Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Nuclear protein import in permeabilized mammalian cells requires soluble cytoplasmic factors.
Adam SA, Marr RS, Gerace L.
???displayArticle.abstract???
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.
Adam,
Identification of specific binding proteins for a nuclear location sequence.
1989, Pubmed
Adam,
Identification of specific binding proteins for a nuclear location sequence.
1989,
Pubmed Akey,
Protein import through the nuclear pore complex is a multistep process.
1989,
Pubmed
,
Xenbase Bittner,
Effects of tetanus toxin on catecholamine release from intact and digitonin-permeabilized chromaffin cells.
1988,
Pubmed Breeuwer,
Facilitated nuclear transport of histone H1 and other small nucleophilic proteins.
1990,
Pubmed Colbeau,
Enzymic characterization and lipid composition of rat liver subcellular membranes.
1971,
Pubmed Dabauvalle,
Inhibition of nuclear accumulation of karyophilic proteins in living cells by microinjection of the lectin wheat germ agglutinin.
1988,
Pubmed
,
Xenbase Davis,
Nuclear pore complex contains a family of glycoproteins that includes p62: glycosylation through a previously unidentified cellular pathway.
1987,
Pubmed Dworetzky,
Translocation of RNA-coated gold particles through the nuclear pores of oocytes.
1988,
Pubmed
,
Xenbase Dworetzky,
The effects of variations in the number and sequence of targeting signals on nuclear uptake.
1988,
Pubmed
,
Xenbase Featherstone,
A monoclonal antibody against the nuclear pore complex inhibits nucleocytoplasmic transport of protein and RNA in vivo.
1988,
Pubmed
,
Xenbase Feldherr,
Evidence for mediated protein uptake by amphibian oocyte nuclei.
1983,
Pubmed Feldherr,
Movement of a karyophilic protein through the nuclear pores of oocytes.
1984,
Pubmed
,
Xenbase Finlay,
Inhibition of in vitro nuclear transport by a lectin that binds to nuclear pores.
1987,
Pubmed
,
Xenbase Finlay,
Reconstitution of biochemically altered nuclear pores: transport can be eliminated and restored.
1990,
Pubmed
,
Xenbase Franke,
The ultrastructure of the nuclear envelope of amphibian oocytes: a reinvestigation. I. The mature oocyte.
1970,
Pubmed Franke,
On the universality of nuclear pore complex structure.
1970,
Pubmed Franke,
The nuclear envelope and the architecture of the nuclear periphery.
1981,
Pubmed Gerace,
Functional organization of the nuclear envelope.
1988,
Pubmed Gerace,
Identification of a major polypeptide of the nuclear pore complex.
1982,
Pubmed Goldfarb,
Synthetic peptides as nuclear localization signals.
,
Pubmed
,
Xenbase Hanover,
O-linked N-acetylglucosamine is attached to proteins of the nuclear pore. Evidence for cytoplasmic and nucleoplasmic glycoproteins.
1987,
Pubmed Holt,
Nuclear pore complex glycoproteins contain cytoplasmically disposed O-linked N-acetylglucosamine.
1987,
Pubmed Holt,
The subcellular distribution of terminal N-acetylglucosamine moieties. Localization of a novel protein-saccharide linkage, O-linked GlcNAc.
1986,
Pubmed Imamoto-Sonobe,
ATP-dependent association of nuclear proteins with isolated rat liver nuclei.
1988,
Pubmed
,
Xenbase Kalderon,
A short amino acid sequence able to specify nuclear location.
1984,
Pubmed Kalinich,
In vitro translocation through the yeast nuclear envelope. Signal-dependent transport requires ATP and calcium.
1989,
Pubmed Kotzin,
Monoclonal anti-histone autoantibodies derived from murine models of lupus.
1984,
Pubmed Lanford,
Effect of basic and nonbasic amino acid substitutions on transport induced by simian virus 40 T-antigen synthetic peptide nuclear transport signals.
1988,
Pubmed Lanford,
Construction and characterization of an SV40 mutant defective in nuclear transport of T antigen.
1984,
Pubmed Lazarovici,
Affinity purified tetanus toxin binds to isolated chromaffin granules and inhibits catecholamine release in digitonin-permeabilized chromaffin cells.
1989,
Pubmed Lee,
Identification and characterization of a nuclear localization sequence-binding protein in yeast.
1989,
Pubmed Li,
Identification of a human protein that interacts with nuclear localization signals.
1989,
Pubmed Lohka,
Roles of cytosol and cytoplasmic particles in nuclear envelope assembly and sperm pronuclear formation in cell-free preparations from amphibian eggs.
1984,
Pubmed
,
Xenbase Markland,
Signal-dependent translocation of simian virus 40 large-T antigen into rat liver nuclei in a cell-free system.
1987,
Pubmed Nelson,
Context affects nuclear protein localization in Saccharomyces cerevisiae.
1989,
Pubmed Newmeyer,
In vitro transport of a fluorescent nuclear protein and exclusion of non-nuclear proteins.
1986,
Pubmed
,
Xenbase Newmeyer,
Nuclear import can be separated into distinct steps in vitro: nuclear pore binding and translocation.
1988,
Pubmed
,
Xenbase Newmeyer,
An N-ethylmaleimide-sensitive cytosolic factor necessary for nuclear protein import: requirement in signal-mediated binding to the nuclear pore.
1990,
Pubmed
,
Xenbase Newmeyer,
Assembly in vitro of nuclei active in nuclear protein transport: ATP is required for nucleoplasmin accumulation.
1986,
Pubmed
,
Xenbase Paine,
Nuclear envelope permeability.
1975,
Pubmed Richardson,
Nuclear protein migration involves two steps: rapid binding at the nuclear envelope followed by slower translocation through nuclear pores.
1988,
Pubmed
,
Xenbase Schickedanz,
Kinetics of nuclear transport and oligomerization of simian virus 40 large T antigen.
1986,
Pubmed Silver,
Yeast proteins that recognize nuclear localization sequences.
1989,
Pubmed
,
Xenbase Snow,
Monoclonal antibodies identify a group of nuclear pore complex glycoproteins.
1987,
Pubmed
,
Xenbase Wolff,
Nuclear protein import: specificity for transport across the nuclear pore.
1988,
Pubmed Yamasaki,
Identification of four nuclear transport signal-binding proteins that interact with diverse transport signals.
1989,
Pubmed
,
Xenbase Yoneda,
Reversible inhibition of protein import into the nucleus by wheat germ agglutinin injected into cultured cells.
1987,
Pubmed
,
Xenbase
