scholarly journals Nuclear protein import in permeabilized mammalian cells requires soluble cytoplasmic factors.

1990 ◽  
Vol 111 (3) ◽  
pp. 807-816 ◽  
Author(s):  
S A Adam ◽  
R S Marr ◽  
L Gerace
Keyword(s):  
Nuclear Import ◽  
Mammalian Cells ◽  
Protein Import ◽  
Nuclear Protein ◽  
T Antigen ◽  
Nuclear Pore ◽  
Permeabilized Cell ◽  
Cytoplasmic Factor ◽  
Nuclear Location ◽  
Nuclear Protein Import

We have developed an in vitro system involving digitonin-permeabilized vertebrate cells to study biochemical events in the transport of macromolecules across the nuclear envelope. While treatment of cultured cells with digitonin permeabilizes the plasma membranes to macromolecules, the nuclear envelopes remain structurally intact and nuclei retain the ability to transport and accumulate proteins containing the SV40 large T antigen nuclear location sequence. Transport requires addition of exogenous cytosol to permeabilized cells, indicating the soluble cytoplasmic factor(s) required for nuclear import are released during digitonin treatment. In this reconstituted import system, a protein containing a nuclear location signal is rapidly accumulated in nuclei, where it reaches a 30-fold concentration compared to the surrounding medium within 30 min. Nuclear import is specific for a functional nuclear location sequence, requires ATP and cytosol, and is temperature dependent. Furthermore, accumulation of the transport substrate within nuclei is completely inhibited by wheat germ agglutinin, which binds to nuclear pore complexes and inhibits transport in vivo. Together, these results indicate that the permeabilized cell system reproduces authentic nuclear protein import. In a preliminary biochemical dissection of the system, we observe that the sulfhydryl alkylating reagent N-ethylmaleimide inactivates both cytosolic factor(s) and also component(s) in the insoluble permeabilized cell fraction required for nuclear protein import. Because this permeabilized cell model is simple, efficient, and works effectively with cells and cytosol fractions prepared from a variety of different vertebrate sources, it will prove powerful for investigating the biochemical pathway of nuclear transport.

scholarly journals An N-ethylmaleimide-sensitive cytosolic factor necessary for nuclear protein import: requirement in signal-mediated binding to the nuclear pore.

1990 ◽  
Vol 110 (3) ◽  
pp. 547-557 ◽  
Author(s):  
D D Newmeyer ◽  
D J Forbes
Keyword(s):  
Nuclear Import ◽  
Protein Import ◽  
Nuclear Protein ◽  
Signal Sequence ◽  
Nuclear Pore ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Sensitive Factor ◽  
Nuclear Protein Import ◽  
Cytosolic Factor

We described previously an assay for authentic nuclear protein import in vitro. In this assay, exogenous nuclei are placed in an extract of Xenopus eggs; a rhodamine-labeled protein possessing a nuclear localization signal is added, and fluorescence microscopy is used to measure nuclear uptake. The requirement in this system for a cytosolic extract suggests that nuclear import is dependent on at least one cytosolic factor. We now confirm this hypothesis. Treatment of the cytosol with N-ethylmaleimide (NEM) abolishes nuclear protein import; readdition of a cytosolic fraction to the NEM-inactivated extract rescues transport. Thus, at least one NEM-sensitive factor required for transport is supplied by the cytosol. This activity, called nuclear import factor-1, or NIF-1, is ammonium-sulfate-precipitable, protease-sensitive, and heat-labile; it is therefore at least partly proteinaceous. NIF-1 stimulates, in a concentration-dependent manner, the rate at which individual nuclei accumulate protein. The effect of NIF-1 is enhanced by a second cytosolic NEM-sensitive factor, NIF-2. Earlier we identified two steps in the nuclear import reaction: (a) ATP-independent binding of a signal-sequence-bearing protein to the nuclear pore; and (b) ATP-dependent translocation of that protein through the pore. We now show that NEM inhibits signal-mediated binding, and that readdition of NIF-1 restores binding. Thus, NIF-1 is required for at least the binding step and does not require ATP for its activity. NIF-1 may act as a cytoplasmic signal receptor that escorts signal-bearing proteins to the pore, or may instead promote signal-mediated binding to the pore in another manner, as discussed.

scholarly journals O-linked glycoproteins of the nuclear pore complex interact with a cytosolic factor required for nuclear protein import.

1992 ◽  
Vol 116 (2) ◽  
pp. 271-280 ◽  
Author(s):  
R Sterne-Marr ◽  
J M Blevitt ◽  
L Gerace
Keyword(s):  
Nuclear Pore Complex ◽  
Mammalian Cells ◽  
Protein Import ◽  
Nuclear Protein ◽  
Nuclear Pore ◽  
Reticulocyte Lysate ◽  
Nuclear Protein Import ◽  
Cytosolic Factor ◽  
Cytosolic Factors

Mediated import of proteins into the nucleus requires cytosolic factors and can be blocked by reagents that bind to O-linked glycoproteins of the nuclear pore complex. To investigate whether a cytosolic transport factor directly interacts with these glycoproteins, O-linked glycoproteins from rat liver nuclear envelopes were immobilized on Sepharose beads via wheat germ agglutinin or specific antibodies. When rabbit reticulocyte lysate (which provides cytosolic factors required for in vitro nuclear import) was incubated with the immobilized glycoproteins, the cytosol was found to be inactivated by up to 80% in its ability to support mediated protein import in permeabilized mammalian cells. Inactivation of the import capacity of cytosol, which was specifically attributable to the glycoproteins, involves stoichiometric interactions and is likely to involve binding and depletion of a required factor from the cytosol. This factor is distinct from an N-ethylmaleimide-sensitive receptor for nuclear localization sequences characterized recently since it is insensitive to N-ethylmaleimide. Cytosol inactivation is suggested to be caused by at least two proteins of the glycoprotein fraction, although substantial capacity for inactivation can be attributed to protein bound by the RL11 antibody, consisting predominantly of a 180-kD glycosylated polypeptide. Considered together, these experiments identify a novel cytosolic factor required for nuclear protein import that directly interacts with O-linked glycoproteins of the pore complex, and provide a specific assay for isolation of this component.

scholarly journals RanBP1 stabilizes the interaction of Ran with p97 nuclear protein import.

1996 ◽  
Vol 135 (3) ◽  
pp. 559-569 ◽  
Author(s):  
N C Chi ◽  
E J Adam ◽  
G D Visser ◽  
S A Adam
Keyword(s):  
Protein Import ◽  
Nuclear Protein ◽  
Gel Filtration ◽  
Nuclear Pore ◽  
Binding Assays ◽  
Permeabilized Cell ◽  
Trimeric Complex ◽  
Cell Extracts ◽  
Nuclear Protein Import ◽  
Ran Gtp

Three factors have been identified that reconstitute nuclear protein import in a permeabilized cell assay: the NLS receptor, p97, and Ran/TC4. Ran/TC4, in turn, interacts with a number of proteins that are involved in the regulation of GTP hydrolysis or are components of the nuclear pore. Two Ran-binding proteins, RanBP1 and RanBP2, form discrete complexes with p97 as demonstrated by immunoadsorption from HeLa cell extracts fractionated by gel filtration chromatography. A > 400-kD complex contains p97, Ran, and RanBP2. Another complex of 150-300 kD was comprised of p97, Ran, and RanBP1. This second trimeric complex could be reconstituted from recombinant proteins. In solution binding assays, Ran-GTP bound p97 with high affinity, but the binding of Ran-GDP to p97 was undetectable. The addition of RanBP1 with Ran-GDP or Ran-GTP increased the affinity of both forms of Ran for p97 to the same level. Binding of Ran-GTP to p97 dissociated p97 from immobilized NLS receptor while the Ran-GDP/RanBP1/p97 complex did not dissociate from the receptor. In a digitonin-permeabilized cell docking assay, RanBP1 stabilizes the receptor complex against temperature-dependent release from the pore. When added to an import assay with recombinant NLS receptor, p97 and Ran-GDP, RanBP1 significantly stimulates transport. These results suggest that RanBP1 promotes both the docking and translocation steps in nuclear protein import by stabilizing the interaction of Ran-GDP with p97.

scholarly journals A T42A Ran Mutation: Differential Interactions with Effectors and Regulators, and Defect in Nuclear Protein Import

1997 ◽  
Vol 8 (12) ◽  
pp. 2591-2604 ◽  
Author(s):  
Gretchen A. Murphy ◽  
Mary Shannon Moore ◽  
George Drivas ◽  
Pablo Pérez de la Ossa ◽  
Alicia Villamarin ◽  
...  
Keyword(s):  
Cell Cycle Progression ◽  
Protein Import ◽  
Nuclear Protein ◽  
Binding Protein ◽  
Nuclear Pore ◽  
Nucleotide Exchange ◽  
Wild Type ◽  
Direct Role ◽  
Permeabilized Cell ◽  
Nuclear Protein Import

Ran, the small, predominantly nuclear GTPase, has been implicated in the regulation of a variety of cellular processes including cell cycle progression, nuclear-cytoplasmic trafficking of RNA and protein, nuclear structure, and DNA synthesis. It is not known whether Ran functions directly in each process or whether many of its roles may be secondary to a direct role in only one, for example, nuclear protein import. To identify biochemical links between Ran and its functional target(s), we have generated and examined the properties of a putative Ran effector mutation, T42A-Ran. T42A-Ran binds guanine nucleotides as well as wild-type Ran and responds as well as wild-type Ran to GTP or GDP exchange stimulated by the Ran-specific guanine nucleotide exchange factor, RCC1. T42A-Ran·GDP also retains the ability to bind p10/NTF2, a component of the nuclear import pathway. In contrast to wild-type Ran, T42A-Ran·GTP binds very weakly or not detectably to three proposed Ran effectors, Ran-binding protein 1 (RanBP1), Ran-binding protein 2 (RanBP2, a nucleoporin), and karyopherin β (a component of the nuclear protein import pathway), and is not stimulated to hydrolyze bound GTP by Ran GTPase-activating protein, RanGAP1. Also in contrast to wild-type Ran, T42A-Ran does not stimulate nuclear protein import in a digitonin permeabilized cell assay and also inhibits wild-type Ran function in this system. However, the T42A mutation does not block the docking of karyophilic substrates at the nuclear pore. These properties of T42A-Ran are consistent with its classification as an effector mutant and define the exposed region of Ran containing the mutation as a probable effector loop.

scholarly journals Identification of cytosolic factors required for nuclear location sequence-mediated binding to the nuclear envelope.

1994 ◽  
Vol 125 (3) ◽  
pp. 547-555 ◽  
Author(s):  
E J Adam ◽  
S A Adam
Keyword(s):  
Nuclear Envelope ◽  
Protein Import ◽  
T Antigen ◽  
Nuclear Pore ◽  
Temperature Dependent ◽  
Permeabilized Cell ◽  
Nuclear Location ◽  
Dependent Modification ◽  
Bovine Erythrocytes ◽  
Cytosolic Factors

Nuclear protein import can be separated into two distinct steps: binding to the nuclear pore complex followed by translocation to the nuclear interior. A previously identified nuclear location sequence (NLS) receptor and a 97-kD protein purified from bovine erythrocytes reconstitute the binding step in a permeabilized cell assay. Binding to the envelope is specific for a functional SV-40 large T antigen NLS and is not ATP or temperature dependent. Modification of p97 with N-ethylmaleimide (NEM) decreases binding to the pore, but interestingly, NEM treatment of the NLS receptor does not. Nuclear envelope binding is inhibited by wheat germ agglutinin suggesting a possible mechanism for the inhibition of transport by the lectin.

scholarly journals p34cdc2-mediated phosphorylation at T124 inhibits nuclear import of SV-40 T antigen proteins.

1991 ◽  
Vol 115 (5) ◽  
pp. 1203-1212 ◽  
Author(s):  
D A Jans ◽  
M J Ackermann ◽  
J R Bischoff ◽  
D H Beach ◽  
R Peters
Keyword(s):  
Nuclear Import ◽  
Protein Import ◽  
Nuclear Protein ◽  
T Antigen ◽  
Regulatory Proteins ◽  
Nuclear Accumulation ◽  
Nuclear Localization Sequence ◽  
Kinetic Measurements ◽  
Nuclear Protein Import

The nuclear import of transcription regulatory proteins appears to be used by the cell to trigger transitions in cell cycle, morphogenesis, and transformation. We have previously observed that the rate at which SV-40 T antigen fusion proteins containing a functional nuclear localization sequence (NLS; residues 126-132) are imported into the nucleus is enhanced in the presence of the casein kinase II (CK-II) site S111/112. In this study purified p34cdc2 kinase was used to phosphorylate T antigen proteins specifically at T124 and kinetic measurements at the single-cell level performed to assess its effect on nuclear protein import. T124 phosphorylation, which could be functionally simulated by a T-to-D124 substitution, was found to reduce the maximal extent of nuclear accumulation whilst negligibly affecting the import rate. The inhibition of nuclear import depended on the stoichiometry of phosphorylation. T124 and S111/112 could be phosphorylated independently of one another. Two alternative mechanisms were considered to explain the inhibition of nuclear import by T124 phosphorylation: inactivation of the NLS and cytoplasmic retention, respectively. Furthermore, we speculate that in vivo T124 phosphorylation may regulate the small but functionally significant amount of cytoplasmic SV-40 T antigen. A sequence comparison showed that many transcription regulatory proteins contain domains comprising potential CK-II-sites, cdc2-sites, and NLS. This raises the possibility that the three elements represent a functional unit regulating nuclear protein import.

scholarly journals A GTPase distinct from Ran is involved in nuclear protein import.

1996 ◽  
Vol 133 (5) ◽  
pp. 971-983 ◽  
Author(s):  
D J Sweet ◽  
L Gerace
Keyword(s):  
Nuclear Import ◽  
Protein Import ◽  
Nuclear Protein ◽  
Inhibitory Effect ◽  
Nuclear Pore ◽  
Mutant Form ◽  
Direct Role ◽  
Nuclear Protein Import ◽  
Dependent Transport

Signal-dependent transport of proteins into the nucleus is a multi-step process mediated by nuclear pore complexes and cytosolic transport factors. One of the cytosolic factors, Ran, is the only GTPase that has a characterized role in the nuclear import pathway. We have used a mutant form of Ran with altered nucleotide binding specificity to investigate whether any other GTPases are involved in nuclear protein import. D125N Ran (XTP-Ran) binds specifically to xanthosine triphosphate (XTP) and has a greatly reduced affinity for GTP, so it is no longer sensitive to inhibition by nonhydrolyzable analogues of GTP such as guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). using in vitro transport assays, we have found that nuclear import supported by XTP-Ran is nevertheless inhibited by the addition of non-hydrolyzable GTP analogues. This in conjunction with the properties of the inhibitory effect indicates that at least one additional GTPase is involved in the import process. Initial characterization suggests that the inhibited GTPase plays a direct role in protein import and could be a component of the nuclear pore complex.

scholarly journals Sequence and characterization of cytoplasmic nuclear protein import factor p97.

1995 ◽  
Vol 130 (2) ◽  
pp. 265-274 ◽  
Author(s):  
N C Chi ◽  
E J Adam ◽  
S A Adam
Keyword(s):  
Nuclear Envelope ◽  
Metal Ion ◽  
Protein Import ◽  
Nuclear Protein ◽  
Binding Activity ◽  
Nuclear Pore ◽  
Permeabilized Cell ◽  
Cytosolic Proteins ◽  
Cell Assay ◽  
Nuclear Protein Import

Nuclear location sequence-mediated binding of karyophilic proteins to the nuclear pore complexes is one of the earliest steps in nuclear protein import. We previously identified two cytosolic proteins that reconstitute this step in a permeabilized cell assay: the 54/56-kD NLS receptor and p97. A monoclonal antibody to p97 localizes the protein to the cytoplasm and the nuclear envelope. p97 is extracted from nuclear envelopes under the same conditions as the O-glycosylated nucleoporins indicating a tight association with the pore complex. The antibody inhibits import in a permeabilized cell assay but does not affect binding of karyophiles to the nuclear pore complex. Immunodepletion of p97 renders the cytosol inactive for import and identifies at least three other cytosolic proteins that interact with p97. cDNA cloning of p97 shows that it is a unique protein containing 23 cysteine residues. Recombinant p97 binds zinc and a bound metal ion is required for the nuclear envelope binding activity of the protein.

scholarly journals Two Isoforms of Npap60 (Nup50) Differentially Regulate Nuclear Protein Import

2010 ◽  
Vol 21 (4) ◽  
pp. 630-638 ◽  
Author(s):  
Yutaka Ogawa ◽  
Yoichi Miyamoto ◽  
Munehiro Asally ◽  
Masahiro Oka ◽  
Yoshinari Yasuda ◽  
...  
Keyword(s):  
Nuclear Import ◽  
Protein Import ◽  
Nuclear Protein ◽  
Time Lapse ◽  
Binding Assays ◽  
Vitro Binding ◽  
Importin Α ◽  
Nuclear Protein Import

Npap60 (Nup50) is a nucleoporin that binds directly to importin α. In humans, there are two Npap60 isoforms: the long (Npap60L) and short (Npap60S) forms. In this study, we provide both in vitro and in vivo evidence that Npap60L and Npap60S function differently in nuclear protein import. In vitro binding assays revealed that Npap60S stabilizes the binding of importin α to classical NLS-cargo, whereas Npap60L promotes the release of NLS-cargo from importin α. In vivo time-lapse experiments showed that when the Npap60 protein level is controlled, allowing CAS to efficiently promote the dissociation of the Npap60/importin α complex, Npap60S and Npap60L suppress and accelerate the nuclear import of NLS-cargo, respectively. These results demonstrate that Npap60L and Npap60S have opposing functions and suggest that Npap60L and Npap60S levels must be carefully controlled for efficient nuclear import of classical NLS-cargo in humans. This study provides novel evidence that nucleoporin expression levels regulate nuclear import efficiency.

Abstract P217: Nuclear Protein Import as the Missing Link in Phenylephrine-Induced Cardiomyocyte Hypertrophy

2011 ◽  
Vol 109 (suppl_1) ◽  
Author(s):  
Mirna N Chahine ◽  
Maxime Mioulane ◽  
Gabor Földes ◽  
Alexander Lyon ◽  
Sian E Harding
Keyword(s):  
Gene Expression ◽  
Nuclear Export ◽  
Protein Import ◽  
Nuclear Protein ◽  
Nuclear Translocation ◽  
Cardiomyocyte Hypertrophy ◽  
Nuclear Pore ◽  
Nucleocytoplasmic Trafficking ◽  
Adult Rat ◽  
Nuclear Protein Import

During cardiac hypertrophy, cardiomyocytes (CM) present alterations in gene expression and increased contractile protein content. Nuclear protein import (NPI) is critical in regulating gene expression, transcription, and subsequently cell hypertrophy. However, it is unknown how the nuclear transport machinery (transport receptors and nuclear pore complex (NPC)) functions to sustain increased demands for nucleocytoplasmic trafficking. The aim of this study was to determine if exposure of adult CM to phenylephrine (PE) affects hypertrophy by altering NPI and NPC density. Comparisons were made to adult failing rat and human CM. Rat myocytes were enzymatically isolated from adult hearts, and used for immunocytochemistry, qPCR and western immunoblotting. Failing CM were obtained from explanted human hearts at the time of transplant and from a rat model of myocardial infarction-induced hypertrophy and failure. Rat adult CM exposed for 48h to PE were injected with a protein import substrate (Alexa488-BSA-NLS) to visually monitor nuclear import with the confocal microscope. The effects of P38 MAPK inhibitor, HDAC inhibitor, Exportin-1 (CRM-1) inhibitor, and GSK-3 β inhibitor were investigated. Cell and nuclear sizes were increased in PE treated-adult rat CM and in the adult failing rat and human CM compared to normal CM. In contrast, PE depressed the rate and maximal NPI (by 65 +/- 3.4 % (3.55 from 5.46), p<0.05) as well as nucleoporin p62 mRNA and protein expression levels in adult rat CM compared to non-treated CM. Nucleoporin p62, cytoplasmic Ranbp1, and nuclear translocation of importins (Imp.α and β) relative densities were also decreased in PE treated-adult rat CM and in adult failing rat CM and human heart tissue compared to normal controls. On the contrary, CRM-1 nuclear export relative density was increased during the same pathological conditions. Thus NPI downregulation is linked to an increased nuclear export required by CM to generate the hypertrophic phenotype. All these effects were P38MAPK, HDAC and CRM-1 dependent but GSK-3Beta independent in rat CM. Our results show that alterations in NPI and NPC density occur in failing CM as well as in CM under hypertrophic stimuli. NPI may represent a critical therapeutic target in hypertrophic conditions.

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