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.
Max von Pettenkofer-Institut, Ludwig-Maximilians-Universität München, Pettenkoferstr. 9a, 80336, München, Germany.
Verena
V
Raschbichler
Diana
D
Lieber
Susanne M
SM
Bailer
D021581:Active Transport, Cell Nucleus; D002467:Cell Nucleus / Q000378:metabolism; D003584:Cytological Techniques; D019281:Dimerization; D049452:Green Fluorescent Proteins / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins; D020123:Sirolimus / Q000737:chemistry / Q000494:pharmacology; D022021:Tacrolimus Binding Proteins / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism; D021601:trans-Golgi Network / Q000187:drug effects / Q000378:metabolism
D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D014764:Viral Proteins; D049452:Green Fluorescent Proteins; D022021:Tacrolimus Binding Proteins; D020123:Sirolimus