We previously purified and characterized a nuclear localization signal (NLS) binding protein, NBP60, in rat liver nuclear envelopes. In this study, we cloned and sequenced the cDNA of rat NBP60, and predicted an amino acid sequence comprising 620 amino acids. The sequence revealed that NBP60 is a rat homologue of lamin B receptor (LBR), and is 79 and 63% identical in amino acids to human and chicken LBR, respectively. Using three fusion proteins containing different parts of the amino-terminal domain of human LBR, it was shown that the stretch comprising amino acids 1 to 89, which contains a Ser-Arg rich region (RS region), binds to nucleoplasmin and that the binding was inhibited by a common NLS-peptide. These results suggested that the amino-terminal domain of LBR contains an NLS-binding site. Furthermore, it was shown that the stretch comprising amino acids 1 to 53, which does not contain the RS region or the predicted DNA-binding site, binds to Xenopus laevis sperm chromatin.

A 47k protein (p47) in a high-salt buffer extract of a rat liver nuclear matrix fraction was purified by means of a wheat germ agglutinin affinity column, reversed phase HPLC, and SDS-PAGE, and partial amino acid sequences were analyzed. Based on these sequences, the mouse cDNA of the protein was cloned and sequenced, and its amino acid sequence was deduced. Mouse p47 consists of 463 amino acid residues with a molecular weight of 51,112. The amino acid sequences of human and Saccharomyces cerevisiae p47s were also deduced from the nucleotide sequences of "expressed sequence tag" fragments and genomic DNA, respectively. These sequences contain helicase motifs and show homology to bacterial RuvB DNA helicases acting in homologous recombination. They also show homology with the putative mammalian helicases p50/TIP49 and RUVBL1. Comparison of the amino acid sequences of p47 group proteins and those of p50/TIP49 group proteins revealed the p47 group proteins to comprise a group distinct from the p50/TIP49 proteins. Ultracentrifugation and gel filtration analyses showed that p47 in the rat liver cytosol fraction exists as large complexes of 697k.

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Based on partial amino acid sequences of p50 purified from a high-salt buffer extract of a rat liver nuclear matrix fraction, p50 cDNA was cloned and sequenced, and its amino acid sequence was predicted. The sequence contained helicase motifs, and showed homology with RuvB DNA helicase of Thermus thermophilus and an open reading frame for an unknown 50.5 k protein of Saccharomyces cerevisiae. p50 was expressed as a GST-fusion protein and antiserum against the protein was generated. p50 was localized to the nuclear matrix by cell fractionation and immunoblotting. p50 bound to ATP-Sepharose beads. Ultracentrifugation and gel filtration analyses showed that p50 in rat liver and Xenopus egg mitotic extracts exists as large complexes corresponding to 697 k and 447 k, respectively. A 50 k protein reactive with p50 antibodies was detected not only in rat liver nuclei, but also in a Xenopus egg cytoplasm fraction and a S. cerevisiae extract. This suggests that this putative DNA helicase is present in a wide variety of species ranging from yeast to mammals.

A nuclear localization signal binding protein in nuclear envelope was studied as the first step to determine the mechanism of nuclear protein recognition by nuclear envelope. The rat liver nuclear envelope extract was resolved by SDS-PAGE and ligand blotted with 125I-labeled nucleoplasmin bearing a strong nuclear localization signal. A nuclear localization signal binding protein with molecular mass of 60 kDa (NBP60) was detected in the extract. NBP60 could be extracted with 2% Triton X-100-1 M KCl but not with 1 M KCl, 2 M urea, or 2% Triton X-100. The protein was partitioned to the lower layer in a two phase system using Triton X-114. These results suggested that the protein is an intrinsic membrane protein and has a hydrophobic surface. This protein was bound to not only nucleoplasmin but also the nuclear localization signal peptide of SV 40 large T-antigen (T-peptide) conjugated to human serum albumin. The binding of NBP60 to nucleoplasmin-Sepharose was inhibited by 50% in the presence of 0.12 mM T-peptide. However, a high concentration of 2.1 mM was necessary, when mutant T-peptide in which the essential amino acid lysine was substituted with threonine was used. These results suggested that NBP60 binds specifically to nuclear localization signals. NBP60 extracted from the nuclear envelope was purified by nucleoplasmin-Sepharose affinity chromatography following hydroxyapatite high performance liquid chromatography.