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Kinetics of Protein Import into Isolated Xenopus Oocyte Nuclei
Thomas Radtke, Dirk Schmalz, Elias Coutavas, Tarik M. Soliman and Reiner Peters
Proceedings of the National Academy of Sciences of the United States of America
Vol. 98, No. 5 (Feb. 27, 2001), pp. 2407-2412
Published by: National Academy of Sciences
Stable URL: http://www.jstor.org/stable/3055069
Page Count: 6
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An in vitro assay for nucleocytoplasmic transport was established in which signal-dependent protein import is reproduced faithfully by isolated purified nuclei. The assay permits the precise quantification of import kinetics and the discrimination between translocation through the nuclear envelope and intranuclear transport. Nuclei were manually isolated from Xenopus oocytes and after manual purification incubated with a medium containing a green fluorescent transport substrate, karyopherins α2 and β1, a red fluorescent control substrate, an energy mix and, for keeping an osmotic balance, 20% (wt/vol) BSA. Import of transport substrates into the nucleus and exclusion of the control substrate were monitored simultaneously by two-color confocal microscopy. Two widely differing import substrates were used: the recombinant protein P4K [480 kDa, four nuclear localization sequences (NLSs) per P4K tetramer], and NLS-BSA (90 kDa, 15 NLSs). The measurements suggested that import, at the specific conditions used in this study, consisted of two consecutive processes: (i) the rapid equilibration of the concentration difference across the nuclear envelope, a process involving binding and translocation of substrate by the nuclear pore complex, and (ii) the dissipation of the intranuclear concentration difference by diffusion.
Proceedings of the National Academy of Sciences of the United States of America © 2001 National Academy of Sciences