Nuclear import of RPA in Xenopus egg extracts requires a novel protein XRIPalpha but not importin alpha.
Replication protein A (RPA) is a eukaryotic single-stranded (ss) DNA-binding protein that is essential for general DNA metabolism. RPA consists of three subunits (70, 33 and 14 kDa). We have identified by two-hybrid screening a novel Xenopus protein called XRIPalpha that interacts with the ssDNA-binding domain of the largest subunit of RPA. XRIPalpha homologues are found in human and in Drosophila but not in yeast. XRIPalpha is complexed with RPA in Xenopus egg extracts together with another 90 kDa protein that was identified as importin beta. We have demonstrated that XRIPalpha, but not importin alpha, is required for nuclear import of RPA. Immunodepletion of XRIPalpha from the egg extracts blocks nuclear import of RPA but not that of nucleoplasmin, a classical import substrate. RPA import can be restored by addition of recombinant XRIPalpha. Conversely, depletion of importin alpha blocks import of nucleoplasmin but not that of RPA. GST-XRIPalpha pull-down assay shows that XRIPalpha interacts directly with recombinant importin beta as well as with RPA in vitro. Finally, RPA import can be reconstituted from the recombinant proteins. We propose that XRIPalpha plays the role of importin alpha in the RPA import scheme: XRIPalpha serves as an adaptor to link RPA to importin beta.
Jullien
D
D
Institute of Cell & Molecular Biology, The University of Edinburgh, Edinburgh EH9 3JR, UK.
Görlich
D
D
Laemmli
U K
UK
Adachi
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D010063:Ovum / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D051716:Replication Protein A; D017386:Sequence Homology, Amino Acid; D014981:Xenopus; D029867:Xenopus Proteins; D028901:alpha Karyopherins; D020931:ran GTP-Binding Protein
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D026901:Membrane Transport Proteins; D009687:Nuclear Proteins; C497167:RPA1 protein, human; D011993:Recombinant Fusion Proteins; D051716:Replication Protein A; D029867:Xenopus Proteins; D028901:alpha Karyopherins; C121149:ripalpha protein, Xenopus; D019204:GTP-Binding Proteins; D020931:ran GTP-Binding Protein