The nuclear import of RCC1 requires a specific nuclear localization sequence receptor, karyopherin alpha3/Qip.
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 alpha2, karyopherin beta1, 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 alpha3 (Kapalpha3/Qip), would support significant nuclear import of pA-RCC1 in permeabilized cells, while members of the other two main classes, Kapalpha1 and Kapalpha2, would not. Accordingly, in vitro binding studies revealed that only Kapalpha3 showed significant binding to RCC1 (unlike Kapalpha1 and Kapalpha2) and that this binding was dependent on the basic amino acids present in the RCC1 NLS. In addition to Kapalpha3, we found that the nuclear import of pA-RCC1 also required both karyopherin beta1 and Ran.
Talcott
B
B
Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA.
Moore
M S
MS
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D018797:Cell Cycle Proteins; D002463:Cell Membrane Permeability; D002467:Cell Nucleus / Q000378:metabolism*; D003600:Cytosol / Q000502:physiology; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D005260:Female; D020662:Guanine Nucleotide Exchange Factors; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D010053:Ovary / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D029867:Xenopus Proteins; D014982:Xenopus laevis; D028901:alpha Karyopherins; D020931:ran GTP-Binding Protein / Q000378:metabolism
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D020662:Guanine Nucleotide Exchange Factors; C106306:KPNA3 protein, human; D009687:Nuclear Proteins; C499304:RCC1 protein, Xenopus; C062521:RCC1 protein, human; D011993:Recombinant Fusion Proteins; D029867:Xenopus Proteins; D028901:alpha Karyopherins; D020931:ran GTP-Binding Protein