lateral sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD) share clinical, neuropathological, and genetic features. This includes common genetic disease-causing mutations such as the expanded G4C2 repeat in the C9orf72 gene (C9-ALS/FTLD) and cytoplasmic and insoluble protein depositions of the TDP-43 in degenerating regions of the brain and spinal cord. Proposed mechanisms of toxicity in C9-ALS/FTLD are the production of repeat expansion transcripts and their dipeptide repeat proteins (DPRs) products which are hypothesized to drive nucleocytoplasmic transport defects. The nuclear pore complex (NPC) regulates nucleocytoplasmic trafficking by creating a selectivity and permeability barrier comprised of phenylalanine glycine nucleoporins (FG nups). However, the relationship between FG nups and TDP-43 pathology remains elusive. Here, we define two mechanisms through which TDP-43 promotes Nup62 nuclear depletion and cytoplasmic in C9-ALS/FTLD and sALS/FTLD. In C9-ALS/FTLD, poly-GR initiates the formation of TDP-43 containing stress granules (SGs) that trigger the nuclear loss and recruitment of Nup62 in vitro and in vivo. When colocalized, cytoplasmic TDP-43:Nup62 assemblies mature into insoluble inclusions through an interaction within the TDP-43 nuclear localization sequence (NLS) suggesting Nup62 promotes deleterious phase transitions. Absent of poly-GR, aberrant TDP-43 phase transitions in the cytoplasm recruits and mislocalizes Nup62 into pathological inclusions. The result of these cytoplasmic Nup62 and TDP-43 interactions are pathological and insoluble TDP-43:Nup62 assemblies that are observed in C9-ALS/FTLD and sALS/FTLD CNS tissue.

Tumorigenicity of cells transformed by Simian virus 40 and of hybrids between such cells and normal diploid cells

1979 ◽  
Vol 36 (1) ◽  
pp. 223-240
Author(s):  
C.J. Gee ◽  
H. Harris
Keyword(s):  
Mouse Embryo ◽  
Simian Virus 40 ◽  
T Antigen ◽  
Transformed Cells ◽  
Sv40 T Antigen ◽  
Malignant Phenotype ◽  
Embryo Cells ◽  
Diploid Cells

A number of newly isolated clonal cell lines derived from diploid mouse embryo cells transformed by SV40 were examined in vitro and in vivo. Although these lines showed the properties that define transformation in vitro, they were not tumorigenic for many passages after their initial isolation. Cells from tumours eventually produced by the SV40-transformed cells were fused with diploid mouse embryo cells. The hybrids formed were initially non-tumorigenic. This indicates that a normal diploid cell can suppress the malignant phenotype of a tumorigenic SV40-transformed cell. The hybrid cells did, however, express the SV40 T antigen and they nad a clearly transformed phenotype in vitro. It thus appears that neither the transformed phenotype nor the expression of the SV40 T antigen are enough to endow a cell with the ability to grow progressively in vivo. The relationship between the transformed phenotype and tumorigenicity was further studied by fusing malignant mouse melanoma cells with non-tumorigenic SV40-transformed cells. The hybrids expressed the transformed phenotype in vitro but unable to form tumours in vivo. The changes that occur in cells after transformation by SV40 do not apparently affect the ability of these cells to suppress the malignant phenotype of tumour cells.

Sign in / Sign up

Export Citation Format

Share Document