Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 275, Issue 14, Pages 10099-10104Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.275.14.10099
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- NIGMS NIH HHS [GM-53678] Funding Source: Medline
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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 alpha 2, karyopherin beta 1, Ran, and p19/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, me found that only one of the NLS receptors, karyopherin alpha 3 (Kap alpha 3/Qip), would support 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 not. Accordingly, in vitro binding studies revealed that only Kap alpha 3 showed significant binding to RCC1 (unlike Kap alpha 1 and Kap alpha 2) and that 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-RCC1 also required both karyopherin beta 1 and Ran.
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