Beta-galactosidase Fusion Protein Research Articles

Streamlining the Detection of Human Thyroid Receptor Ligand Interactions with XL1-Blue Cell-Free Protein Synthesis and Beta-Galactosidase Fusion Protein Biosensors.

Thyroid receptor signaling controls major physiological processes and disrupted signaling can cause severe disorders that negatively impact human life. Consequently, methods to detect thyroid receptor ligands are of great toxicologic and pharmacologic importance. Previously, we reported thyroid receptor ligand detection with cell-free protein synthesis of a chimeric fusion protein composed of the human thyroid receptor beta (hTRβ) receptor activator and a β-lactamase reporter. Here, we report a 60% reduction in sensing cost by reengineering the chimeric fusion protein biosensor to include a reporter system composed of either the full-length beta galactosidase (β-gal), the alpha fragment of β-gal (β-gal-α), or a split alpha fragment of the β-gal (split β-gal-α). These biosensor constructs are deployed using E. coli XL1-Blue cell extract to (1) avoid the β-gal background activity abundant in BL21 cell extract and (2) facilitate β-gal complementation reporter activity to detect human thyroid receptor ligands. These results constitute a promising platform for high throughput screening and potentially the portable detection of human thyroid receptor ligands.

Folding of a misfolding‐prone β‐galactosidase in absence of DnaK

In absence of chaperone DnaK, bacterially produced misfolding-prone proteins aggregate into large inclusion bodies, but still a significant part of these polypeptides remains in the soluble cell fraction. The functional analysis of the model beta-galactosidase fusion protein VP1LAC produced in DnaK(-) cells has revealed that the soluble version exhibits important folding defects and that it is less stable and less active than when produced in wild-type DnaK(+) cells. In addition, we have observed that the induction of gene expression at the very late exponential phase enhances twofold the stability of VP1LAC, a fact that in DnaK(-) background results in a dramatic increase of its specific activity up to phenotypically detectable levels. These results indicate that the chaperone DnaK is critical for the folding of misfolding-prone proteins and also that the soluble form reached in its absence by a fraction of polypeptides is not necessarily supportive of biological activity. In the case of E. coli beta-galactosidase, the catalytic activity requires assembling into tetramers and the fine organization of the activating interfaces holding the active sites, what might not be properly reached in absence of DnaK.

Establishment of cloned homogeneous immunoassay for detection of P53 protein in serum and stool samples from patients with gastric carcinoma

目的 评价检测P53蛋白的克隆酶均相免疫分析方法的可行性.方法通过基因重组技术构建β-半乳糖苷酶亚单位EA、ED及ED-P53融合蛋白的原核表达质粒,建立克隆酶均相免疫技术,测定胃癌、非胃癌和正常对照组血清、粪便P53蛋白含量, 并与酶联免疫吸附试验(ELISA)比较.结果 31例胃癌患者的血清、粪便P53蛋白阳性率高于39例非胃癌患者和17名健康献血员(均P<0.01).11例胃癌患者血清P53和免疫组化均阳性,且免疫复合物均以P53-IgG形式存在,对5例胃癌血清动态观察表明,术后1周P53蛋白仍阳性,术后1个月显著下降,并在术后3个月消失.检测10份不同浓度的样本, 表明克隆酶均相免疫法与ELISA具有较好的相关性.结论建立的克隆酶均相免疫技术可用于P53蛋白的测定,P53蛋白可作为判断胃癌预后的一项辅助诊断指标。

Global analysis of gene expression in yeast.

In the past decade, there has been an intense effort to comprehensively catalogue the expressed genes in the yeast Saccharomyces cerevisiae and to determine the absolute and relative abundance of transcript and protein levels under different cellular conditions. Several methods have been developed to monitor gene expression: DNA microarray analysis, Serial Analysis of Gene Expression (SAGE), kinetic RT-PCR and monitoring expression of beta-galactosidase fusion proteins. These techniques have been used to measure transcript and protein abundance in different developmental states and under different environmental stimuli. A wealth of expression data for yeast is now publicly available through several web sites. The expression information that exists has the obvious benefits of providing a better understanding of the gene expression patterns that accompany changes in a yeast cell's environmental and developmental states. This data has also, however, provided clues to unraveling the complicated questions surrounding gene regulation: why and how is gene expression controlled?

The Interdomain Region of Dengue NS5 Protein That Binds to the Viral Helicase NS3 Contains Independently Functional Importin β1 and Importin α/β-Recognized Nuclear Localization Signals

Dengue virus NS5 protein is a multifunctional RNA-dependent RNA polymerase that is essential for virus replication. We have shown previously that the 37- amino acid interdomain spacer sequence (residues (369)X(2)KKX(14)KKKX(11)RKX(3)405) of Dengue2 NS5 contains a functional nuclear localization signal (NLS). In this study, beta-galactosidase fusion proteins carrying point mutations of the positively charged residues or truncations of the interdomain linker region (residues 369-389 or residues 386-405) were analyzed for nuclear import and importin binding activities to show that the N-terminal part of the linker region (residues 369-389, a/bNLS) is critical for nuclear localization and is recognized with high affinity by the conventional NLS-binding importin alpha/beta heterodimeric nuclear import receptor. We also show that the importin beta-binding site (residues 320-368, bNLS) adjacent to the a/bNLS, previously identified by yeast two-hybrid analysis, is functional as an NLS, recognized with high affinity by importin beta, and able to target beta-galactosidase to the nucleus. Intriguingly, the bNLS is highly conserved among Dengue and related flaviviruses, implying a general role for the region and importin beta in the infectious cycle.

Patients with neoplastic and nonneoplastic hematologic diseases acquire CTLA-4 antibodies after blood transfusion.

The presence of antibodies to CTLA-4, a negative regulator of T-cell activation, was investigated in multiply transfused patients with malignant and non- malignant hematologic diseases. A previous study showed that, in multiply transfused patients, an immune response against nuclear matrix proteins can be induced by WBCs undergoing apoptosis during RBC unit storage. This study evaluated whether the same phenomenon could be involved in the induction of CTLA-4 antibodies in the patients analyzed. Patient sera were tested for binding to the recombinant full-length CTLA-4 beta-galactosidase fusion protein by an ELISA. Immuno-fluorescence stainings were performed to analyze the CTLA-4 epitopes recognized by the antibodies and to detect such epitopes in the apoptotic cells present in the RBC units. CTLA-4 antibodies were found in multiply transfused patients with beta-thalassemia (40%) and with other hemolytic diseases (33%) including leukemias (42%). A higher incidence of CTLA-4 antibodies was found in patients receiving non-WBC-reduced blood (88%) than in those receiving WBC-reduced blood (26%). Immunofluorescence staining showed that WBCs undergoing apoptosis in the RBC unit expressed CTLA-4 epitopes. The apoptotic WBCs present in the RBC units, after cold storage, express CTLA-4 epitopes. These epitopes can be released and induce formation of CTLA-4 antibodies with profound implications in the development of autoimmune disorders and in facilitating tumor dissemination and metastasis.

The endogenous retroviral insertion in the human complement C4 gene modulates the expression of homologous genes by antisense inhibition.

Intron 9 contains the complete endogenous retrovirus HERV-K(C4) as a 6.4-kb insertion in 60% of human C4 genes. The retroviral insertion is in reverse orientation to the C4 coding sequence. Therefore, expression of C4 could lead to the transcription of an antisense RNA, which might protect against exogenous retroviral infections. To test this hypothesis, open reading frames from the HERV sequence were subcloned in sense orientiation into a vector allowing expression of a beta-galactosidase fusion protein. Mouse L cells which had been stably transfected with either the human C4A or C4B gene both carrying the HERV insertion (LC4 cells), and L(Tk-) cells without the C4 gene were transiently transfected either with a retroviral construct or with the wild-type vector. Expression was monitored using an enzymatic assay. We demonstrated that (1) HERV-K(C4) antisense mRNA transcripts are present in cells constitutively expressing C4, (2) expression of retroviral-like constructs is significantly downregulated in cells expressing C4, and (3) this downregulation is further modulated in a dose-dependent fashion following interferon-gamma stimulation of C4 expression. These results support the hypothesis of a genomic antisense strategy mediated by the HERV-K(C4) insertion as a possible defense mechanism against exogenous retroviral infections.

The major tegumental antigen of Fasciola hepatica contains repeated elements.

In order to provide a better understanding of the interaction between the liver fluke (Fasciola hepatica) and the immune system of its mammalian host immunoreactive lambda bacteriophage clones containing F. hepatica cDNA have been isolated. Plasmids from these clones were sequenced and found to encode a family of proteins containing certain common elementst. All the clones contained a coding repeating sequence (RRRXCA) which is conserved at the nucleic acid level followed by a non-repeating element coding for the C terminal used by the proteins which shows conservation of amino acids at certain positions. Antisera raised against a beta-galactosidase fusion protein with one of these sequences as a terminal extension was used to localize the immunoreactive antigens. Binding was predominantly in the tegument of the juvenile fluke but was reduced in the adult tegument. The wall of the uterus showed strong reactivity in the adult. Rats immunized with the beta-galactosidase fusion protein showed enhanced resistance to challenge infections. The role of these antigens in the host response to infection by F. hepatica is discussed.

Characterization of a dodecapeptide containing a dominant epitope of Par j 1 and Par o 1, the major allergens of P. judaica and P. officinalis pollen

The pollen of Parietaria, a weed of the Urticaceae family, is a major cause of respiratory allergy in Europe, where the most common species are P. judaica and P. officinalis. Previously, we reported that a beta-galactosidase fusion protein (6a-BG) expressing a 26-bp cDNA fragment (6a cDNA) contained a dominant IgE-binding epitope (6a epitope) of the major allergens Par o 1 and Par j 1. The present study aimed to define the amino-acid sequence containing the 6a epitope. We analyzed the reactivity of anti-Par o 1 antibodies affinity purified from allergic patient sera with: 1) a panel of synthetic peptides deduced from the 6a nucleotide sequence using different reading frames 2) glutathione S-transferase (GST) fusion proteins containing selected peptides. The peptide NSARARADSCRI (p102) specifically bound anti-Par o 1 antibodies affinity purified from allergic patient sera or from rabbit anti-Par o 1 antiserum (ELISA). The related peptide NSARAGTSSCRI (p101) reacted to human but not to rabbit, anti-Par o 1 antibodies. GST fusion proteins containing p101 (GST 3.5) or p102 (GST 3.2) extensively inhibited the binding between Par o 1 and IgE or IgG antibodies from an allergic patient serum pool according to a dose-response curve. Percent inhibition of IgE antibodies binding obtained by absorbing a solution (50 microl) of affinity-purified antibodies with 5 microg of GST 3.2 or with 1.2 mg of GST 3.5 was 69% and 66%, respectively. In conclusion, the results of the present study indicate that the amino-acid sequences NSARARADSCRI (p102) and NSARAGTSSCRI (p101) contain the dominant epitope of Par o 1 and Par j 1 for human IgE and IgG antibodies indicated as 6a epitope. Moreover, the study shows that the epitope is conserved in recombinant molecules containing these peptides, irrespective of the fused polypeptide (beta-galactosidase or GST). The knowledge of the amino-acid sequence of this dominant epitope is important in therapeutic approaches to the development of allergen-derived haptens.

The efficient export of NADP-containing glucose-fructose oxidoreductase to the periplasm of Zymomonas mobilis depends both on an intact twin-arginine motif in the signal peptide and on the generation of a structural export signal induced by cofactor binding.

The periplasmic, NADP-containing glucose-fructose oxidoreductase of the gram-negative bacterium Zymomonas mobilis belongs to a class of redox cofactor-dependent enzymes which are exported with the aid of a signal peptide containing a so-called twin-arginine motif. In this paper we show that the replacement of one or both arginine residues results in drastically reduced translocation of glucose-fructose oxidoreductase to the periplasm, showing that this motif is essential. Mutant proteins which, in contrast to wild-type glucose-fructose oxidoreductase, bind NADP in a looser and dissociable manner, were severely affected in the kinetics of plasma membrane translocation. These results strongly suggest that the translocation of glucose-fructose oxidoreductase into the periplasm uses a Sec-independent apparatus which recognizes, as an additional signal, a conformational change in the structure of the protein, most likely triggered by cofactor binding. Furthermore, these results suggest that glucose-fructose oxidoreductase is exported in a folded form. A glucose-fructose oxidoreductase:beta-galactosidase fusion protein is not lethal to Z. mobilis cells and leads to the accumulation of the cytosolic preform of wild-type glucose-fructose oxidoreductase expressed in trans but not of a typical Sec-substrate (OmpA), indicating that the glucose-fructose oxidoreductase translocation apparatus can be blocked without interfering with the export of essential proteins via the Sec pathway.

Adenoviruses synergize with nuclear localization signals to enhance nuclear delivery and photodynamic action of internalizable conjugates containing chlorin e6.

Photosensitizers, molecules that produce active oxygen species upon activation by visible light, are currently being used in photodynamic therapy (PDT) to treat cancer and other conditions, where limitations include normal cells and tissue damage and associated side effects, and the fact that cytotoxic effects are largely restricted to the plasma and other peripheral membranes. In this study, we used insulin-containing conjugates to which variants of the simian-virus-SV40 large-tumor antigen (T-ag) nuclear localization signal (NLS) were linked in order to target the photosensitizer chlorin e6 to the nucleus. NLSs were included either as peptides coupled co-valently to the carrier bovine serum albumin, or within the coding sequence of beta-galactosidase fusion proteins. The most potent photosensitizing conjugate was the NLS-containing T-ag beta-galactosidase fusion protein (P10)-(chlorin e6)-insulin, exhibiting an EC50 more than 2400-fold lower than the value for free chlorin e6, and more than 15-fold lower than that of an NLS-deficient beta-galactosidase-(chlorin e6)-insulin construct, thus demonstrating that NLSs can increase the photosensitizing activity of chlorin e6. Attenuated adenoviruses were used to increase the nuclear delivery of conjugates through its endosomal-membrane-disrupting activity. In the case of the NLS-containing P10-conjugate, co-incubation with adenovirus increased the proportion of cells whose nuclear photosensitizing activity was higher than that in the cytoplasm by 2.5-fold. This use of adenoviruses in conjunction with photosensitizers has clear implications for achieving efficient cell-type-specific PDT.

Identification of a nuclear localization signal in activin/inhibin betaA subunit; intranuclear betaA in rat spermatogenic cells.

Activin is a dimeric glycoprotein hormone that was initially characterized by its ability to stimulate pituitary FSH secretion and was subsequently recognized as a growth factor with diverse biological functions in a large variety of tissues. In the testis, activin has been implicated in the auto/paracrine regulation of spermatogenesis through its cognate cell membrane receptors on Sertoli and germ cells. In this study we provide evidence for intranuclear activin/inhibin betaA subunit and show its distribution in the rat seminiferous epithelium. We have shown by transient expression in HeLa cells of beta-galactosidase fusion proteins that the betaA subunit precursor contains a functional nuclear localization signal within the lysine-rich sequence corresponding to amino acids 231-244. In all stages of the rat seminiferous epithelial cycle, an intense immunohistochemical staining of nuclear betaA was demonstrated in intermediate or type B spermatogonia or primary spermatocytes in their initial stages of the first meiotic prophase, as well as in pachytene spermatocytes and elongating spermatids primarily in stages IX-XII. In some pachytene spermatocytes, the pattern of betaA immunoreactivity was consistent with the characteristic distribution of pachytene chromosomes. In the nuclei of round spermatids, betaA immunoreactivity was less intense, and in late spermatids it was localized in the residual cytoplasm, suggesting disposal of betaA before spermatozoal maturation. Immunoblot analysis of a protein extract from isolated testicular nuclei revealed a nuclear betaA species with a molecular mass of approximately 24 kDa, which is more than 1.5 times that of the mature activin betaA subunit present in activin dimers. These results suggest that activin/inhibin betaA may elicit its biological functions through two parallel signal transduction pathways, one involving the dimeric molecule and cell surface receptors and the other an alternately processed betaA sequence acting directly within the nucleus. According to our immunohistochemical data, betaA may play a significant role in the regulation of nuclear functions during meiosis and spermiogenesis.

A Toxic Fusion Protein Accumulating between the Mitochondrial Membranes Inhibits Protein Assembly in Vivo

When overexpressed in Saccharomyces cerevisiae, beta-galactosidase fusion proteins directed to the mitochondria are toxic, preventing growth of yeast cells on non-fermentable carbon sources (Emr, S. D., Vassarotti, A., Garrett, J., Geller, B. L., Takeda, M., and Douglas, M. G. (1986) J. Cell Biol. 102, 523-533). We show that such fusion proteins interfere with the assembly of respiratory complexes in the mitochondrial inner membrane, without blocking protein translocation. The gene YME1, encoding an ATP-dependent metalloprotease of the mitochondrial inner membrane, acts as a suppressor of this defect; a 3-fold overexpression of Yme1p is sufficient to restore respiratory complex assembly and mitochondrial function. Detailed knowledge of the topology and effect of the toxic beta-galactosidase fusion proteins will permit the identification and characterization of components that control protein sorting and protein assembly within the mitochondrial inner membrane.

Dynamics of in vivo protein aggregation: building inclusion bodies in recombinant bacteria.

Time-dependent aggregation of a plasmid-encoded beta-galactosidase fusion protein, VP1LAC, has been carefully monitored during its high-rate synthesis in Escherichia coli. Immediately after recombinant gene induction, the full-length form of the protein steadily accumulates into rapidly growing cytoplasmic inclusion bodies. Their volume increases during at least 5 h at a rate of 0.4 micron3 h-1, while the average density remains constant. Protein VP1LAC accounts for about 90% of the aggregated protein throughout the building process. Minor components, such as DnaK and GroEL chaperones, have been identified in variable, but low concentrations. The homogeneous distribution of inclusion bodies among the cell population and the coexistence of large, still growing bodies with newly appearing aggregates indicate that the aggregation cores are mutually exclusive, this fact being a main determinant of the in vivo dynamics of protein aggregation.

Immunodiagnosis of clonorchiasis using a recombinant antigen.

A cDNA expression library of Clonorchis sinensis adult worm was constructed, and screened out immunologically. One clone, pBCs31, was selected in view of its predominant reactivity with an experimentally infected rabbit serum. Recombinant C. sinensis antigen with 28 kDa as a beta-galactosidase fusion protein produced in Escherichia coli was identified by immunoblot analysis. The cloned gene was composed of 16 copies of a 30 base pair repeat and an additional 320 bases. The deduced amino acid sequence of the tandem repeat was AQPPKSGDGG. On RNA slot blot analysis. C. sinensis adult worm RNA showed a positive reaction with the cloned gene. Enzyme-linked immunosorbent assay using a purified recombinant antigen of pBCs31 showed high specificity for diagnosis of clonorchiasis.

Glypican and biglycan in the nuclei of neurons and glioma cells: presence of functional nuclear localization signals and dynamic changes in glypican during the cell cycle.

We have investigated the expression patterns and subcellular localization in nervous tissue of glypican, a major glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan that is predominantly synthesized by neurons, and of biglycan, a small, leucine-rich chondroitin sulfate proteoglycan. By laser scanning confocal microscopy of rat central nervous tissue and C6 glioma cells, we found that a significant portion of the glypican and biglycan immunoreactivity colocalized with nuclear staining by propidium iodide and was also seen in isolated nuclei. In certain regions, staining was selective, insofar as glypican and biglycan immunoreactivity in the nucleus was seen predominantly in a subpopulation of large spinal cord neurons. The amino acid sequences of both proteoglycans contain potential nuclear localization signals, and these were demonstrated to be functional based on their ability to target beta-galactosidase fusion proteins to the nuclei of transfected 293 cells. Nuclear localization of glypican beta-galactosidase or Fc fusion proteins in transfected 293 cells and C6 glioma cells was greatly reduced or abolished after mutation of the basic amino acids or deletion of the sequence containing the nuclear localization signal, and no nuclear staining was seen in the case of heparan sulfate and chondroitin sulfate proteoglycans that do not possess a nuclear localization signal, such as syndecan-3 or decorin (which is closely related in structure to biglycan). Transfection of COS-1 cells with an epitope-tagged glypican cDNA demonstrated transport of the full-length proteoglycan to the nucleus, and there are also dynamic changes in the pattern of glypican immunoreactivity in the nucleus of C6 cells both during cell division and correlated with different phases of the cell cycle. Our data therefore suggest that in certain cells and central nervous system regions, glypican and biglycan may be involved in the regulation of cell division and survival by directly participating in nuclear processes.

Kinetic Characterization of the Human Retinoblastoma Protein Bipartite Nuclear Localization Sequence (NLS) in Vivo andin Vitro: A COMPARISON WITH THE SV40 LARGE T-ANTIGEN NLS

The retinoblastoma (RB) tumor suppressor is a nuclear phosphoprotein important for cell growth control and able to bind specifically to viral oncoproteins such as the SV40 large tumor antigen (T-ag). Human RB possesses a bipartite nuclear localization sequence (NLS) consisting of two clusters of basic amino acids within amino acids 860-877, also present in mouse and Xenopus homologs, which resembles that of nucleoplasmin. The T-ag NLS represents a different type of NLS, consisting of only one stretch of basic amino acids. To compare the nuclear import kinetics conferred by the bipartite NLS of RB to those conferred by the T-ag NLS, we used beta-galactosidase fusion proteins containing the NLSs of either RB or T-ag. The RB NLS was able to target beta-galactosidase to the nucleus both in vivo (in microinjected cells of the HTC rat hepatoma line) and in vitro (in mechanically perforated HTC cells). Mutational substitution of the proximal basic residues of the NLS abolished nuclear targeting activity, confirming its bipartite character. Nuclear accumulation of the RB fusion protein was half-maximal within about 8 min in vivo, maximal levels being between 3-4-fold those in the cytoplasm, which was less than 50% of the maximal levels attained by the T-ag fusion protein, while the initial rate of nuclear import of the RB protein was also less than half that of T-ag. Nuclear import conferred by both NLSs in vitro was dependent on cytosol and ATP and inhibited by the nonhydrolyzable GTP analog GTPgammaS. Using an ELISA-based binding assay, we determined that the RB bipartite NLS had severely reduced affinity, compared with the T-ag NLS, for the high affinity heterodimeric NLS-binding protein complex importin 58/97, this difference presumably representing the basis of the reduced maximal nuclear accumulation and import rate in vivo. The results support the hypothesis that the affinity of NLS recognition by NLS-binding proteins is critical in determining the kinetics of nuclear protein import.

Nuclear Targeting of Chlorin e6 Enhances Its Photosensitizing Activity

Although photosensitizers, molecules that produce active oxygen species upon activation by visible light, are being extensively used in photodynamic therapy to treat cancer and other clinical conditions, problems include normal cell and tissue damage and associated side effects, which are attributable in part to the fact that cytotoxic effects are largely restricted to the plasma membrane. We have previously shown that the photosensitizer chlorin e6 has significantly higher photosensitizing activity when present in conjugates containing specific ligands and thus able to be internalized by receptor-expressing cells. In this study we use insulin-containing conjugates to which variants of the simian virus SV40 large tumor antigen nuclear localization signal (NLS) were linked to target chlorin e6 to the nucleus, a hypersensitive site for active oxygen species-induced damage. NLSs were either included as peptides cross-linked to the carrier bovine serum albumin or encoded within the sequence of a beta-galactosidase fusion protein carrier. The results for photosensitization demonstrate clearly for the first time that NLSs increase the photosensitizing activity of chlorin e6, maximally reducing the EC50 by a factor of over 2000-fold. This has wide-reaching implications for achieving efficient cell type-specific photodynamic therapy.

A retroviral vector that allows efficient co-expression of two genes and the versatility of alternate selection markers.

pZIG(hGCSFR) is a retroviral vector that can co-express two genes and also provides alternative selection markers. This retroviral vector has been constructed to incorporate an internal ribosome entry site (IRES) element to co-express two exogenous genes in mammalian cells. Two marker/selection genes have been cloned into the vector that not only allow the efficiency of co-expression to be quantified but also provide versatility of selection criteria. A cDNA encoding the hGCSFR (signaling deficient) was cloned as the 5' cistron, and a gene encoding a neomycin-resistance and beta-galactosidase (beta geo) fusion protein was cloned as the 3' cistron. The two marker genes cloned in this retroviral vector allow the selection and isolation of mammalian cells following either transient (by fluorescence-activating cell sorting analysis of hGCSFR-positive cells) or stable (neomycin resistance) infection with the certainty that over 93% of clones will co-express both genes. This novel vector offers a versatile retroviral vector that can be potentially used in a wide range of gene therapy applications as the gene of interest can be coexpressed with either of the marker genes depending on whether the retroviral infection is to be performed in vitro, in vivo, or ex vivo.

Nuclear entry, oligomerization, and DNA binding of the Drosophila heat shock transcription factor are regulated by a unique nuclear localization sequence.

In normally growing Drosophila cultured cells the Drosophila heat shock transcription factor (dHSF) is localized in the cytosol and translocates into the nucleus after heat shock. In the cytosol of nonshocked cells, the dHSF is present as a monomer that cannot bind DNA. Upon stress, the dHSF enters the nucleus where it is observed to be a trimer. A novel nuclear localization sequence (NLS) in the dHSF was found to be responsible for stress-dependent nuclear entry. Deletion of the NLS prevents nuclear entry, as expected, yet surprisingly also allows constitutive oligomerization and DNA binding in the cytosol. Further analysis of the NLS by mutagenesis suggests that the two functions of nuclear entry and oligomerization are separable in that distinct residues present in the NLS are responsible for each. Mutations in certain basic residues completely block nuclear entry, as expected for a constitutive NLS. In addition, two residues were found in the NLS that, when altered, allowed constitutive nuclear entry of dHSF independent of stress. These residues may interact with a putative cellular component or possibly other domains of the HSF to prevent nuclear entry in normally growing cells. The NLS can also function autonomously to target a beta-galactosidase fusion protein into the nucleus in a heat shock-dependent fashion.