Stimulation of nuclear export and inhibition of nuclear import by a Ran mutant deficient in binding to Ran-binding protein 1
| Autor: | Ralph H. Kehlenbach, Jörg Becker, Ralf Assheuer, Larry Gerace, Angelika Kehlenbach |
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| Rok vydání: | 2001 |
| Předmět: |
Cytoplasm
Guanine Blotting Western Receptors Cytoplasmic and Nuclear Importin Biology Karyopherins environment and public health Biochemistry Importin-alpha Humans Nuclear protein Nuclear export signal Fluorescent Antibody Technique Indirect Molecular Biology Glutathione Transferase Cell Nucleus Binding Sites Dose-Response Relationship Drug Nuclear Proteins Cell Biology Precipitin Tests Recombinant Proteins Cell biology Nuclear Pore Complex Proteins Kinetics Protein Transport ran GTP-Binding Protein Ran Mutation Mutagenesis Site-Directed Nucleoporin Nuclear transport Carrier Proteins Nuclear localization sequence HeLa Cells Protein Binding |
| Zdroj: | The Journal of biological chemistry. 276(17) |
| ISSN: | 0021-9258 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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