A genetic system for detection of protein nuclear import and export

Yoon Rhee; Filiz Gurel; Yedidya Gafni; Colin Dingwall; Vitaly Citovsky

doi:10.1038/74500

A genetic system for detection of protein nuclear import and export

Yoon Rhee, Filiz Gurel, Yedidya Gafni, Colin Dingwall, Vitaly Citovsky

Research output: Contribution to journal › Article › peer-review

3 Scopus citations

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.

Original language	English
Pages (from-to)	433-437
Number of pages	5
Journal	Nature Biotechnology
Volume	18
Issue number	4
DOIs	https://doi.org/10.1038/74500
State	Published - 2000
Externally published	Yes

Bibliographical note

Funding Information:
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.

Keywords

Agrobacterium
Geminivirus
Nucleotoplasmic shuttle protein
TYLCV
VirD2/VirE2

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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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author = "Yoon Rhee and Filiz Gurel and Yedidya Gafni and Colin Dingwall and Vitaly Citovsky",

note = "Funding Information: 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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A genetic system for detection of protein nuclear import and export

Abstract

Bibliographical note

Keywords

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