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Vol. 18, Issue 11, 4365-4376, November 2007

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Hyperosmotic Stress Signaling to the Nucleus Disrupts the Ran Gradient and the Production of RanGTP

Center for Cell Signaling, Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, VA 22908

Submitted January 31, 2007; Revised August 2, 2007; Accepted August 17, 2007
Monitoring Editor: Karsten Weis

The RanGTP gradient depends on nucleocytoplasmic shuttling of Ran and its nucleotide exchange in the nucleus. Here we show that hyperosmotic stress signaling induced by sorbitol disrupts the Ran protein gradient and reduces the production of RanGTP. Ran gradient disruption is rapid and is followed by early (10–20 min) and late (30–60 min) phases of recovery. Results from SB203580 and siRNA experiments suggest the stress kinase p38 is important for Ran gradient recovery. NTF2 and Mog1, which are transport factors that regulate the nuclear localization of Ran, showed kinetics of delocalization and recovery similar to Ran. Microinjection of a nuclear localization signal reporter protein revealed that sorbitol stress decreases the rate of nuclear import. Sorbitol stress also slowed RCC1 mobility in the nucleus, which is predicted to reduce RCC1 dissociation from chromatin and RanGTP production. This was tested using a FRET biosensor that registers nuclear RanGTP levels, which were reduced in response to sorbitol stress. Although sorbitol alters nucleotide levels, we show that inverting the GTP/GDP ratio in cells is not sufficient to disrupt the Ran gradient. Thus, the Ran system is a target of hyperosmotic stress signaling, and cells use protein localization–based mechanisms as part of a rapid stress response.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

Address correspondence to: Bryce M. Paschal (paschal{at}virginia.edu)

Abbreviations used: FRAP, fluorescence recovery after photobleaching; FRET, fluorescence resonance energy transfer; YIC, yellow fluorescent protein, importin-–binding domain, cyan fluorescent protein; GGNLS, GST-GFP-NLS; YFP, yellow fluorescent protein; CFP, cyan fluorescent protein; IBB, importin-–binding domain; N/C, ratio of mean nuclear fluorescence to mean cytoplasmic fluorescence.