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Rhee, Y., Dept. of Biochem. and Cell Biology, Inst. for Cell and Devmtl. Biology, State University of New York, Stony Brook, NY 11794-5215, United States
Gurel, F., Dept. of Biochem. and Cell Biology, Inst. for Cell and Devmtl. Biology, State University of New York, Stony Brook, NY 11794-5215, United States
Gafni, Y., Department of Genetics, Agricultural Research Organization, P.O. Box 6, Bet Dagan 50250, Israel
Dingwall, C., Department of Pharmacology, State University of New York, Stony Brook, NY 11794-8651, United States, Neurosciences Research Department, SmithKline-Beecham Pharmaceutical, Harlow, Essex, United Kingdom
Citovsky, V., Dept. of Biochem. and Cell Biology, Inst. for Cell and Devmtl. Biology, State University of New York, Stony Brook, NY 11794-5215, United States
We have developed a simple genetic assay to detect active nuclear localization (NLS) and export signals (NES) on the basis of their function within yeast cells. The bacterial LexA protein was modified (mLexA) to abolish its intrinsic NLS and fused to the activation domain of the yeast Gal4p (Gal4AD) with or without the SV40 large T-antigen NLS. In the import assay, if a tested protein fused to mLexA-Gal4AD contains a functional NLS, it will enter the cell nucleus and activate the reporter gene expression. In the export assay, if a tested protein fused to mLexA-SV40 NLS-Gal4AD contains a functional NES, it will exit into the cytoplasm, decreasing the reporter gene expression. We tested this system with known NLS and NES and then used it to demonstrate a NES activity of the capsid protein of a plant geminivirus. This approach may help to identify, analyze, and select for proteins containing functional NLS and NES.
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A genetic system for detection of protein nuclear import and export
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Rhee, Y., Dept. of Biochem. and Cell Biology, Inst. for Cell and Devmtl. Biology, State University of New York, Stony Brook, NY 11794-5215, United States
Gurel, F., Dept. of Biochem. and Cell Biology, Inst. for Cell and Devmtl. Biology, State University of New York, Stony Brook, NY 11794-5215, United States
Gafni, Y., Department of Genetics, Agricultural Research Organization, P.O. Box 6, Bet Dagan 50250, Israel
Dingwall, C., Department of Pharmacology, State University of New York, Stony Brook, NY 11794-8651, United States, Neurosciences Research Department, SmithKline-Beecham Pharmaceutical, Harlow, Essex, United Kingdom
Citovsky, V., Dept. of Biochem. and Cell Biology, Inst. for Cell and Devmtl. Biology, State University of New York, Stony Brook, NY 11794-5215, United States
A genetic system for detection of protein nuclear import and export
We have developed a simple genetic assay to detect active nuclear localization (NLS) and export signals (NES) on the basis of their function within yeast cells. The bacterial LexA protein was modified (mLexA) to abolish its intrinsic NLS and fused to the activation domain of the yeast Gal4p (Gal4AD) with or without the SV40 large T-antigen NLS. In the import assay, if a tested protein fused to mLexA-Gal4AD contains a functional NLS, it will enter the cell nucleus and activate the reporter gene expression. In the export assay, if a tested protein fused to mLexA-SV40 NLS-Gal4AD contains a functional NES, it will exit into the cytoplasm, decreasing the reporter gene expression. We tested this system with known NLS and NES and then used it to demonstrate a NES activity of the capsid protein of a plant geminivirus. This approach may help to identify, analyze, and select for proteins containing functional NLS and NES.
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