RNA1P, A RAN TC4 GTPASE-ACTIVATING PROTEIN, IS REQUIRED FOR NUCLEAR IMPORT/

The Saccharomyces cerevisiae gene, RNA1, encodes a protein with extens ive homology to the mammalian Ran/TC4 GTPase activating protein, Using indirect immunofluorescence microscopy, we have demonstrated that rna 1-1 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 o f proteins across the nuclear membrane. To investigate this role more directly, an in vitro import assay that monitors the import of a fluor escently labeled substrate into the nuclei of semi-intact yeast cells was used. Import to the nucleus requires the addition of exogenous cyt osol. Results indicate that, in contrast to wild-type cytosols, extrac ts made from rna1-1 mutant cells are unable to support import of the f luorescently labeled substrate into competent nuclei. Immunoblotting d emonstrates that these mutant-derived extracts are depleted of Rna1p. However, when purified Rna1p is added back to these extracts the impor t activity is restored in a dose-dependent manner. These results demon strate that Rna1p plays a direct role in the import of proteins into t he nucleus.