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Nuclear import of RPA in Xenopus egg extracts requires a novel protein XRIPα but not importin α
Author(s) -
Jullien Denis,
Görlich Dirk,
Laemmli Ulrich K.,
Adachi Yasuhisa
Publication year - 1999
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1093/emboj/18.15.4348
Subject(s) - biology , importin , xenopus , nuclear transport , microbiology and biotechnology , alpha (finance) , nuclear protein , cell nucleus , computational biology , biochemistry , nucleus , gene , transcription factor , construct validity , medicine , nursing , patient satisfaction
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 XRIPα that interacts with the ssDNA‐binding domain of the largest subunit of RPA. XRIPα homologues are found in human and in Drosophila but not in yeast. XRIPα is complexed with RPA in Xenopus egg extracts together with another 90 kDa protein that was identified as importin β. We have demonstrated that XRIPα, but not importin α, is required for nuclear import of RPA. Immunodepletion of XRIPα 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 XRIPα. Conversely, depletion of importin α blocks import of nucleoplasmin but not that of RPA. GST–XRIPα pull‐down assay shows that XRIPα interacts directly with recombinant importin β as well as with RPA in vitro . Finally, RPA import can be reconstituted from the recombinant proteins. We propose that XRIPα plays the role of importin α in the RPA import scheme: XRIPα serves as an adaptor to link RPA to importin β.