Traglia H M, O'Connor J P, Tung K S, Dallabrida S, Shen W C, Hopper A K
Department of Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, Hershey 17033, USA.
Proc Natl Acad Sci U S A. 1996 Jul 23;93(15):7667-72. doi: 10.1073/pnas.93.15.7667.
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.
Rna1p是Ran/TC4的GTP酶激活酶，Ran/TC4是一种核质交换所必需的类Ras GTP酶。尽管大多数野生型Rna1p位于细胞质中，但我们发现绝大多数突变型Rna1-1p，以及在适当生理条件下的一小部分野生型Rna1p与酵母细胞核共分级分离。亚核分级分离研究表明，大多数Rna1p与核成分紧密结合，并且一部分活性蛋白可以通过不能溶解无活性Rna1-1p的处理而溶解。为了了解Rna1蛋白在细胞核中的精确位置，我们研究了它们在HeLa细胞中的亚细胞分布。通过间接免疫荧光，我们表明野生型Rna1p有三个亚细胞位置。大多数蛋白质分布在整个细胞质中，但一部分蛋白质与细胞核相关，位于细胞质表面和核质内。突变型Rna1-1p存在于核外表面和细胞质中。我们提出一小部分野生型Rna1p位于核内部，支持Ran/TC4 GTP酶循环的相同成分存在于核膜两侧的模型。
