Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA
Author for corresondence (e-mail: csp{at}csp.caltech.edu)
Accepted June 15, 2001
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SUMMARY |
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Key words: Drosophila melanogaster, dKap-3, Heat shock factor, Nuclear transport
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INTRODUCTION |
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Nuclear restriction also plays a role in the regulation of the heat shock response during early development in Drosophila (Wang and Lindquist, 1998). Although many heat shock proteins (HSPs) of Drosophila are maternally supplied, including Hsp83 and the small HSPs, Hsp70 is not (Arrigo and Tanguay, 1991; Zimmerman and Cohill, 1991). In fact Hsp70 is not inducible by heat shock in the early Drosophila embryo, nor is any other HSP gene (Graziosi et al., 1980). Indeed Hsp70 is not inducible in embryos of a wide variety of organisms in addition to flies including mice, frogs and sea urchins (Heikkila et al., 1985; Morange et al., 1984; Roccheri et al., 1982). Despite the fact that in the Drosophila embryo the heat shock transcription factor (dHSF; which is responsible for the heat-induced transcription activation of HSP genes) is maternally supplied and abundant, the embryo remains refractory to heat shock until cycle 13. Wang and Lindquist have shown that the dHSF does not enter the nucleus until cycle 13 at which point Hsp70 induction can occur (Wang and Lindquist, 1998). Developmental regulation of the nuclear localization of dHSF, therefore, plays a key role in the establishment of the heat shock response in the early embryo.
The nuclear localization sequence of the dHSF has been identified and characterized (Zandi et al., 1997b). To identify protein(s) that bind to the NLS and may be involved in the nuclear transport of dHSF, a yeast two-hybrid screen was conducted using the NLS as bait. Several positive Drosophila cDNAs were identified of which one belongs to the karyopherin family of nuclear transport proteins, and has been designated Drosophila karyopherin-3 (dKap-
3) (Gorlich et al., 1994; Kohler et al., 1997). Biochemical analysis of dKap-
3 demonstrates specific and functional interactions of the nuclear transporter with dHSF in vitro. Examination of the temporal and spatial expression pattern of dKap-
3 revealed that it is not expressed until cycle 13 of embryogenesis. These observations strongly support the notion that dKap-
3 is the nuclear transporter of the dHSF in vivo, and that developmental regulation of dKap-
3 synthesis determines the time at which the heat shock response can be activated in the early embryo.
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MATERIALS AND METHODS |
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dKap-3 was expressed in E. coli cells by subcloning the full-length cDNA into a GST fusion protein vector pGEX-2t. The GST-tagged dKap-
3 was then bound to a GST-affinity resin (Stratagene) and eluted with 10 mM reduced glutathione in 50 mM Tris (pH 8). The GST tag was cleaved with thrombin for 60 minutes at a ratio of 3 units protease per mg of recombinant protein in 50 mM Tris (pH 8), 150 mM NaCl and 2.5 mM CaCl2.
Colony lift ß-galactosidase filter assay
The primary His+ transformants were grown in 5 ml selective SD medium at 30°C to OD 0.5. Cells were then re-streaked onto a 150 mm SD/-His/-Trp/-Leu agar plate and incubated for 72 hours at 30°C. A sterile Whatman no. 5 filter was placed over the surface of the agar plate. As soon as the filter was completely wet it was carefully lifted off the plate and quick frozen in liquid nitrogen for 1 minute. A second sterile Whatman no. 5 filter was pre-soaked in 3.5 ml of Z buffer/X-gal solution. The frozen filter was thawed at room temperature and carefully placed on the second filter, colony side up. Both filters were incubated at room temperature for up to 8 hours until the appearance of blue colonies.
In vitro cross-linking assay
A 191 aa 6HIS-tagged polypeptide from dHSF (mini-probe) containing the NLS was over-expressed in DE3 cells and purified by Ni-NTA affinity column (Qiagen). In addition, a similar 154 aa polypeptide with the NLS deleted (mini-NLS probe) was also over-expressed in DE3 as were all polypeptides with point mutant NLS (mini-mNLS probes). All of the protein probes were labeled with [
-32P]ATP by MAPK phosphorylation in vitro.
dKap-3 (1 µg/µl) was incubated in D buffer (25 mM Hepes, pH 7.9, 100 mM KCl, 1 mM EDTA and 0.2% Triton X-100) with 2 µl of
-32P-mini probes (1 ng/µl, approx. 80000 cpm/µl) for 20 minutes at 25°C in a 20 µl reaction volume. Cross-linking was subsequently carried out by addition of 2 µl of 20 mM DSS and incubation for 15 minutes at 25°C. The reaction was quenched by the addition of 2 µl of 200 mM lysine for 10 minutes. Protein-protein adducts were analyzed by 6% SDS-PAGE and autoradiography.
Cloning, expression and purification of Drosophila karyopherin (importin) ß
Four PCR primers were designed according to the partial genomic sequence published in GenBank (accession number g92598391):
5'GCGCGCGAATTCCATATAGAGAGGAAAAGAG3'
5'GCGCGCCTCGAGCATAGTGCTTGGACAC3'
5'GCGCGCCTCGAGGTGCTCTGCAGTTCCTG3'
5'GCGCGCTCTAGACTACTGTGCGATGGACCTGGGT3'
Two amplified fragments corresponding to 26 to 962 and 963 to 2655 of the cDNA sequence of karyopherin ß were obtained using the above primers and a Drosophila embryonic cDNA library (t11) as PCR template. The two fragments were ligated into pBluescript (Stratagene) and sequenced. The full-length karyopherin ß was fused to a GST tag using vector pGEX-2t (Smith and Johnson, 1988), expressed in E. coli, and purified with GST affinity resin (Stratagene). The GST tag was removed with thrombin.
In vitro nuclear docking assays
In vitro nuclear docking assays were performed according to the methods developed by (Stochaj and Silver, 1992). To study the binding of Drosophila heat shock factor NLS, Schneider cells were allowed to attach to polylysine-coated slides for 20 minutes on ice. Then the cells were permeabilized with 45 µg/ml digitonin in Buffer A (20 mM Hepes (pH 7.3), 110 mM potassium acetate, 5 mM sodium acetate, 2 mM magnesium acetate, 1 mM EGTA, 2 mM DTT, 1 mM phenylmethylsulphonyl fluoride (PMSF), and complete protease inhibitors from Boehringer for 20 minutes on ice. NLS-EGFP fusion proteins, dKap-3 and dKap-ß were pre-incubated on ice for 20 minutes, then were incubated with permeabilized cells at room temperature for 20 minutes. Cells were washed with Buffer A and fixed with Histochoice Tissue Fixative MB (Amresco). Slides were mounted in Buffer A/90% glycerol containing 1 mg/ml o-phenylenediamine.
Immunofluorescence staining
Mouse monoclonal antibodies were raised against recombinant dKap-3 using standard immunization procedures. Three monoclonal lines were characterized: 5E3, 5F6, 6G7 and all three reacted with an epitope present in the N-terminal 100 amino acids of dKap-
3. This domain is unique among the dKap-
family members and therefore should eliminate any cross reactivity. Western analysis with these antibodies reveal only a single strongly reactive protein species of the correct molecular mass.
18x18 mm no. 1 cover slips were coated in 1 mg/ml poly-L-lysine for 15 minutes and air dried. 0.5 ml of Drosophila SL2 cells with a density of 4x105 cells/ml were placed onto each cover slide and incubated for 15 minutes at room temperature. Cells were then heat shocked at 37°C for various times, washed with PBST, and fixed with Histochioce tissue fixative MB (Amresco) for 15 minutes on ice. After several washes to remove the fixative, the fixed cells were incubated with 1:1000 dilution of monoclonal anti-dKap-3 antibody (5E3) in PBS buffer containing 0.5% bovine serum albumin (BSA) for 2 hours at room temperature. Cells were then washed with PBST 4 times for 10 minutes each to remove unbound first antibody. Fluorescein-coupled goat anti-mouse IgG (Pierce) secondary antibody was then added at 1:100 dilution in 0.5% BSA in PBS and incubated at 4°C overnight. Cell nuclei were visualized by co-staining with DAPI (4', 6-diamidine-2'-phenylindole dihydrochloride) for 10 minutes. Finally, the cover slips were washed with PBST 4 times and mounted onto microscope slides in 90% glycerol/PBS containing 2.5% DABCO (1,4-diazabicyclo [2,2,2] octane; Sigma). The fluorescent images were viewed and photographed using a Zeiss Axioplan microscope with UV irradiation and appropriate filters.
Fixed Drosophila embryos were rehydrated in a methanol/PBST series: 15 minutes each in 75%, 50%, 25% and 30 minutes in PBST. Immunofluorescence staining was then carried out as described previously (Patel, 1994). All antibodies were pre-incubated with 0- to 12-hour embryos overnight at 4°C. Anti-dKap-3 antibodies were diluted 1:500 and anti-dHSF antibodies were diluted 1:250. Fluorescent dye-labeled secondary antibodies were diluted 1:100. All primary antibody incubations were at 4°C overnight and secondary antibody incubations were at room temperature for 90 minutes. Images were taken using confocal microscopy (Zeiss, LSM310) or Axioplan microscopy. The embryonic stages were determined by co-staining with DAPI (4', 6-Diamindine-2'-phenylinedole dihydrochloride).
Developmental western blots
0- to 2-hour, 0- to 4-hour and 0- to 6-hour embryos were collected and washed with 0.03% Triton X-100/0.9% NaCl. Non-shocked or heat shocked (37°C for 15 minutes) embryos were rinsed twice with homogenization buffer (50 mm Tris (pH 7.5), 140 mM NaCl, 5 mM MgCl2, 0.05% NP-40, 1 mM PMSF, 1 µg/ml pepstain A, 1-2 µg/ml aprotinin, 1 µg/ml leupeptin), and then homogenized in 2 volumes of homogenization buffer. Extracts were then centrifuged to remove cell debris and the supernatants were mixed with SDS-PAGE gel loading buffer and electrophoresed in 8% SDS-PAGE. The separated proteins were then transferred to nitrocellulose and blocked with 5% non-fat dry milk overnight. The blot was then probed with anti-dDKap-3 monoclonal antibody 5E3 and developed with anti-mouse alkaline phosphatase conjugated antibodies.
Embryo preparation
Drosophila embryos were collected in the population cages with different time span. No-shock embryos were processed immediately, while heat-shock samples were incubated in a 37°C water bath for 15 minutes. All embryos were washed with NaCl-Triton (0.9%NaCl, 0.03%Triton X-100), dechorionated in 50% bleach for 3 minutes and fixed in n-heptane/formaldehyde/PBS (5:1:5) for 30 minutes. After removing vitelline membranes in n-heptane/methanol (1:1) by vigorous shaking, embryos were washed three times with methanol and stored in ethanol at 20°C. For western blotting, embryonic extracts were made without the fixation step and stored at 70°C.
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RESULTS |
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To identify components of this regulatory system, a 43 amino acid segment of the Drosophila HSF which includes the NLS (Fig. 1A) was used as bait in a yeast two-hybrid system screen (Bartel et al., 1993; Chien et al., 1991; Fields and Song, 1989). This screen allowed a search of a Drosophila cDNA embryonic library for proteins capable of specifically binding to the NLS. The screen revealed one primary class of NLS-binding proteins with sequence similarity to nuclear transport proteins.
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Karyopherin-3 binds specifically to the Drosophila HSF NLS
In vitro protein cross-linking was used to determine the capability and specificity of dKap-3 binding to the Drosophila HSF NLS. A 191 amino acid protein segment derived from the dHSF containing a centrally located NLS was cloned into pET11a and expressed in E. coli (Fig. 1A). The purified protein was labeled in vitro with MAP kinase and [
-32P]ATP at fortuitous MAP kinase sites present within the protein segment; this probe was termed the mini-NLS. An otherwise identical protein segment containing a deletion of the NLS was similarly labeled with MAP kinase and termed mini-
NLS (Fig. 1A). Mini-NLS was incubated with recombinant dKap-
3, molecularly cross-linked using disuccinimidyl suberate (DSS) and the product of this reaction analyzed by SDS-PAGE (Fig. 2 lane 1). A complex of approximately 90 kDa was identified by autoradiography of the gel. This complex is only observed when dKap-
3 was present in the reactions, and immunoprecipitation with anti-Kap-
3 antibodies demonstrates that Kap-
3 is present in the complex (data not shown). Similar analysis with the mini-
NLS revealed no complex formation (Fig. 2 lane 9). These results demonstrate that the NLS is required for dKap-
3 binding to the mini probes.
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Karyopherin-ß enhances dKap-3 binding to the NLS of Drosophila HSF
Active nuclear transport complexes in vivo include an /ß-karyopherin heterodimer with the
subunit bound to the NLS of the cargo (Enenkel et al., 1995; Rexach and Blobel, 1995). It has been shown that in vitro binding of recombinant yeast Kap-
(kap 60) to NLS domains is cooperatively enhanced by Kap-ß (kap 95) (Rexach and Blobel, 1995). The cross-linking experiments described above show that in the absence of dKap-ß the NLS and dKap-
3 bind specifically to each other. To determine what quantitative role dKap-ß may have in the dNLS-dKap-
3 interaction we cloned the Drosophila Kap-ß cDNA using primers derived from the known genomic sequence (see Materials and Methods). dKap-ß was expressed in E. coli and purified using the GST-tag system and the GST-tag removed prior to use (Smith and Johnson, 1988).
and ß proteins were combined and incubated with 32P-labeled mini probes cross-linked with DSS and examined by SDS-PAGE. The addition of dKap-ß to the reactions modestly stimulated dKap-
3 binding to the NLS (Fig. 3A; compare lanes 1, 2 and 3). Interestingly, the molecular mass of the complex was not altered by the presence of dKap-ß, suggesting that either the dKap-ß association is transient in vitro or the interaction is such that it cannot be cross-linked with DSS.
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To investigate the role of these domains in dKap-3 binding to the Drosophila HSF NLS, N-terminal deletions of dKap-
3 were examined. One deletion removed the internal NLS (deleting residues 1-47 of the N terminus) and the other had the entire N-terminal 99 amino acid ß-binding domain deleted. These proteins were cross-linked to 32P-labeled mini-NLS and mini-
NLS probes and the results are shown in Fig. 3B, lanes 3-6. Drosophila Kap-
3, deleted of its internal NLS, bound the mini-NLS probe very well but not the mini-
NLS probe (lanes 3 and 4, respectively). Deletion of the entire ß-binding domain, however, eliminates mini-NLS probe binding (lane 5). This observation suggests that the ß-binding domain of Drosophila Kap-
3 is necessary for dHSF-NLS binding. This is unexpected because previous biochemical and structural studies with yeast and human proteins have demonstrated that karyopherin-
3 binds to its target NLS-peptide cargo within the arm repeats. Indeed, an N-terminal ß-binding domain-deleted form of the yeast
3 protein was sufficient for crystallization with an SV-40 NLS peptide (Conti et al., 1998). It is possible that the use of a significantly larger cargo in our experiments requires a more significant portion of dKap-
3 for docking.
Karyopherin-3 is required for nuclear docking in vitro
Nuclear docking experiments were performed to determine if dKap-3 and dKap-ß can target the dHSF-NLS to the nuclear pore complex. These experiments employed Schneider (SL2) cells, which were depleted of nuclear transport factors along with other cytosolic proteins by digitonin permeabilization (Smith and Johnson, 1988). Nuclear transport factors are added to the cells as purified recombinant proteins along with NLS-EGFP fusion proteins to serve as the cargo protein. As shown in Fig. 4, wild-type NLS fused to EGFP was effectively docked on the nuclear periphery in the presence of both
and ß proteins (Fig. 4B). The NLS-EGFP protein alone was not able to dock on the nuclear membrane (Fig. 4A), indicating that dKap-
3 and dKap-ß are required for docking. Consistent with the biochemical crosslinking experiments, and in vivo localization studies, the K405M NLS mutant did not show nuclear docking in the presence of dKap-
3 and dKap-ß (Fig. 4C). The constitutive nuclear-localized mutant, Q403L NLS, was able to dock on the nuclear pore complexes in the presence of dKap-
3 and ß, as expected (Fig. 4D).
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Similar analyses of the mRNA distribution of dHSF and dKap-3 are in agreement with the protein distribution patterns both temporally and spatially. The absence of appreciable dKap-
3 mRNA in the early embryo is evident in Fig. 7M (panel 1), where a cycle 10-11 embryo is compared with a cycle 13-14 embryo that shows considerable mRNA accumulation. It is interesting to note, however, the presence of a small amount of dKap-
3 mRNA in the posterior of the cycle 10-11 embryo where the pole cells will arise (indicated by the arrow; Fig. 7M, panel 1). These observations demonstrate that dKap-
3 expression during the first 12 cycles of embryogenesis is restricted to the very posterior of the embryo. Examination of the posterior region of cycle 12 embryos with anti-dHSF antibodies shows clear localization of dHSF protein within the nuclei of the poll cells (Fig. 7M, panel 2). These data further strengthen the correlation between the presence of dKap-
3 and the nuclear localization of dHSF.
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DISCUSSION |
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Biochemical analysis of dKap-3
dKap-3 is likely to be the bona fide nuclear transporter for dHSF for a number of reasons. First, the two-hybrid system used to screen for NLS binding proteins selected dKap-
3 as the primary interacting protein. Second, dKap-
3 was the only gene isolated from this family; indeed, neither dKap-
1 nor dKap-
2 were identified in this screen although both cDNAs are present in the embryonic library that was used (unpublished observations; Kussel and Frasch, 1995; Torok et al., 1995). Third, point mutations in the NLS of dHSF, which block nuclear entry in vivo, also prevent dKap-
3 binding in vitro. Finally, dKap-ß promotes binding of the NLS to dKap-
3 in vitro and together the
3 and ß proteins allow NLS-EGFP fusion proteins to dock to nuclei in digitonin-treated cells. Successful docking to the nuclei is also sensitive to mutations in the NLS that prevent nuclear entry in vivo.
Localization of dKap-3
In normally growing SL-2 cells, dKap-3 protein is uniformly localized throughout the cells. After heat shock, the transporter relocates to the nuclear membrane and within 15 minutes is entirely excluded from the nucleus. The significance of this localization may be to prevent interactions of dHSF with the transporter while it is involved in the activation of transcription. Alternatively, the dKap-
3 may become associated with the nuclear membrane and not be able to undergo the normal nuclear transporter recycling events during heat stress.
The spatial and temporal aspects of dKap-3 expression in the early embryo demonstrate that the dKap-
3 protein is not expressed until 13 cycle, at which point significant RNA and protein accumulation is observed. Remarkably, this correlates precisely with that of dHSF nuclear entry and Hsp70 heat inducibility. The absence of dKap-
3 expression is coincident with the refractory period of Hsp70 induction and the nuclear exclusion of dHSF. Taken together with the biochemical analysis, these data present a compelling case that dKap-
3 is the nuclear transporter of the dHSF in vivo. Further genetic analysis will be necessary to demonstrate that mutations in dKap-
3 alter dHSF subcellular localization and function.
The role of dKap-3
Western blotting and immunoprecipitation of dKap-3 in cultured Drosophila Kc cells have shown that dKap-
3 is present in significantly greater quantities than dHSF (data not shown). It is therefore reasonable to suppose that dKap-
3 transports a number of other proteins into the nucleus. Indeed, a recent report using similar methods to identify proteins interacting with the Drosophila germ cell-less protein identified the same dKap-
3 described in this report (Dockendorff et al., 1999). Interestingly, the Gcl protein also contains a bi-partite NLS of approximately 30 amino acids. Comparison of the two NLSs revealed essentially no sequence similarity other than the presence of basic residues. Computer projections of the putative structure of the dHSF NLS suggests that it is
-helical (Zandi et al., 1997b), whereas the Gcl NLS contains two proline residues that would interrupt an
-helical structure.
Previous northern analysis and whole-mount in situ hybridization results have suggested that dKap-3 is ubiquitously expressed throughout early development (Dockendorff et al., 1999). These observations do not agree with our analysis of protein and RNA expression. Although we do not observe any appreciable accumulation of either dKap-
3 protein or mRNA until cycle 13, there is a small amount mRNA in the posterior of cycle 10-11 embryos as described in the Results section. This RNA may provide dKap-
3 for the developing pole cells and hence transport Gcl; it is clear that in the cycle 12 pole cells dHSF is nuclear. Thus, early expression of dKap-
3 in the posterior of the embryo may facilitate nuclear entry of critical proteins like Gcl into the developing pole cells.
Domains of dKap-3
Remarkably, deletion of the dKap-3 ß-binding domain eliminates binding of the dHSF NLS to dKap-
3 in vitro. Previous structural studies have shown that for a fragment of the yeast dKap-
3 protein, which lacks the ß-domain, two binding sites exist for an SV-40 NLS peptide within the arm-repeat domain (Conti et al., 1998). A recent structural study of mouse importin
using full-length protein shows that the N-terminal ß-binding domain is capable of interacting intramolecularly with the arm repeats to form a self-inhibitory structure (Kobe, 1999). In this case no exogenous NLS was present in the crystals. It is likely that the significant size difference between the dHSF mini-NLS cargo used in this report and the SV-40 peptide may explain why other domains of the
-3 protein are needed for binding.
Early embryonic transcription and nuclear transport
Early development in Drosophila is characterized by series of rapid zygotic nuclear divisions without appreciable transcription until cycles 8 and 9 (Erickson and Cline, 1993). It has been demonstrated that components of the basal transcription machinery are transported into the nuclei at different division cycles. The RNA polymerase IIC subunit is found within the nucleus at cycle 7 whereas TFIIDs TATA-binding protein (TBP) is localized within the nucleus between cycles 8 and 9 (Wang and Lindquist, 1998). The timing of dHSF entry into the nucleus is independent of these two general factors and this is probably due to the requirement of other nuclear transport molecules for the nuclear localization of these basal factors.
Developmental regulation of the heat shock response by a nuclear transporter represents a novel form of transcription regulation for a specific group of genes. It is possible that the absence of transcription during early embryonic stages may, in general, be due to the absence of specific nuclear transporters, at least for those transcription factors that are maternally provided. Indeed, this mechanism could represent a general explanation for the lack of transcription of early acting genes in embryonic nuclei. It will be very interesting to determine whether the nuclear entry of specific transcription factors as well as members of the basal transcription machinery correlates with the presence of specific nuclear transporters.
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ACKNOWLEDGMENTS |
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