In vitro analysis of nuclear transport mediated by the C-terminal shuttle domain of tap

The Tap protein of higher eukaryotes is implicated in the nuclear export of type D retroviral mRNA and some cellular mRNAs. Here we have developed an in vitro assay to study nuclear export mediated by the C-terminal shuttle d omain of Tap involving the rapamycin-induced attachment of this transport d omain to a nuclear green fluorescent protein-containing reporter. We found that export by the Tap transport domain does not involve cytosolic transpor t factors including the GTPase Ran. The transport domain directly binds to several nucleoporins positioned in different regions of the nuclear pore co mplex. These results argue that a direct interaction of the Tap transport d omain with nucleoporins is responsible for its nucleocytoplasmic translocat ion. We found that the karyopherin beta -related export receptor CRM1 compe tes with the Tap transport domain for binding to Nup214 but not for binding to Nup62 or Nup153, suggesting that the Tap and CRM1 nuclear export pathwa ys converge at the cytoplasmic periphery of the nuclear pore complex. Becau se the rates of in vitro nuclear import and export by the Tap transport dom ain are very similar, the directionality of mRNA export mediated by Tap pro bably is determined by mechanisms other than simple binding of the Tap tran sport domain to nucleoporins.