Rna1p, a Ran/TC4 GTPase activating protein, is required for nuclear import

J Cell Biol. 1995 Sep;130(5):1017-26. doi: 10.1083/jcb.130.5.1017.

Abstract

The Saccharomyces cerevisiae gene, RNA1, encodes a protein with extensive homology to the mammalian Ran/TC4 GTPase activating protein. Using indirect immunofluorescence microscopy, we have demonstrated that rna1-1 mutant cells are defective in nuclear import of several proteins. The same result is obtained when nuclear import is examined in living cells using a nuclear protein fused to the naturally green fluorescent protein. These findings suggest a role for the Rna1p in trafficking of proteins across the nuclear membrane. To investigate this role more directly, an in vitro import assay that monitors the import of a fluorescently labeled substrate into the nuclei of semi-intact yeast cells was used. Import to the nucleus requires the addition of exogenous cytosol. Results indicate that, in contrast to wild-type cytosols, extracts made from rna1-1 mutant cells are unable to support import of the fluorescently labeled substrate into competent nuclei. Immunoblotting demonstrates that these mutant-derived extracts are depleted of Rna1p. However, when purified Rna1p is added back to these extracts the import activity is restored in a dose-dependent manner. These results demonstrate that Rna1p plays a direct role in the import of proteins into the nucleus.

Publication types

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

MeSH terms

  • Biological Transport / physiology
  • Cell Nucleus / metabolism*
  • Cytosol / metabolism
  • Fluorescent Dyes
  • Fungal Proteins / genetics
  • Fungal Proteins / metabolism*
  • Fungal Proteins / pharmacology
  • GTP-Binding Proteins / metabolism*
  • GTPase-Activating Proteins
  • Genes, Fungal
  • Mutation / physiology
  • Nuclear Envelope / metabolism
  • Nuclear Proteins / metabolism*
  • Proteins / metabolism*
  • Saccharomyces cerevisiae / chemistry
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae Proteins
  • Substrate Specificity
  • Temperature
  • ran GTP-Binding Protein

Substances

  • Fluorescent Dyes
  • Fungal Proteins
  • GTPase-Activating Proteins
  • Nuclear Proteins
  • Proteins
  • RNA1 protein, S cerevisiae
  • Saccharomyces cerevisiae Proteins
  • GTP-Binding Proteins
  • ran GTP-Binding Protein