Kap104p-mediated nuclear import. Nuclear localization signals in mRNA-binding proteins and the role of Ran and Rna.
Kap104p is a Saccharomyces cerevisiae nuclear import receptor for two essential mRNA-binding proteins, Nab2p and Nab4p/Hrp1p. We demonstrate direct binding of Kap104p to each of these substrates. We have defined the nuclear localization signals in both Nab2p and Nab4p/Hrp1p by Kap104p binding in vitro and KAP104-dependent nuclear import in vivo. The nuclear localization signals map to similar arginine/glycine-rich RNA-binding domains in both proteins and are thus termed rg-nuclear localization signals to distinguish them from classical nuclear localization signals. We also demonstrate that Kap104p, like other known beta-karyopherins (or importins), interacts directly with the small GTPase Ran/Gsp1. However, unlike other known import factors, Ran binding is not sufficient to mediate release of substrates from Kap104p; efficient Ran-GTP-mediated substrate release requires RNA. Also, addition of Kap104p to Nab2p and Nab4p/Hrp1p prebound to single-stranded DNA-cellulose stimulated release of both proteins from the resin. We suggest a simple cycle in which Nab2p and Nab4p/Hrp1p, upon import, are released in the nucleus at sites of transcription by the concerted action of Ran-GTP and binding to newly synthesized mRNA. The resulting ribonucleoprotein complexes are exported to the cytoplasm, where Kap104p rebinds to Nab2p and Nab4p/Hrp1p, contributing to their release from mRNA.
Lee
D C
DC
Department of Cell Biology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
Aitchison
J D
JD
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism*; D028884:Karyopherins; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals; D029543:Nucleocytoplasmic Transport Proteins; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D028961:beta Karyopherins; D039221:mRNA Cleavage and Polyadenylation Factors; D020931:ran GTP-Binding Protein / Q000378:metabolism
D002352:Carrier Proteins; D005656:Fungal Proteins; C101820:KAP104 protein, S cerevisiae; D028884:Karyopherins; C080773:NAB2 protein, S cerevisiae; D019913:Nuclear Localization Signals; D029543:Nucleocytoplasmic Transport Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D012261:Ribonucleoproteins; D029701:Saccharomyces cerevisiae Proteins; D028961:beta Karyopherins; D039221:mRNA Cleavage and Polyadenylation Factors; D020931:ran GTP-Binding Protein