Nucleocytoplasmic shuttling factors including Ran and CRM1 mediate nuclear export of NFAT In vitro.
We have developed a permeabilized cell assay to study the nuclear export of the shuttling transcription factor NFAT, which contains a leucine-rich export signal. The assay uses HeLa cells that are stably transfected with NFAT fused to the green fluorescent protein (GFP). Nuclear export of GFP-NFAT in digitonin-permeabilized cells occurs in a temperature- and ATP-dependent manner and can be quantified by flow cytometry. In vitro NFAT export requires the GTPase Ran, which is released from cells during the digitonin permeabilization. At least one additional rate-limiting export factor is depleted from permeabilized cells by a preincubation at 30 degrees C in the absence of cytosol. This activity can be provided by cytosolic or nucleoplasmic extracts in a subsequent export step. Using this assay, we have purified a second major export activity from cytosol. We found that it corresponds to CRM1, a protein recently reported to be a receptor for certain leucine-rich export sequences. CRM1 appears to be imported into the nucleus by a Ran-dependent mechanism that is distinct from conventional signaling pathways. Considered together, our studies directly demonstrate by fractionation and reconstitution that nuclear export of NFAT is mediated by multiple nucleocytoplasmic shuttling factors, including Ran and CRM1.
Kehlenbach
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RH
Department of Cell Biology and Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Dickmanns
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Gerace
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D000255:Adenosine Triphosphate / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D002463:Cell Membrane Permeability; D002467:Cell Nucleus / Q000502:physiology*; D003593:Cytoplasm / Q000502:physiology; D003600:Cytosol / Q000502:physiology; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism*; D005434:Flow Cytometry; D019204:GTP-Binding Proteins / Q000378:metabolism; D005982:Glutathione Transferase / Q000096:biosynthesis; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D028884:Karyopherins; D007700:Kinetics; D008164:Luminescent Proteins / Q000096:biosynthesis; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014157:Transcription Factors / Q000096:biosynthesis / Q000378:metabolism*; D014162:Transfection; D020931:ran GTP-Binding Protein; D000097606:Exportin 1 Protein
D000255:Adenosine Triphosphate; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D019204:GTP-Binding Proteins; D005982:Glutathione Transferase; D049452:Green Fluorescent Proteins; D028884:Karyopherins; D008164:Luminescent Proteins; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D020931:ran GTP-Binding Protein; D000097606:Exportin 1 Protein