Identification of a novel cytoplasmic protein that specifically binds to nuclear localization signal motifs.
Active transport of proteins into the nucleus is mediated by interaction between the classical nuclear localization signals (NLSs) of the targeted proteins and the NLS receptor (importin) complex. This nuclear transport system is highly regulated and conserved in eukaryotes and is essential for cell survival. Using a fragment of BRCA1 containing the two NLS motifs as a bait for yeast two-hybrid screening, we have isolated four clones, one of which is importin alpha. Here we characterize one of the other clones identified, BRAP2, which is a novel gene and expressed as a 2-kilobase mRNA in human mammary epithelial cells and some but not all tissues of mice. The isolated full-length cDNA encodes a novel protein containing 600 amino acid residues with pI 6.04. Characteristic motifs of C2H2 zinc fingers and leucine heptad repeats are present in the middle and C-terminal regions of the protein, respectively. BRAP2 also shares significant homology with a hypothetical protein from yeast Saccharomyces cerevisiae, especially in the zinc finger region. Antibodies prepared against the C-terminal region of BRAP2 fused to glutathione S-transferase specifically recognize a cellular protein with a molecular size of 68 kDa, consistent with the size of the in vitro translated protein. Cellular BRAP2 is mainly cytoplasmic and binds to the NLS motifs of BRCA1 with similar specificity to that of importin alpha in both two-hybrid assays in yeast and glutathione S-transferase pull-down assays in vitro. Other motifs such as the SV40 large T antigen NLS motif and the bipartite NLS motif found in mitosin are also recognized by BRAP2. Similarly, the yeast homolog of BRAP2 also binds to these NLS motifs in vitro. These results imply that BRAP2 may function as a cytoplasmic retention protein and play a role in regulating transport of nuclear proteins.
Li
S
S
Department of Molecular Medicine and Institute of Biotechnology, The University of Texas Health Science Center, San Antonio, Texas 78245-3207, USA.
Ku
C Y
CY
Farmer
A A
AA
Cong
Y S
YS
Chen
C F
CF
Lee
W H
WH
D000595:Amino Acid Sequence; D000818:Animals; D019313:BRCA1 Protein / Q000378:metabolism*; D001483:Base Sequence; D001693:Biological Transport, Active; D001940:Breast / Q000166:cytology; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002451:Cell Compartmentation; D003593:Cytoplasm / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D004847:Epithelial Cells; D005260:Female; D006801:Humans; D008321:Mammary Glands, Animal / Q000166:cytology; D051379:Mice; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D044767:Ubiquitin-Protein Ligases
D019313:BRCA1 Protein; D002352:Carrier Proteins; D018076:DNA, Complementary; D019913:Nuclear Localization Signals; D010446:Peptide Fragments; C111164:BRAP protein, human; D044767:Ubiquitin-Protein Ligases