Journal
TRAFFIC
Volume 13, Issue 10, Pages 1326-1334Publisher
WILEY
DOI: 10.1111/j.1600-0854.2012.01389.x
Keywords
automated imaging; HSV-1; NES; NEX-TRAP; nuclear export assay; p53; RanBP3; rapamycin-induced dimerization; UL31; UL4; UL42
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Funding
- Deutsche Forschungsgemeinschaft [BA1165/5-1]
- Baygene
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Transport of proteins between cytoplasm and nucleus is mediated by transport factors of the importin a- and beta-families and occurs along a gradient of the small GTPase Ran. To date, in vivo analysis as well as prediction of protein nuclear export remain tedious and difficult. We generated a novel bipartite assay called NEX-TRAP (Nuclear EXport Trapped by RAPamycin) for in vivo analysis of protein nuclear export. The assay is based on the rapamycin-induced dimerization of the modules FRB (FK506-rapamycin (FR)-binding domain) and FKBP (FK506-binding protein-12): a potential nuclear export cargo is fused to FRB, to EYFP for direct visualization as well as to an SV40-derived nuclear localization signal (NLS) for constitutive nuclear import. An integral membrane protein that resides at the trans Golgi network (TGN) is fused to a cytoplasmically exposed FKBP and serves as reporter. EYFP-NLS-FRB fusion proteins with export activity accumulate in the nucleus at steady state but continuously shuttle between nucleus and cytoplasm. Rapamycin-induced dimerization of FRB and FKBP at the TGN traps the shuttling protein outside of the nucleus, making nuclear export permanent. Using several example cargoes, we show that the NEX-TRAP is superior to existing assays owing to its ease of use, its sensitivity and accuracy. Analysis of large numbers of export cargoes is facilitated by recombinational cloning. The NEX-TRAP holds the promise of applicability in automated fluorescence imaging for systematic analysis of nuclear export, thereby improving in silico prediction of nuclear export sequences.
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