Facilitated nucleocytoplasmic shuttling of the Ran binding protein RanBP1.
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 beta. 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.
Plafker
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Markey Center for Cell Signaling and Department of Pharmacology, University of Virginia, Charlottesville, Virginia 22908, USA.
Macara
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D001693:Biological Transport, Active / Q000187:drug effects; D002352:Carrier Proteins / Q000378:metabolism; D002451:Cell Compartmentation; D018797:Cell Cycle Proteins; D002467:Cell Nucleus / Q000378:metabolism*; D003080:Cold Temperature; D003593:Cytoplasm / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004734:Energy Metabolism; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D020662:Guanine Nucleotide Exchange Factors; D006160:Guanosine Triphosphate / Q000378:metabolism; D028884:Karyopherins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D020931:ran GTP-Binding Protein / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000097606:Exportin 1 Protein
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D005231:Fatty Acids, Unsaturated; D049452:Green Fluorescent Proteins; D020662:Guanine Nucleotide Exchange Factors; D006160:Guanosine Triphosphate; D028884:Karyopherins; D008164:Luminescent Proteins; D009687:Nuclear Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D020931:ran GTP-Binding Protein; D000097606:Exportin 1 Protein; C091775:ran-binding protein 1; C038753:leptomycin B