The nuclear import of RCC1 requires a specific nuclear localization sequence receptor, karyopherin α3/Qip

Abstract

RCC1 is the only known guanine nucleotide exchange factor for the small GTPase Ran and is normally found inside the nucleus bound to chromatin. In order to analyze in more detail the nuclear import of RCC1, we created a fusion construct in which four IgG binding domains of protein A were fused to the amino terminus of human RCC1 (pA-RCC1). Surprisingly, we found that neither Xenopus ovarian cytosol nor a mixture of recombinant import factors (karyopherin α2, karyopherin β1, Ran, and p10/NTF2) were able to support the import of pA-RCC1 into the nuclei of digitonin-permeabilized cells. Both, in contrast, were capable of supporting the import of a construct containing another classical nuclear localization sequence (NLS), glutathione S- transferase-green fluorescent protein-NLS. Subsequently, we found that only one of the NLS receptors, karyopherin α3 (Kapα3/Qip), would support significant nuclear import of pA-RCC1 in permeabilized cells, while members of the other two main classes, Kapα1 and Kapα2, would not. Accordingly, in vitro binding studies revealed that only Kapα3 showed significant binding to RCC1 (unlike Kapα1 and Kapα2) and that this binding was dependent on the basic amino acids present in the RCC1 NLS. In addition to Kapα3, we found that the nuclear import of pA-RCC1 also required both karyopherin β1 and Ran.