The translocation of transportin-cargo complexes through nuclear pores is independent of both Ran and energy

Active transport between nucleus and cytoplasm proceeds through nuclear por e complexes (NPCs) and is mediated largely by shuttling transport receptors that use direct RanGTP binding to coordinate loading and unloading of carg o [1-4]. Import receptors such as importin beta or transportin bind their s ubstrates at low RanGTP levels in the cytoplasm and release them upon encou ntering RanGTP in the nucleus, where a high RanGTP concentration is predict ed. This substrate release is, in the case of import by the importin alpha/ beta heterodimer, coupled directly to importin beta release from the NPCs. If the importin beta-RanGTP interaction is prevented, import intermediates arrest at the nuclear side of the NPCs [5,6]. This arrest makes it difficul t to probe directly the Ran and energy requirements of the actual transloca tion from the cytoplasmic to the nuclear side of the NPC, which immediately precedes substrate release. Here, we have shown that in the case of transp ortin, dissociation of transportin-substrate complexes is uncoupled from tr ansportin release from NPCs. This allowed us to dissect the requirements of translocation through the NPC, substrate release and transportin recycling , Surprisingly, translocation of transportin-substrate complexes into the n ucleus requires neither Ran nor nucleoside triphosphates (NTPs), It is only nuclear RanGTP, not GTP hydrolysis, that is needed for dissociation of tra nsportin-substrate complexes and for re-export of transportin to the cytopl asm, GTP hydrolysis is apparently required only to restore the import compe tence of the re-exported transportin and, thus, for multiple rounds of tran sportin-dependent import. In addition, we provide evidence that at least on e type of substrate can also complete NPC passage mediated by importin beta independently of Ran and energy.