Ran-dependent signal-mediated nuclear import does not require GTP hydrolysis by Ran

Nuclear import of classical nuclear localization sequence-containing protei ns involves the assembly of an import complex at the cytoplasmic face of th e nuclear pore complex (NPC) followed by movement of this complex through t he NPC and release of the import substrate into the nuclear interior. This process has historically been thought to require nucleotide hydrolysis as a source of energy. We found, using hydrolysis-resistant GTP analogs and a m utant Ran unable to hydrolyze GTP, that transport of classical nuclear loca lization sequence containing substrate through the NPC and release of the s ubstrate into the nucleus did not require hydrolysis of GTP by Ran. In fact , for movement of this type of import substrate into the nuclear interior w e did not observe a requirement for hydrolysis of any nucleotide triphospha te. We did, however, find that a pool of free GTP (or its structural equiva lent) must be added, probably because the GDP Ran that is added must be con verted to GTP Ran during the import process. We found that a requirement fo r GTP hydrolysis can be restored to an import mixture consisting of recombi nant import factors by the addition of RCC1, the Ran guanine nucleotide exc hange factor.