NUCLEUS-ASSOCIATED POOLS OF RNA1P, THE SACCHAROMYCES-CEREVISIAE RAN TC4 GTPASE-ACTIVATING PROTEIN INVOLVED IN NUCLEUS/CYTOSOL TRANSIT/

Rna1p is the GTPase activating enzyme for Ran/TC4, a Ras-like GTPase n ecessary for nuclear/cytosolic exchange. Although most wild-type Rna1p is located in the cytosol, we found that the vast majority of the mut ant Rna1-1p and, under appropriate physiological conditions, a small p ortion of the wild-type Rna1p cofractionate with yeast nuclei, Subnucl ear fractionation studies show that most of the Rna1p is tightly assoc iated with nuclear components, and that a portion of the active protei n can be solubilized by treatments that fail to solubilize inactive Rn a1-1p. To learn the precise nuclear locations of the Rna1 proteins, we studied their subcellular distributions in HeLa cells, By indirect im munofluorescence we show that wild-type Rna1p has three subcellular lo cations, The majority of the protein is distributed throughout the cyt osol, but a portion of the protein is nucleus-associated, located at b oth the cytosolic surface and within the nucleoplasm, Mutant Rna1-1p i s found at the outer nuclear surface and in the cytosol, We propose th at a small pool of the wild-type Rna1p is located in the nuclear inter ior, supporting the model that the same components of the Ran/TC4 GTPa se cycle exist on both sides of the nuclear membrane.