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A genetic system for detection of protein nuclear import and export

Nature Biotechnology volume 18, pages 433–437 (2000)Cite this article

Abstract

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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Figure 1: A schematic representation of pNIA and pNEA plasmids.
Figure 2: Nuclear import assay.
Figure 3: Effects of NLS and NLS-like or NES and NES-like motifs on induction of β-galactosidase expression in cells grown on minimal medium without tryptophan.
Figure 4: Nuclear export assay.
Figure 5: Detection of NES within TYLCV capsid protein.

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Acknowledgements

We thank Aaron Neiman for his help in devising the one-hybrid assay, Rolf Sternglanz for critical reading of this manuscript, and Hangil Park, Ann Sutton, and Nancy Hollingsworth for guidance with yeast techniques. This work was supported by grants from NIH, NSF, and USDA to V.C., and from BARD to V.C. and Y.G., by a scholarship from the Council for Tobacco Research to C.D., and by a scholarship from TUBITAK-NATO Science Program to F.G.

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Author notes

  1. Colin Dingwall

    Present address: Neurosciences Research Department, SmithKline-Beecham Pharmaceuticals, Harlow, Essex, UK

Authors and Affiliations

  1. Department of Biochemistry Cell Biology, Institute for Cell and Developmental Biology, State University of New York, Stony Brook, 1794-5215, NY

    Yoon Rhee, Filiz Gurel & Vitaly Citovsky

  2. Department of Genetics, Agricultural Research Organization, P.O. Box 6, Bet Dagan, 50250, Israel

    Yedidya Gafni

  3. Department of Pharmacology, State University of New York, Stony Brook, 11794-8651, NY

    Colin Dingwall

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Correspondence to Vitaly Citovsky.

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Rhee, Y., Gurel, F., Gafni, Y. et al. A genetic system for detection of protein nuclear import and export. Nat Biotechnol 18, 433–437 (2000). https://doi.org/10.1038/74500

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