A switching system regulating subcellular localization of nuclear proteins using a viral protease.
We explored a novel approach to the functional regulation of nuclear proteins; altering their subcellular localization. To anchor a nuclear protein, beta-galactosidase with the nuclear localization signal of SV40 (nbeta-gal), within the cytoplasm, nbeta-gal was fused to the transmembrane domain of granulocyte colony-stimulating factor receptor (G-CSFR), a membrane protein. To liberate the nbeta-gal portion from the fusion protein, we used a protease derived from a plant virus, whose recognition sequence was inserted between the G-CSFR and nbeta-gal. Western analysis showed that the chimeric protein was cleaved in the presence of the protease in 293 cells and that the fusion protein without the recognition sequence remained intact. This chimeric protein was localized exclusively in the cytoplasm as visualized by X-gal staining and immunofluorescence microscopy. In contrast, when expressed together with the protease, beta-gal was predominantly detected in the nuclei. Moreover, we isolated 293-cell clones constitutively expressing the protease, indicating that this protease is not cytotoxic. These results suggest that the viral protease-mediated alteration of subcellular localization can potentially regulate the function of nuclear proteins.
Urabe
M
M
Division of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical School, Tochigi, Japan. murabe@jichi.ac.jp
Kume
A
A
Takahashi
T
T
Serizawa
N
N
Tobita
K
K
Ozawa
K
K
D001692:Biological Transport; D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D003593:Cytoplasm / Q000378:metabolism*; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity; D005455:Fluorescent Antibody Technique; D016179:Granulocyte Colony-Stimulating Factor / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008970:Molecular Weight; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D016179:Granulocyte Colony-Stimulating Factor; D001616:beta-Galactosidase; D010450:Endopeptidases; C099488:TEV protease; C059992:nuclear inclusion protein a, mosaic viruses