Nuclear import of RPA in Xenopus egg extracts requires a novel protein XRIP alpha but not importin alpha

Replication protein A (RPA) is a eukaryotic single-stranded (ss) DNA-bindin g protein that is essential for general DNA metabolism. RPA consists of thr ee subunits (70, 33 and 14 kDa), We have identified by two-hybrid screening a novel Xenopus protein called XRIP alpha that interacts with the ssDNA-bi nding domain of the largest subunit of RPA. XRIP alpha homologues are found in human and in Drosophila but not in yeast, XRIP alpha is complexed with RPA in Xenopus egg extracts together with another 90 kDa protein that was i dentified as importin beta. We have demonstrated that XRIP alpha, but not i mportin alpha, is required for nuclear import of RPA, Immunodepletion of XR IP alpha 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 XRIP alpha. Conversely, depletion of importin alph a blocks import of nucleoplasmin but not that of RPA. GST-XRIP alpha pull-d own assay shows that XRIP alpha interacts directly with recombinant importi n beta as well as with RPA irt vitro, Finally, RPA import can be reconstitu ted from the recombinant proteins. We propose that XRIP alpha plays the rol e of importin alpha in the RPA import scheme: XRIP alpha serves as an adapt or to link RPA to importin beta.