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Question: To test the hypothesis that the directionality of transport across the nuclear membrane is determ…


To test the hypothesis that the directionality of transport across the nuclear membrane is determined primarily by the gradient of the Ran-GDP outside the nucleus and Ran-GTP inside the nucleus, you decide to reverse the gradient to see if you can force the import of a protein that is normally exported from the nucleus. You add a well-defined nuclear export substrate, fluorescent BSA coupled with a nuclear export signal (NES-BSA), to the standard permeabilized cell assay. Sure enough, it is excluded from the nuclei (Figure A). Now you add Crm1, the nuclear export receptor that recognizes the export signal, and RanQ69L-GTP, the mutant formm of Ran that cannot hydrolyze GTP. With these additions, the tagged BSA now enters the nuclei (Figure B). Unlike conventional nuclear import, the maximum nuclear concentration of NES-BSA is no higher than in the cytoplasm surrounding the nucleus.

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To test the hypothesis that the directionality of transport across the nuclear membrane is determined primarily by the gradient of the Ran-GDP outside the nucleus and Ran-GTP inside the nucleus, you decide to reverse the gradient to see if you can force the import of a protein that is normally exported from the nucleus. You add a well-defined nuclear export substrate, fluorescent BSA coupled with a nuclear export signal (NES-BSA), to the standard permeabilized cell assay. Sure enough, it is excluded from the nuclei (Figure A). Now you add Crm1, the nuclear export receptor that recognizes the export signal, and RanQ69L-GTP, the mutant formm of Ran that cannot hydrolyze GTP. With these additions, the tagged BSA now enters the nuclei (Figure B). Unlike conventional nuclear import, the maximum nuclear concentration of NES-BSA is no higher than in the cytoplasm surrounding the nucleus.

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