The nuclear import of RCC1 requires a specific nuclear localization sequence receptor, karyopherin alpha 3/Qip

RCC1 is the only known guanine nucleotide exchange factor for the small GTP ase Ran and is normally found inside the nucleus bound to chromatin, In ord er to analyze in more detail the nuclear import of RCC1, we created a fusio n construct in which four IgG binding domains of protein A were fused to th e amino terminus of human RCC1 (pA-RCC1), Surprisingly, we found that neith er Xenopus ovarian cytosol nor a mixture of recombinant import factors (kar yopherin alpha 2, karyopherin beta 1, Ran, and p19/NTF2) were able to suppo rt the import of pA-RCC1 into the nuclei of digitonin permeabilized cells. Both, in contrast, were capable of supporting the import of a construct con taining another classical nuclear localization sequence (NLS), glutathione S-transferase-green fluorescent protein-NLS. Subsequently, me found that on ly one of the NLS receptors, karyopherin alpha 3 (Kap alpha 3/Qip), would s upport significant nuclear import of pA-RCC1 in permeabilized cells, while members of the other two main classes, Kap alpha 1 and Kap alpha 2, would n ot. Accordingly, in vitro binding studies revealed that only Kap alpha 3 sh owed significant binding to RCC1 (unlike Kap alpha 1 and Kap alpha 2) and t hat this binding was dependent on the basic amino acids present in the RCC1 NLS. In addition to Kap alpha 3, we found that the nuclear import of pA-RC C1 also required both karyopherin beta 1 and Ran.