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 domain of Tap involving the rapamycin-induced attachment of this transport domain to a nuclear green fluorescent protein-containing reporter. We found that export by the Tap transport domain does not involve cytosolic transport factors including the GTPase Ran. The transport domain directly binds to several nucleoporins positioned in different regions of the nuclear pore complex. These results argue that a direct interaction of the Tap transport domain with nucleoporins is responsible for its nucleocytoplasmic translocation. We found that the karyopherin beta-related export receptor CRM1 competes 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 pathways converge at the cytoplasmic periphery of the nuclear pore complex. Because the rates of in vitro nuclear import and export by the Tap transport domain are very similar, the directionality of mRNA export mediated by Tap probably is determined by mechanisms other than simple binding of the Tap transport domain to nucleoporins.
Schmitt
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I
Department of Cell, The Scripps Research Institute, La Jolla, California 92037, USA.
Gerace
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L
D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000502:physiology; D021581:Active Transport, Cell Nucleus; D001665:Binding Sites; D002352:Carrier Proteins / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D028884:Karyopherins / Q000502:physiology; D028861:Nuclear Pore Complex Proteins / Q000502:physiology; D029543:Nucleocytoplasmic Transport Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D020931:ran GTP-Binding Protein / Q000502:physiology; D000097606:Exportin 1 Protein
D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D028884:Karyopherins; D028861:Nuclear Pore Complex Proteins; D029543:Nucleocytoplasmic Transport Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D020931:ran GTP-Binding Protein; D000097606:Exportin 1 Protein; C498546:NXT1 protein, human; C073119:TAP1 protein, human