Stimulation of nuclear export and inhibition of nuclear import by a Ran mutant deficient in binding to Ran-binding protein 1.
Receptor-mediated nucleocytoplasmic transport is dependent on the GTPase Ran and Ran-binding protein 1 (RanBP1). The acidic C terminus of Ran is required for high affinity interaction between Ran and RanBP1. We found that a novel Ran mutant with four of its five acidic C-terminal amino acids modified to alanine (RanC4A) has an approximately 20-fold reduced affinity for RanBP1. We investigated the effects of RanC4A on nuclear import and export in permeabilized HeLa cells. Although RanC4A promotes accumulation of the nuclear export receptor CRM1 at the cytoplasmic nucleoporin Nup214, it strongly stimulates nuclear export of GFP-NFAT. Since RanC4A exhibits an elevated affinity for CRM1 and other nuclear transport receptors, this suggests that formation of the export complex containing CRM1, Ran-GTP, and substrate is a rate-limiting step in export, not release from Nup214. Conversely, importin alpha/beta-dependent nuclear import of bovine serum albumin, coupled to a classical nuclear localization sequence is strongly inhibited by RanC4A. Inhibition can be reversed by additional importin alpha, which promotes the formation of an importin alpha/beta complex. These results provide physiological evidence that release of Ran-GTP from importin beta by RanBP1 and importin alpha is critical for the recycling of importin beta to a transport-competent state.
Kehlenbach
R H
RH
Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA. Ralph.Kehlenbach@med.uni-heidelberg.de
Assheuer
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R
Kehlenbach
A
A
Becker
J
J
Gerace
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D001665:Binding Sites; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D019084:Fluorescent Antibody Technique, Indirect; D005982:Glutathione Transferase / Q000378:metabolism; D006147:Guanine / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D028884:Karyopherins; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D011233:Precipitin Tests; D011485:Protein Binding; D021381:Protein Transport; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins / Q000378:metabolism; D020931:ran GTP-Binding Protein / Q000235:genetics* / Q000378:metabolism*; D000097606:Exportin 1 Protein
D002352:Carrier Proteins; D005982:Glutathione Transferase; D006147:Guanine; D028884:Karyopherins; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins; D020931:ran GTP-Binding Protein; D000097606:Exportin 1 Protein; C085914:NUP214 protein, human; C091775:ran-binding protein 1