Nucleus-associated pools of Rna1p, the Saccharomyces cerevisiae Ran/TC4 GTPse activating protein involved in nucleus/cytosol transit.
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.
Traglia
H M
HM
Department of Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, Hershey 17033, USA.
O'Connor
J P
JP
Tung
K S
KS
Dallabrida
S
S
Shen
W C
WC
Hopper
A K
AK
D001483:Base Sequence; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D017931:DNA Primers; D019084:Fluorescent Antibody Technique, Indirect; D019204:GTP-Binding Proteins / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism*; D020690:GTPase-Activating Proteins; D006367:HeLa Cells; D006801:Humans; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000378:metabolism*; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins; D016324:Sequence Tagged Sites; D014162:Transfection; D020931:ran GTP-Binding Protein; D020703:ras GTPase-Activating Proteins
D017931:DNA Primers; D020690:GTPase-Activating Proteins; D009687:Nuclear Proteins; D011506:Proteins; C060997:RNA1 protein, S cerevisiae; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D020703:ras GTPase-Activating Proteins; D019204:GTP-Binding Proteins; D020931:ran GTP-Binding Protein