Abstract
The targeting of karyophilic proteins to nuclear pores is mediated via the formation of a nuclear pore-targeting complex, through the interaction of nuclear localization signal (NLS) with its NLS receptor. Recently, a novel human protein, Qip1, was identified from a yeast two-hybrid system with DNA helicase Q1. This study demonstrates that Qip1 is a novel third class of NLS receptor that efficiently recognizes the NLS of the helicase Q1. Moreover, the data obtained in this study show that the specific interaction between Qip1 and the NLS of the helicase Q1 requires its upstream sequence of the minimal essential NLS. By using purified recombinant proteins alone in the digitonin-permeabilized cell-free transport system, it was demonstrated that the two known human NLS receptors, Rch1 and NPI-1, are able to transport all the tested NLS substrates into the nucleus, while Qip1 most efficiently transports the helicase Q1-NLS substrates, which contain its upstream sequence in so far as we have examined the system. Furthermore, in HeLa cell crude cytosol, it was found that endogenous Rch1 binds to all the tested NLS substrates, while the binding of endogenous NPI-1 is restricted to only some NLSs, despite the fact that NPI-1 itself shows binding activity to a variety of NLSs. These results indicate that at least three structurally and functionally distinct NLS receptors exist in the human single cell population, and suggest that the nuclear import of karyophilic proteins may be controlled in a complex manner at the NLS recognition step by the existence of a variety of NLS receptors with various specificities to each NLS.
Highlights
In eukaryotic cells, the selective transport of karyophilic proteins to the nuclei is mediated by short amino acid sequences, which are commonly referred to as nuclear localization signals (NLSs)1 and which are characteristically rich in basic amino acids [1,2,3]
Since another basic amino acid cluster (KK626) was located 16 amino acids upstream from this C-terminal basic cluster, we considered the possibility that the helicase Q1-NLS is a bipartite type
The primary sequences of helicase Q1 suggest that the NLS may be a bipartite type, as shown in Fig. 1, its C-terminal 15 amino acids containing only a single basic amino acid cluster (KKRK645) (ShortQ1) and its C-terminal 27 amino acids, which included amino acid substitutions (K625,626A) (LongQ1(KK-AA)) were found to act as an NLS in living cells
Summary
Cell Culture—Madin-Darby bovine kidney (MDBK) cells were cultured in 5% CO2 at 37 °C in Dulbecco’s modified Eagle’s essential medium (Life Technologies, Inc.) supplemented with 5% fetal bovine serum (Dainippon Pharmaceutical Co., Ltd). GST and thrombin were separated from recombinant proteins by ion-exchange chromatography (MonoQ) column at a flow rate of 0.5 ml/min, with a linear gradient from 0.05 to 1.0 M NaCl in 20 mM Hepes-NaOH, pH 7.3, 2 mM dithiothreitol (DTT), 1 g/ml each of aprotinin, leupeptin, and pepstatin A, and checked on 10% SDS-polyacrylamide gel electrophoresis. Solution Binding Assay for Recombinant or Endogenous NLS Receptor—Ten l of each peptide-conjugated bBSA (1 mg/ml) was immobilized on 15 l of avidin-agarose gel, and mixed with affinity-purified recombinant GST-PTAC97 (100 pmol) in addition to GST-Qip, GSTPTAC58, or GST-NPI-1 (100 pmol), respectively, and the total reaction volume was adjusted to 100 l with TB. One ml of HeLa cell cytosol (8 mg/ml) containing a final concentration of 2 g/ml cytochalasin B was added to 10 l of peptide-bBSA conjugates (1 mg/ml), and the mixture was incubated with 15 l of avidin-agarose gel for 1 h at 4 °C.
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