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The karyopherin Kap142p/Msn5p mediates nuclear import and nuclear export of different cargo proteins
1Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, New York 10021, USA.
Abstract:
We have identified a novel pathway for protein import into the nucleus. Although the product of Saccharomyces cerevisiae gene MSN5 was previously shown to function as a karyopherin (Kap) for nuclear export of various proteins, we discovered a nuclear import pathway mediated by Msn5p (also referred to as Kap142p). We have purified from yeast cytosol a complex containing Kap142p and the trimeric replication protein A (RPA), which is required for multiple aspects of DNA metabolism, including DNA replication, DNA repair, and recombination. In wild-type cells, RPA was localized primarily to the nucleus but, in a KAP142 deletion strain, RPA was mislocalized to the cytoplasm and the strain was highly sensitive to bleomycin (BLM). BLM causes DNA double-strand breaks and, in S. cerevisiae, the DNA damage is repaired predominantly by RPA-dependent homologous recombination. Therefore, our results indicate that in wild-type cells a critical portion of RPA was imported into the nucleus by Kap142p. Like several other import-related Kap-substrate complexes, the endogenous RPA-Kap142p complex was dissociated by RanGTP, but not by RanGDP. All three RPA genes are essential for viability, whereas KAP142 is not. Perhaps explaining this disparity, we observed an interaction between RPA and Kap95p in a strain lacking Kap142p. This interaction could provide a mechanism for import of RPA into the nucleus and cell viability in the absence of Kap142p. Together with published results (Kaffman, A., N.M. Rank, E.M. O'Neill, L.S. Huang, and E.K. O'Shea. 1998. Nature. 396:482-486; Blondel, M., P.M. Alepuz, L.S. Huang, S. Shaham, G. Ammerer, and M. Peter. 1999. Genes Dev. 13:2284-2300; DeVit, M.J., and M. Johnston. 1999. Curr. Biol. 9:1231-1241; Mahanty, S.K., Y. Wang, F.W. Farley, and E.A. Elion. 1999. Cell. 98:501-512) our data indicate that the karyopherin Kap142p is able to mediate nuclear import of one set of proteins and nuclear export of a different set of proteins.
Insights
Researchers discovered a new nuclear import pathway for Replication Protein A (RPA) mediated by Kap142p, a protein previously known for nuclear export. This finding is crucial for DNA repair and cell viability in Saccharomyces cerevisiae.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- Karyopherins (Kap) are crucial for nucleocytoplasmic transport, with Msn5p (Kap142p) previously characterized for nuclear export functions.
- Replication Protein A (RPA) is essential for DNA metabolism, including replication, repair, and recombination, and is typically localized to the nucleus.
Purpose of the Study:
- To identify and characterize novel pathways for protein import into the nucleus.
- To investigate the role of Msn5p/Kap142p in the nuclear import of RPA.
Main Methods:
- Purification of a complex containing Kap142p and RPA from yeast cytosol.
- Analysis of RPA localization in wild-type and KAP142 deletion strains.
- Assessment of strain sensitivity to bleomycin (BLM) and interaction studies with Kap95p.
Main Results:
- A novel nuclear import pathway for RPA mediated by Kap142p was identified.
- Deletion of KAP142 caused RPA mislocalization to the cytoplasm and sensitivity to DNA-damaging agent bleomycin.
- The RPA-Kap142p complex dissociates in the presence of RanGTP, and an interaction with Kap95p may compensate for Kap142p absence.
Conclusions:
- Kap142p functions as a nuclear importin for RPA, in addition to its known nuclear export roles.
- Kap142p-mediated nuclear import of RPA is essential for DNA repair via homologous recombination and cell survival.
- The dual role of Kap142p in both nuclear import and export highlights its versatility in regulating nucleocytoplasmic transport.