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 journalArticlepeer-review

97 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 languageEnglish
Pages (from-to)433-437
Number of pages5
JournalNature Biotechnology
Volume18
Issue number4
DOIs
StatePublished - Apr 2000
Externally publishedYes

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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