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Molecular and Cellular Biology, May 2000, p. 3510-3521, Vol. 20, No. 10
Markey Center for Cell Signaling and
Department of Pharmacology, University of Virginia,
Charlottesville, Virginia 22908
Received 7 September 1999/Returned for modification 12 October
1999/Accepted 21 February 2000
The Ran binding protein RanBP1 is localized to the cytosol of
interphase cells. A leucine-rich nuclear export signal (NES) near the C
terminus of RanBP1 is essential to maintain this distribution. We now
show that RanBP1 accumulates in nuclei of cells treated with the export
inhibitor, leptomycin B, and collapse of the
nucleocytoplasmic Ran:GTP gradient leads to equilibration of
RanBP1 across the nuclear envelope. Low temperature prevents nuclear
accumulation of RanBP1, suggesting that import does not occur via
simple diffusion. Glutathione S-transferase
(GST)-RanBP1(1-161), which lacks the NES, accumulates in the nucleus
after cytoplasmic microinjection. In permeabilized cells,
nuclear accumulation of GST-RanBP1(1-161) requires nuclear Ran:GTP but
is not inhibited by a dominant interfering G19V mutant of Ran. Nuclear
accumulation is enhanced by addition of exogenous karyopherins/importins or RCC1, both of which also enhance nuclear Ran
accumulation. Import correlates with Ran concentration. Remarkably, an
E37K mutant of RanBP1 does not import into the nuclei under any
conditions tested despite the fact that it can form a ternary complex
with Ran and importin
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Facilitated Nucleocytoplasmic Shuttling of the Ran
Binding Protein RanBP1
. These data indicate that RanBP1 translocates
through the pores by an active, nonclassical mechanism and requires
Ran:GTP for nuclear accumulation. Shuttling of RanBP1 may function to
clear nuclear pores of Ran:GTP, to prevent premature release of import
cargo from transport receptors.
*
Corresponding author. Mailing address: Room 7191 Hospital West, P.O. Box 800577, HSC, University of Virginia School of
Medicine, Charlottesville, VA 22908. Phone: (804) 982-0074. Fax: (804)
924-1236. E-mail: imacara{at}virginia.edu.
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