Rna1p, a Ran/TC4 GTPase activating protein, is required for nuclear import

doi:10.1083/jcb.130.5.1017

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Rna1p, a Ran/TC4 GTPase activating protein, is required for nuclear import

AH Corbett, DM Koepp, G Schlenstedt, MS Lee, AK Hopper and PA Silver
Division of Cellular and Molecular Biology, Dana Farber Cancer Institute, Boston, MA 02115.

The Saccharomyces cerevisiae gene, RNA1, encodes a protein with extensive homology to the mammalian Ran/TC4 GTPase activating protein. Using indirect immunofluorescence microscopy, we have demonstrated that rna1-1 mutant cells are defective in nuclear import of several proteins. The same result is obtained when nuclear import is examined in living cells using a nuclear protein fused to the naturally green fluorescent protein. These findings suggest a role for the Rna1p in trafficking of proteins across the nuclear membrane. To investigate this role more directly, an in vitro import assay that monitors the import of a fluorescently labeled substrate into the nuclei of semi- intact yeast cells was used. Import to the nucleus requires the addition of exogenous cytosol. Results indicate that, in contrast to wild-type cytosols, extracts made from rna1-1 mutant cells are unable to support import of the fluorescently labeled substrate into competent nuclei. Immunoblotting demonstrates that these mutant-derived extracts are depleted of Rna1p. However, when purified Rna1p is added back to these extracts the import activity is restored in a dose-dependent manner. These results demonstrate that Rna1p plays a direct role in the import of proteins into the nucleus.
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