RESUMEN
Transport receptors of the importin beta superfamily account for many of the nuclear import and export events in eukaryotic cells. They mediate translocation through nuclear pore complexes, shuttle between nucleus and cytoplasm and co-operate with the RanGTPase system to regulate their interactions with cargo molecules in a compartment-specific manner. We used affinity chromatography on immobilized RanGTP to isolate further candidate nuclear transport receptors and thereby identified exportin 4 as the most distant member of the importin beta family so far. Exportin 4 appears to be conserved amongst higher eukaryotes, but lacks obvious orthologues in yeast. It mediates nuclear export of eIF-5A (eukaryotic translation initiation factor 5A) and possibly that of other cargoes. The export signal in eIF-5A appears to be complex and to involve the hypusine modification that is unique to eIF-5A. We discuss possible cellular roles for nuclear export of eIF-5A.
Asunto(s)
Proteínas Portadoras/fisiología , Núcleo Celular/metabolismo , Lisina/análogos & derivados , Proteínas de Unión al ARN/fisiología , Secuencia de Aminoácidos , Animales , Cromatografía de Afinidad , Clonación Molecular , Citoplasma/metabolismo , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , GTP Fosfohidrolasas/metabolismo , Células HeLa , Humanos , Carioferinas , Cinética , Lisina/metabolismo , Ratones , Microscopía Fluorescente , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Factores de Iniciación de Péptidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , ARN/metabolismo , Factores de Tiempo , Proteína de Unión al GTP ran/metabolismo , Factor 5A Eucariótico de Iniciación de TraducciónRESUMEN
In eukaryotes, tRNAs are synthesized in the nucleus and after several maturation steps exported to the cytoplasm. Here, we identify exportin-t as a specific mediator of tRNA export. It is a RanGTP-binding, importin beta-related factor with predominantly nuclear localization. It shuttles rapidly between nucleus and cytoplasm and interacts with nuclear pore complexes. Exportin-t binds tRNA directly and with high affinity. Its cellular concentration in Xenopus oocytes was found to be rate-limiting for export of all tRNAs tested, as judged by microinjection experiments. RanGTP regulates the substrate-exportin-t interaction such that tRNA can be preferentially bound in the nucleus and released in the cytoplasm.