Rna1p, a Ran/TC4 GTPase activating protein, is required for nuclear import.
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.
Corbett
A H
AH
Division of Cellular and Molecular Biology, Dana Farber Cancer Institute, Boston, MA 02115.
Koepp
D M
DM
Schlenstedt
G
G
Lee
M S
MS
Hopper
A K
AK
Silver
P A
PA
D001692:Biological Transport / Q000502:physiology; D002467:Cell Nucleus / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D005456:Fluorescent Dyes; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D020690:GTPase-Activating Proteins; D005800:Genes, Fungal; D009154:Mutation / Q000502:physiology; D009685:Nuclear Envelope / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity; D013696:Temperature; D020931:ran GTP-Binding Protein
D005456:Fluorescent Dyes; D005656:Fungal Proteins; D020690:GTPase-Activating Proteins; D009687:Nuclear Proteins; D011506:Proteins; C060997:RNA1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D019204:GTP-Binding Proteins; D020931:ran GTP-Binding Protein