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 ␣. 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 ␣ 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.The passage of macromolecules between the nucleus and the cytoplasm occurs through nuclear pores. Small macromolecules can diffuse through the nuclear pores at a rate inversely proportional to their mass. Proteins with molecular masses greater than 40 -60 kDa are actively transported through the nuclear pores. To be transported into the nucleus, the protein must either contain a nuclear localization signal or, if not, be bound to another protein that does (1, 2). This process requires at least four different factors acting in two distinct steps. The first step is mediated by importin ␣ (also termed karyopherin ␣) and importin ␤ (also termed karyopherin ␤). The ␣ subunit is primarily responsible for NLS 1 recognition, whereas the ␤ subunit appears to mediate docking to the nuclear pore complex. The second translocation step requires the small G protein Ran/TC4 and an interacting partner, p15 (3-13).The presence of a nuclear localization signal may not be sufficient to direct nuclear import. The target efficiency of NLS motifs can be modified by the presence of multiple NLS motifs within a protein, by modifications of the flanking sequences, and by the accessibility of the NLSs to the import machinery (1...

The hepatitis B virus X protein is a promiscuous transcriptional transactivator. Transactivation by the X protein is most likely mediated through binding to different cellular factors. Using the yeast two-hybrid method, we have isolated a clone that encodes a novel X-associated cellular protein: XAP2. X and XAP2 interactions also occur in vitro. Antiserum raised against XAP2 recognizes a cytoplasmic protein with an apparent molecular mass of 36 kDa. The interaction between X and XAP2 requires a small region on X containing amino acids 13-26. From Northern blot analyses, XAP2 is ubiquitously expressed in both liver-derived and non-liver-derived cell lines as well as in normal non-liver tissues. In contrast, XAP2 is expressed in very low level in the normal human liver. In transfection assays, overexpression of XAP2 abolishes transactivation by the X protein. Based on these results, we suggest that XAP2 is an important cellular negative regulator of the X protein, and that X-XAP2 interaction may play a role in HBV pathology.

The hepatitis B virus X protein induces transcriptional activation of a wide variety of viral and cellular genes. In addition to its ability to interact directly with many nuclear transcription factors, several reports indicate that the X protein stimulates different cytoplasmic kinase signal cascades. Using the yeast two-hybrid screen, we have isolated a clone designated X-associated protein 3 (XAP3) that encodes a human homolog of the rat protein kinase C-binding protein. One of the activation domains of X (amino acids 90 -122) is required for binding to XAP3, while the NH 2 -terminal part of XAP3 is necessary for binding to X. Both X and XAP3 bound specifically to the PKC isoenzyme synthesized in rabbit reticulocyte lysates. Overexpression of XAP3 enhanced X transactivation activity. These results support earlier findings that one of the mechanisms of transactivation by X is through involvement with the cellular protein kinase C pathway.Transcriptional activation is a widespread phenomenon among mammalian viral systems. Mammalian viral proteins that increase the rate of transcription can be divided into two groups based on whether they exhibit sequence-specific DNA binding. For example, the herpes simplex virus 1 (HSV-1) 1 Vmw175 (1, 2), the Epstein-Barr virus BZLF1 (3), the papilloma virus E2 (4), and the simian virus 40 and polyoma virus large T antigens (5) bind specific DNA sequence motifs, whereas HSV-1 VP16 (6, 7), the pseudorabies virus immediate early protein (8,9), and the adenovirus E1A protein (10, 11) do not. During the last decade, many studies have shown that non-DNA binding viral transactivators achieve their task by direct interaction with different cellular sequence-specific DNA binding transcription factors. For example, VP16 interacts with the Oct-1 protein, thereby positioning the VP16 activating domain at a promoter to enhance transcription (7,(12)(13)(14)(15)(16)(17). Similarly, E1A interacts with a number of cellular proteins including ATF-2 (18).The hepatitis B virus (HBV) X protein is a promiscuous transcriptional transactivator (reviewed in Ref. 19). This conclusion is derived from a large number of studies using mostly transient cotransfection of the bacterial chloramphenicol acetyltransferase (CAT) gene under control of a potential target promoter/enhancer and the X gene under the control of a heterologous promoter in mammalian cells. Induction of transcription by X usually ranges from 2-to 20-fold depending on the target promoter and cell type; whether this transactivation activity contributes to viral function, however, remains to be determined. Fusion of the X protein to the DNA-binding domain of the bacterial LexA repressor resulted in a protein that can activate transcription from a reporter plasmid bearing lexA operator sequences fused to a minimal promoter (20). Similarly, fusion of the X protein to the DNA-binding domain of transcription factor C/EBP increased the ability of X to activate a reporter containing C/EBP binding sites (21). Attempts to demonstrate sequence-speci...

Linear DNA plasmids were found in the following yeasts: four strains of Kluyveromyces lactis, one of Debaryomyces hansenii, one of Wingea robettsiae and four of Pichia etchellsii. In each case, the plasmids were present as a pair of DNA molecules of different sizes. The plasmids of K. lactis strains were associated with a killer activity and their structure was similar to the known killer plasmids pGKLl and 2. The plasmids from the other three species were different from pGKL plasmids and showed no killer activity against the yeast species tested so far. In all cases, the linear molecules possessed terminal (probably inverted) repeats and their 5' ends had a protected structure insensitive to 1 exonuclease, while the 3' ends were accessible to exonuclease 111. All these strains could be efficiently cured of the plasmids by ultraviolet irradiation. The plasmids from D. hansenii (pDH1A and B) and from W. robettsiae (pWR1A and B) shared related sequences with some of the K. lactis killer plasmid genes (encoding the supposed DNA polymerases, RNA polymerase and the chitinase), suggesting related genome organization of these plasmids. The pair of plasmids from P. etchellsii (pPE1A and B) appear to be a distantly related member of the group. This pair showed no sequence homology with other plasmids, except weak homology with the putative RNA polymerase gene of pGKL2. None of the plasmids contained the sequences homologous to ORF3 and ORF4 of pGKLl encoding the toxin resistance determinant and the toxin y subunit, respectively.