Access

You are not currently logged in.

Access your personal account or get JSTOR access through your library or other institution:

login

Log in to your personal account or through your institution.

If You Use a Screen Reader

This content is available through Read Online (Free) program, which relies on page scans. Since scans are not currently available to screen readers, please contact JSTOR User Support for access. We'll provide a PDF copy for your screen reader.

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
Stable URL: http://www.jstor.org/stable/3055069
Page Count: 6
  • Read Online (Free)
  • Subscribe ($19.50)
  • Cite this Item
Since scans are not currently available to screen readers, please contact JSTOR User Support for access. We'll provide a PDF copy for your screen reader.
Kinetics of Protein Import into Isolated Xenopus Oocyte Nuclei
Preview not available

Abstract

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.

Page Thumbnails

  • Thumbnail: Page 
2407
    2407
  • Thumbnail: Page 
2408
    2408
  • Thumbnail: Page 
2409
    2409
  • Thumbnail: Page 
2410
    2410
  • Thumbnail: Page 
2411
    2411
  • Thumbnail: Page 
2412
    2412