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

Abstract

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 completly 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.

Dynamic equilibrium between calcineurin and kinase activities regulates the phosphorylation state and localization of the nuclear factor of activated T-cells

“…Protein import into the nucleus has been studied in permeabilized cells by using model proteins, allowing the characterization both of factors required for import and of the nuclear pore complex itself [25,26,34,35]. Less is known about protein nuclear export, although it utilizes the same pore complex [36].…”

Ku80 can translocate to the nucleus independent of the translocation of Ku70 using its own nuclear localization signal

“…Digitonin-permeabilized MDBK cells were prepared as described previously (Ikuta et al, 1998) based on the method of Adam et al (1990). The testing solution (10 ml) consisted of various GST-Ku80-GFP vectors and transport bu er (20 mM HEPES, pH 7.3, 110 mM potassium acetate, 2 mM magnesium acetate, 5 mM sodium acetate, 1 mM EGTA, and 2 mM dithiothreitol) containing 1 mg/ml each of aprotinin, leupeptin, and pepstatin.…”

Importin 7 and Importin α/Importin β Are Nuclear Import Receptors for the Glucocorticoid Receptor

“…Cells were lyzed by Dounce homogenization with a type A pestle (20 strokes). After addition of 1/30 volume of 3 M KOAc, homogenate was clarified at 23,500 ϫ g for 20 min and centrifuged at 100,000 ϫ g for 1 h. Supernatant was dialyzed into transport buffer: 20 mM HEPES, pH 7.4, 110 mM KOAc, 2 mM MgOAc, 0.5 mM EGTA, protease inhibitors, and 2 mM DTT and flash-frozen in liquid nitrogen.Nuclear import assays were performed as described (Adam et al, 1990;Nachury and Weis, 1999;Takizawa et al, 1999;Moore and Schwoebel, 2000). Cells were grown on poly-dl-lysine-coated glass coverslips for 48 h. Cells were washed twice with transport buffer, incubated with 40 g/ml digitonin in transport buffer for 6 min on ice, washed twice in transport buffer, and kept on ice for 10 min.…”