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NEX‐TRAP, a Novel Method for In Vivo Analysis of Nuclear Export of Proteins

Transport of proteins between cytoplasm and nucleus is mediated by transport factors of the importin α‐ and β‐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.