Nucleus-associated pools of Rna1p, the Saccharomyces cerevisiae Ran/TC4 GTPse activating protein involved in nucleus/cytosol transit

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 93; no. 15; pp. 7667 - 7672
• Main Authors: Traglia, H M, O'Connor, J P, Tung, K S, Dallabrida, S, Shen, W C, Hopper, A K
• Format: Journal Article
• Language: English
• Published: United States National Acad Sciences 23.07.1996
• Subjects: Base Sequence; Cell Fractionation; Cell Nucleus - metabolism; Cytosol - metabolism; DNA Primers; Fluorescent Antibody Technique, Indirect; GTP-Binding Proteins - analysis; GTP-Binding Proteins - biosynthesis; GTP-Binding Proteins - metabolism; GTPase-Activating Proteins; HeLa Cells; Humans; Models, Biological; Molecular Sequence Data; Mutagenesis, Site-Directed; Nuclear Proteins - metabolism; Open Reading Frames; Polymerase Chain Reaction; Proteins - metabolism; ran GTP-Binding Protein; ras GTPase-Activating Proteins; Recombinant Proteins - analysis; Recombinant Proteins - biosynthesis; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - physiology; Saccharomyces cerevisiae Proteins; Sequence Tagged Sites; Transfection
• DOI: 10.1073/pnas.93.15.7667
• ISSN: 0027-8424

Rna1p is the GTPase activating enzyme for Ran/TC4, a Ras-like GTPase necessary for nuclear/cytosolic exchange. Although most wild-type Rna1p is located in the cytosol, we found that the vast majority of the mutant Rna1-1p and, under appropriate physiological conditions, a small portion of the wild-type Rna1p cofractionate with yeast nuclei. Subnuclear fractionation studies show that most of the Rna1p is tightly associated with nuclear components, and that a portion of the active protein can be solubilized by treatments that fail to solubilize inactive Rna1-1p. To learn the precise nuclear locations of the Rna1 proteins, we studied their subcellular distributions in HeLa cells. By indirect immuno-fluorescence we show that wild-type Rna1p has three subcellular locations. The majority of the protein is distributed throughout the cytosol, but a portion of the protein is nucleus-associated, located at both the cytosolic surface and within the nucleoplasm. Mutant Rna1-1p is found at the outer nuclear surface and in the cytosol. We propose that a small pool of the wild-type Rna1p is located in the nuclear interior, supporting the model that the same components of the Ran/TC4 GTPase cycle exist on both sides of the nuclear membrane.