Abstract
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.
Highlights
In eukaryotic cells, the physical separation of nuclear DNA from cytoplasmic protein synthesis necessitates the bidirectional transport of a diverse set of macromolecules between the nucleus and the cytoplasm
Identification of an rg-nuclear localization signal (NLS) in Nab2p and Nab4p/Hrp1p— Immunopurification studies showed that Kap104p isolated from the yeast cytoplasm copurified with its two import substrates, Nab2p and Nab4p/Hrp1p [18]
Because this domain is both necessary and sufficient for Kap104p binding and acts as a KAP104-dependent nuclear import signal in yeast, we term this domain an rg-NLS to distinguish it from cNLSs recognized by Kap60p/Kap95p
Summary
Yeast strains were derived from DF5 (Mat a/Mat ␣ ura3–52/ ura his3⌬200/his3⌬200 trp1–1/trp leu2–3, 112/leu 112 lys2– 801/lys2– 801) or W303 (Mat a/Mat ␣ ade2–1/ade ura3–1/ ura his3–11, 15/his trp1–1/trp leu2–3, 112/leu 112 can1–100/can1–100) [44]. KAP104 and NAB2 open reading frames were amplified from yeast genomic DNA by polymerase chain reaction, cloned into the BamHI site of pGEX-2TK (Amersham Pharmacia Biotech), and termed Nab2pGEX2TK. NAB4/HRP1 was cloned into the BamHI site of pGEX-4T1 and termed Nab4-pGEX4T1. Full-length Nab2p tagged with green fluorescent protein (GFP) was a gift from C. Guthrie (University of California at San Francisco, CA). Nab2p deletion constructs ⌬8 –188, ⌬200 –264, and ⌬306 – 499 were constructed by polymerase chain reaction. Nab4p fragments encoding amino acid residues 492–534, 392–534, and 241– 400 were amplified from yeast genomic DNA (Promega, Madison, WI) using polymerase chain reaction and oligonucleotides containing restriction sites for cloning as described below. Weis (University of California at San Francisco, CA). The carboxyl-terminal deletion of Nab4p was constructed by digesting full-length Nab4-pGEX4T1 with NdeI and SmaI. T4 DNA polymerase was used to create blunt ends, and the sample was ligated
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