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

Proc Natl Acad Sci U S A. 1996 Jul 23;93(15):7667-72. doi: 10.1073/pnas.93.15.7667.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 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*
  • Recombinant Proteins / analysis
  • Recombinant Proteins / biosynthesis
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / physiology*
  • Saccharomyces cerevisiae Proteins
  • Sequence Tagged Sites
  • Transfection
  • ran GTP-Binding Protein
  • ras GTPase-Activating Proteins

Substances

  • DNA Primers
  • GTPase-Activating Proteins
  • Nuclear Proteins
  • Proteins
  • RNA1 protein, S cerevisiae
  • Recombinant Proteins
  • Saccharomyces cerevisiae Proteins
  • ras GTPase-Activating Proteins
  • GTP-Binding Proteins
  • ran GTP-Binding Protein