A genetic system for detection of protein nuclear import and export

• Published in: Nature biotechnology Vol. 18; no. 4; pp. 433 - 437
• Main Authors: Citovsky, Vitaly, Rhee, Yoon, Gurel, Filiz, Gafni, Yedidya, Dingwall, Colin
• Format: Journal Article
• Language: English
• Published: New York, NY Nature 01-04-2000; Nature Publishing Group
• Subjects: Amino Acid Sequence; Amino acids; Bacterial infections; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Base Sequence; beta-Galactosidase - analysis; beta-Galactosidase - genetics; Biological and medical sciences; Biotechnology; Cell Nucleus - metabolism; Cloning, Molecular - methods; Cytoplasm; Diverse techniques; DNA-Binding Proteins - metabolism; Fundamental and applied biological sciences. Psychology; Fungal Proteins - genetics; Fungal Proteins - metabolism; Gal4p protein; Geminivirus; Gene expression; LexA protein; Localization; Molecular and cellular biology; Molecular Sequence Data; nuclear export; nuclear import; nuclear localization signal; Plasmids; Proteins; Recombinant Fusion Proteins - metabolism; Repressor Proteins - metabolism; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - growth & development; Saccharomyces cerevisiae - physiology; Saccharomyces cerevisiae Proteins; Serine Endopeptidases - genetics; Serine Endopeptidases - metabolism; Simian virus 40; Transcription Factors - genetics; Transcription Factors - metabolism; Yeast; Yeasts; Yeast; Plant pathogen; Nuclear localization signal; Gene expression; Protein; Nuclear export signal; Fungi; Virus; Reporter gene; Analysis method; Geminivirus; Ascomycetes; Geminiviridae; Nucleocytoplasmic transport; Capsid; Fusion protein; Saccharomyces cerevisiae; Thallophyta
• ISSN: 1087-0156; 1546-1696
• DOI: 10.1038/74500

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.