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Question: Nuclear import and export are typically studied in cells treated with mild non-ionic detergents u…


Nuclear import and export are typically studied in cells treated with mild non-ionic detergents under carefully controlled conditions, so that the plasma membrane is freely permeable to proteins, but the nuclear envelope remains intact. Because the cytoplasm leaks out, nuclei take up proteins only when the cell remnants are incubated with fresh cytoplasm (i.e, extract or lysate) as a source of critical soluble proteins. One common substrate for uptake studies is serum albumin that is cross-linked to a nuclear localization signal peptide and tagged with a fluorescent label such as fluorescein, so that its uptake into nuclei can be observed by microscopy The small GTPase, Ran, was shown to be required for nuclear uptake using this assay, and not surprisingly, it requires GTP to function. Since neither Ran-GDP nor Ran-GTP binds to nuclear localization signals, other factors must be responsible for identifying proteins to be imported into the nucleus. Using this experimental system, you purify a protein, which you name importin, that in the presence of Ran and GTP promotes uptake of the labeled substrate into nuclei to about the same extent as crude cytosol (Figure A). No uptake occurs in the absence of GTP, and Ran alone is unable to promote nuclear uptake. Importin by itself causes a GTP-independent accumulation of substrate at the nuclear periphery, but does not promote nuclear uptake (Figure A) To define the steps in the uptake pathway, you first incubate nuclei with substrate in the presence of importin. You then wash away free importin and substrate and incubate a second time with Ran and GTP (Figure B)

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Nuclear import and export are typically studied in cells treated with mild non-ionic detergents under carefully controlled conditions, so that the plasma membrane is freely permeable to proteins, but the nuclear envelope remains intact. Because the cytoplasm leaks out, nuclei take up proteins only when the cell remnants are incubated with fresh cytoplasm (i.e, extract or lysate) as a source of critical soluble proteins. One common substrate for uptake studies is serum albumin that is cross-linked to a nuclear localization signal peptide and tagged with a fluorescent label such as fluorescein, so that its uptake into nuclei can be observed by microscopy The small GTPase, Ran, was shown to be required for nuclear uptake using this assay, and not surprisingly, it requires GTP to function. Since neither Ran-GDP nor Ran-GTP binds to nuclear localization signals, other factors must be responsible for identifying proteins to be imported into the nucleus. Using this experimental system, you purify a protein, which you name importin, that in the presence of Ran and GTP promotes uptake of the labeled substrate into nuclei to about the same extent as crude cytosol (Figure A). No uptake occurs in the absence of GTP, and Ran alone is unable to promote nuclear uptake. Importin by itself causes a GTP-independent accumulation of substrate at the nuclear periphery, but does not promote nuclear uptake (Figure A) To define the steps in the uptake pathway, you first incubate nuclei with substrate in the presence of importin. You then wash away free importin and substrate and incubate a second time with Ran and GTP (Figure B)

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