INTRODUCTION

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The vast majority of eukaryotic heat stress (HS)-inducible genes share conserved palindromic promoter elements with the consensus motif (AGAAn)(nTTCT) (2, 23, 32). They represent the recognition sites for the corresponding HS transcription factors (Hsfs), which are characterized by an N-terminal DNA-binding domain with a central helix-turn-helix motif (11, 46, 52), an adjacent domain with heptad hydrophobic repeats (HR-A/B) involved in oligomerization, and a C-terminal activator domain (for reviews, see references 27, 30, and 54).

Hsfs are encoded by small multigene families (see the summary by Nover et al. [30]). So far, four types of Hsfs have been characterized for plants (5, 8, 12, 43, 44) and vertebrates (19, 21, 34, 41, 45). In tomato, a constitutively expressed HsfA1 is accompanied by two HS-inducible forms, HsfA2 and HsfB1. By use of tobacco protoplasts as a transient-expression system, all three were shown to function as transcriptional activators (15, 51).

In contrast to those in plants, none of the four Hsfs in vertebrates is expressed in a stress-dependent manner. Hsf1 is the major form expressed in all cells. Its activity and intracellular localization are under stress control. Hsf2 is evidently involved in developmental control of chaperone gene expression, whereas Hsf3 may be considered a cell-specific variant of Hsf1 (13, 16, 19, 20, 40, 49). A new member of the vertebrate Hsf family is the recently described Hsf4 (21). It lacks an activator region and probably functions as a repressor of the basal-level expression of HS genes. This multiplicity may be increased by additional variants of Hsf1 and Hsf2 created by alternative splicing adding or eliminating a small, 66-bp exon close to the C-terminal HR-C region (6, 9).

Two reports from R. Morimoto's group present evidence for a functional cooperation of different Hsfs in vertebrate cells. (i) Both Hsf1 and Hsf2 are expressed in human erythroleukemia cells; Hsf1 is activated by HS, whereas Hsf2 is probably involved in chaperone gene expression during hemin-induced differentiation of these cells. Both proteins trimerize and translocate into the nucleus. Interestingly, a synergistic effect on hsp70 gene transcription was observed if hemin treatment was followed by HS (48). (ii) In the avian erythroblast HD6 cell line, Hsf1 and Hsf3 can be activated by HS to undergo trimerization and transport to the nucleus. However, Hsf1 activation precedes that of Hsf3 (20). In both studies, the active Hsf forms were found to be homotrimers. There is no evidence for a physical interaction of different Hsfs.

In the course of our investigations of the intracellular localization of Hsfs in tobacco protoplasts and the characterization of putative nuclear localization signal (NLS) motifs in tomato HsfA1 and HsfA2, we observed an unexpected property of HsfA2. Despite having a functional NLS, it is defective in nuclear import, forming large cytoplasmic aggregates under HS conditions. This defect can be relieved by deletion of short C-terminal peptide motifs, and efficient nuclear import of this truncated versions is connected with a considerable increase of their activator potential as determined by an Hsf-dependent reporter assay (15).

In order to mimic the physiological basis for the HsfA2 function and to reconstruct the situation of the native tomato cells, we coexpressed HsfA2 with HsfA1 and/or HsfB1 in tobacco protoplasts. The results presented in this paper demonstrate efficient nuclear import of HsfA2 in the presence of HsfA1 but not of HsfB1. Using immunofluorescence and immunoelectron microscopy, we document the dynamic changes of the intracellular localization of HsfA2 in tomato cells and present evidence for the direct physical interaction of HsfA1 and HsfA2 by means of coimmunoprecipitation and a Saccharomyces cerevisiae two-hybrid test.

	MATERIALS AND METHODS

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General materials and methods. On the basis of an international agreement (30), the nomenclature of Hsfs and of their functional parts was revised. Following this, tomato Hsf8, Hsf30, and Hsf24 (44) are now designated HsfA1, HsfA2, and HsfB1, respectively.

Antisera. Rabbit and chicken antisera against tomato HsfA1, HsfA2, and HsfB1 and pea Hsp18 class I were generated by Eurogentec SA (Seraing, Belgium) by using purified recombinant proteins prepared in our laboratory. For detection of Hsp90 and the Hsp-Hsc70 complex and detection of tubulin, commercial antisera from StressGen and Amersham, respectively, were used.

Coimmunoprecipitation. Tomato cells (200 mg [fresh weight]) were collected on filter discs and homogenized by sonication in 400 µl of lysis buffer containing 25 mM HEPES (pH 8.0), 500 mM NaCl, 5 mM MgCl₂, 1 mM EDTA, 10 mM NaF, 0.2% Nonidet P-40, 10 mM -mercaptoethanol, and protease inhibitors (10 µg of Pefabloc per ml, 1 µg of pepstatin per ml, 2 µg of aprotinin per ml, 1 µg of leupeptin per ml, 50 µg of TLCK [N-p-tosyl-L-lysyl chloromethyl ketone] per ml, and 100 µg of TPCK [N-p-tosyl-L-phenylalanyl chloromethyl ketone] per ml). Prior to addition of antiserum, protein levels were adjusted to about 10 µg/µl, and 50-µl samples were diluted 10-fold with modified lysis buffer containing 100 mM NaCl but no -mercaptoethanol. Tobacco protoplast lysates from 4 × 10⁵ cells were prepared by sonication in 400 µl of modified lysis buffer containing 150 mM NaCl and 1 mM -mercaptoethanol. The resulting lysates were precleared with protein A-Sepharose and incubated for 1 h at 4°C with 3 µl of HsfA2 antiserum in bovine serum albumin (BSA)-coated reaction tubes. Protein A-Sepharose beads (Pharmacia) were added as a 1:1 slurry in lysis buffer, and samples were further incubated at 4°C for 1 h with gentle agitation. The beads were pelleted and washed three times with 500 µl of 10 mM Tris buffer (pH 8.0) supplied with 140 mM NaCl and 500 mM LiCl and once with 10 mM Tris buffer (pH 8.0) containing 140 mM NaCl. Bound proteins were eluted with 30 µl of sodium dodecyl sulfate (SDS) sample buffer by being heated for 5 min to 95°C. Ten-microliter samples were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and processed for Western analysis using a chicken HsfA1 antiserum.

Yeast plasmids. Plasmids were constructed and transformed into Escherichia coli DH5 by standard techniques (38). The 2µm vectors pADGal4 (Gal4p activator domain [Gal4p-AD] amino acids [aa] 768 to 881; LEU2) and pBDGal4 (Gal4p DNA-binding domain [Gal4p-DBD] aa 1 to 147; TRP1) were obtained from Stratagene, La Jolla, Calif. Yeast transformation procedures were performed according to the Stratagene two-hybrid manual.

Library construction, screening, and interaction studies. A library of hybrid proteins between the yeast Gal4p-AD and oligo(dT)-primed cDNA fragments, derived from an HS tomato cell culture, was constructed in pADGal4 with a HybriZAP Two-Hybrid cDNA Gigapack cloning kit (Stratagene). The average insert size was 1.5 kb. The S. cerevisiae YRG-2 reporter strain carrying the HIS3 and lacZ genes, both under control of a Gal4p-inducible promoter, was sequentially transformed with the bait plasmid (pRL123) and this plasmid library. Of the estimated 4 × 10⁶ transformants, 174 were histidine prototrophs. They were recovered and tested by retransformation. Of 69 hybrid constructs proved to be positive, 25 were representative of L. peruvianum HsfA2 (LpHsfA2).

Quantification of -galactosidase activity. Yeast cultures were grown overnight in 20 ml of yeast extract-peptone-dextrose medium. The cells were washed with ice-cold 100 mM potassium phosphate (pH 6.5) (KPP) and centrifuged for 5 min at 4°C at 4,500 × g. The pellet was resuspended in 6 volumes of KPP buffer and vortexed for 10 min at 4°C with 0.3 to 0.5 g of glass beads (Sigma). Aliquots (20 µl) of the cell extracts in the wells of a microtiter plate were incubated with 100 µl of Galacton-Star substrate (Galacto-Star kit; Tropix, Bedford, Mass.). Immediately after addition of the substrate, the light emission was registered with a luminometer (Microlumat 2400; EG&G Berthold). The activity is expressed as relative light units (RLU) per second and milligram of protein.

Indirect immunofluorescence. Cells from rapidly growing tomato cultures, partially protoplasted by a 2-h incubation with 1% mazerozyme-1% cellulase (22), or tobacco protoplasts were fixed for 30 min in 3% paraformaldehyde-50 mM piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES) (pH 6.0). After sedimentation, the cells were washed twice in phosphate-buffered saline (PBS) containing 1% Nonidet NP-40 and transferred to poly-L-lysine-coated coverslips. After being washed twice with PBS and once with PBS-1% BSA (Sigma), the coverslips were incubated for 1.5 h at 37°C with the indicated antiserum diluted 1:1,000 in PBS-1% BSA. After being washed with PBS and PBS-1% BSA, the cells were incubated with fluorescein isothiocyanate-labeled polyclonal goat anti-rabbit immunoglobulin G (Sigma) diluted 1:200 in PBS-1% BSA and then subjected to extensive washing with PBS and mounting in PBS-glycerol (1:3) containing 0.1% phenylenediamine. The immunostaining was analyzed with a Zeiss Axiophot microscope, and micrographs were taken with Ilford HP5 film. For a control, all samples were also stained with 4',6-diamidino-2-phenylindole (DAPI) to localize the nuclei (pictures not shown) (see Fig. 1).

Electron microscopy. L. peruvianum cells were initially fixed in 50 mM cacodylate buffer (pH 7.4) containing 1.5% glutaraldehyde (4 h at 4°C) and then fixed with 1% OsO₄ in 50 mM cacodylate buffer, pH 7.4 (2 h at 4°C). After a washing, the cell material was en bloque contrasted overnight with 2% uranyl acetate, washed again, dehydrated in ethanol and propylene oxide, and embedded in Spurr (TAAB Laboratories, Aldermaston, United Kingdom) according to standard procedures. Blocks were sectioned with a Reichert Ultracut S ultramicrotome. Micrographs were taken with a Zeiss 902 electron microscope using Agfa Scientia EM film.

	RESULTS

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Nuclear cotransport of HsfA1 and HsfA2 in tobacco protoplasts. During earlier experiments on the functional characterization of tomato Hsfs in a transient-expression system using tobacco protoplasts, an interesting peculiarity of the HS-inducible HsfA2 was noticed (15). Despite the presence of a functional NLS, HsfA2 is defective in nuclear transport irrespective of HS or control conditions. Instead, it forms large cytoplasmic complexes during HS (Fig. 1A and B). As demonstrated previously (15), this cytoplasmic retention can be overcome by deletion of 8 or 28 aa residues from the C-terminal activator domain (HsfA2C343 or HsfA2C323).

View larger version (57K): [in this window] [in a new window]   FIG. 1.   Immunofluorescence of tobacco protoplasts demonstrating nuclear transport of HsfA2 by coexpression with HsfA1. A total of 105 tobacco protoplasts were transformed with the indicated Hsf expression plasmids and incubated for 10 h under control (CO) (A, C, E, and G) and HS (B, D, F, and H) conditions (see Materials and Methods). Samples were processed for immunofluorescence with an antiserum against HsfA2. HsfA2 was expressed alone (A and B) or with wild-type HsfA1 (C and D), the NLS-defective mutant M3 of HsfA2 was expressed with HsfA1 (E and F), and wild-type HsfA2 was coexpressed with a C-terminally truncated version of HsfA1 (C396) (G and H). The position of the nucleus as deduced from the corresponding staining with DAPI is indicated (arrowheads). A control sample showing the background fluorescence of protoplasts transformed with the empty vector only is also shown (I and K). Bar, 10 µm. endog., endogenous.

View larger version (26K): [in this window] [in a new window]   FIG. 2.   Interaction of HsfA1 and HsfA2 in tobacco protoplasts: intracellular localization (A) and transactivation assay (B). Protoplasts were transformed with 5 µg of the phsp17gus reporter (51) and the indicated Hsf expression plasmids. After 10 h of incubation under control (co) and HS (hs) conditions (see Materials and Methods), cells were harvested and processed for immunofluorescence (A) and the Gus assay (B). For evaluation of the intracellular localization of Hsfs, protoplasts were assigned to three classes (see examples in Fig. 1) with dominant nuclear (Nucleus), nuclear-cytoplasmic (Nucl./Cytopl.), and dominant cytoplasmic (Cytoplasm) localization of the Hsf. Samples 2 and 3 were probed with antiserum against HsfA1, and samples 4 to 6 were probed with antiserum against HsfA2. The total number of protoplasts evaluated is given to the right of the bars in panel A. The effective nuclear import of HsfA2 in the presence of HsfA1 (samples 5 and 6) is also evident from the increased relative Gus expression levels shown in panel B. To optimize the detection of the synergistic effect, we reduced the amount of the HsfA1 expression plasmids in this assay to 0.25 µg per 100,000 protoplasts. Under these conditions, the intracellular level of the carrier Hsf is barely detectable by Western analysis (n.d., not detected). In contrast to this, HsfA2 expression in samples 4 to 6 was monitored by Western blotting. endog., endogenous Hsf of tobacco protoplasts.

Expression and localization of Hsfs in tomato cell cultures. The intriguing observation of the interaction of HsfA1 and HsfA2 during nuclear transport in tobacco protoplasts led us to investigate the natural Hsf system in tomato cell cultures. In contrast to the former, the latter is characterized by a continuously changing composition and intracellular distribution of Hsfs in the course of an HS and recovery period. As described earlier (26, 29), our standard procedure for study of such dynamic changes comprises temperature shift experiments, as indicated in the pictograph of Fig. 3. Samples of tomato cell suspension cultures were harvested at the indicated time points and analyzed for expression levels of Hsfs and Hsps (Fig. 3).

View larger version (47K): [in this window] [in a new window]   FIG. 3.   Expression levels of Hsfs and Hsps in tomato cell cultures. Cell suspension cultures were subjected to the HS treatment indicated in the pictograph at the top. Total protein extracts were analyzed by Western blotting using the indicated antisera. Two samples were supplemented with 10 µg of cycloheximide (CH) at the beginning of the HS regimen (4*) or before the recovery (6*). Mrs are indicated in thousands.

View larger version (74K): [in this window] [in a new window]   FIG. 4.   Intracellular localization of HsfA1 and HsfA2 in tomato cell cultures. Cells were harvested in the course of an HS treatment as indicated by the numbers, which refer to the pictograph in Fig. 3. Samples were processed for immunofluorescence with antiserum against HsfA1 (A to E), antiserum against HsfA2 (F to J), and antiserum against HsfB1 (K to O). The speckled cytoplasmic fluorescence in panels I and J results from the accumulation of HsfA2 in HSG (see Fig. 5). Bar, 10 µm.

HsfA2 becomes part of the HSG. The coarse, speckled fluorescence generated by antibody staining of HsfA2 (Fig. 4I and J) indicates formation of large Hsf-containing protein aggregates in the cytoplasm. As described previously, a general peculiarity of HS plants is the formation of 40-nm-diameter ribonucleoprotein (RNP) aggregates in the cytoplasm (HSG) which represent major sites of Hsp accumulation (25, 28, 29). Using immunogold labeling and cell fractionation techniques, we investigated the possible colocalization of Hsp17 as a marker of the HSG fraction with HsfA2. Figure 5 shows ultrathin sections of tomato cells after a preinduction period followed by a 2-h HS. The typical, highly contrasted 40-nm-diameter particles in the cytoplasm (Fig. 5A and B) can be immunolabeled with antibodies against HsfA2 (Fig. 5A) and Hsp17 (Fig. 5C). In Fig. 5D, we show the result of a double immunolabeling using anti-Hsp17-protein A-15-nm-diameter gold in the first step and anti-HsfA2-protein A-5-nm-diameter gold in the second step. Both proteins are evidently part of the same type of HSG complex. In fact, as a control for specificity, the ultrathin section shown in Fig. 5C was also treated with the double labeling procedure, but with preimmune serum used for the second step. In this case, no protein A-5-nm-diameter gold was bound.

View larger version (172K): [in this window] [in a new window]   FIG. 5.   Colocalization of HsfA2 and Hsp17 in cytoplasmic HSG. HS tomato cell cultures corresponding to sample 5 in Fig. 3 were processed for electron microscopy as described in Materials and Methods. (A, C, and D) Cells embedded in LR Gold. Ultrathin sections were used for immunolabeling with anti-HsfA2 (A) and with anti-Hsp17 (C); both were detected with protein A-15-nm-diameter gold. The ultrathin section in panel D was double labeled, first with anti-Hsp17-protein A-15-nm-diameter gold and then with anti-HsfA2-protein A-5-nm-diameter gold. (B) Cells embedded in Epon and contrasted with uranyl acetate show the 40-nm-diameter particles (arrowheads) characteristic for HSG. Bars, 500 (A) and 100 (B to D) nm.

View larger version (40K): [in this window] [in a new window]   FIG. 6.   Cofractionation of HsfA2 and Hsp17 in tomato cell cultures. Cells were harvested at the indicated time points of an HS regimen. After disruption by sonication, the crude homogenate was fractionated by centrifugation to give fractions with soluble proteins (S100) and structure-bound proteins (P100). The proteins were dissolved in SDS sample buffer and processed for Western analysis using the indicated antisera for detection. Under HS conditions (samples 4 and 5), substantial proportions of the HsfA2 and Hsp17, but not of the other proteins tested, are found in a high-molecular-weight sedimentable form (HSG), which dissociates in the recovery (samples 6 and 6*).

Physical interaction of HsfA1 and HsfA2. We confirmed the direct physical interaction of the two Hsfs by two independent methods and expression systems: (i) coimmunoprecipitation of whole-cell protein extracts from tomato cells and tobacco protoplasts and (ii) two-hybrid interaction tests in yeast.

View larger version (46K): [in this window] [in a new window]   FIG. 7.   Coimmunoprecipitation of HsfA1 and HsfA2 from lysates of tomato cell suspension cultures. Total cell lysates were prepared for coimmunoprecipitation with HsfA2 antiserum (see Materials and Methods). Protein samples were from control cells expressing HsfA1 only (sample 1) and from cultures subjected to the HS treatment indicated at the top (A) (samples 3 to 5). Western blots of the selected samples with antisera detecting HsfA1 and HsfA2 (C) and the results from the corresponding coimmunoprecipitation with anti-HsfA2 using chicken anti-HsfA1 for detection (B) are shown. HsfA1 is precipitated only in samples 3 to 5 containing HsfA2. Ab, position of immunoglobulins.

View larger version (59K): [in this window] [in a new window]   FIG. 8.   Coimmunoprecipitation experiment with anti-HsfA2 using tobacco protoplast lysates. Samples of 4 × 105 protoplasts transformed with 5 µg of the indicated expression plasmids for HsfA2 (lane 1), HsfA1 (lane 4), and HsfA27/8 (lane 6) and for neomycin phosphotransferase as a control (lane 7) per 105 protoplasts were harvested after 20 h of expression at 25°C and processed for immunoprecipitation as described in Materials and Methods. Results for protoplasts coexpressing HsfA1 with HsfA2 (lane 2) and HsfA27/8 (lane 6) are also shown. For lane 3, lysates from samples 1 and 4 were mixed 5 min prior to the addition of the anti-HsfA2. Ab, position of immunoglobulins.

Two-hybrid test. Initially, we used a C-terminally truncated HsfA1 fused to Gal4p-DBD (Fig. 9, bait B) as bait for detection of interacting proteins expressed from an oligo(dT)-primed HS tomato library. Among the positive clones, we identified a considerable number of clones coding for C-terminal parts of HsfA2. The longest clone started at codon 95 (Fig. 9, clone 3); the shortest started at codon 168 (clone 7). Evidently, clone 7 defines the minimum part of the HR-A/B region required for interaction with HsfA1. To verify this conclusion and extend the results, we created an additional clone (clone 8) lacking the entire HR-A/B region. As expected, it was unable to interact with baits B and C. The latter contains the HR-A/B region of HsfA2. It interacts with all constructs of HsfA2 identified by the initial screening (clones 3 to 7). In this context, it is surprising and unexplained that all heterologous interactions (A1-A2) as well as the homologous combination A2-A2 are positive in the two-hybrid test on the basis of His prototrophy (Fig. 9). In contrast to this, yeast strains with the homologous combination A1-A1 are nonviable on His-free medium. In support of this, we did not find any HsfA1-specific clone in the initial screening. Essential controls for the specificity of the protein interactions are the nonviable strains containing Gal4p-DBD alone (bait A) and the combinations of baits B and C with Gal4p-AD alone (clone 1) or with the fusion construct of HsfA2 lacking the entire HR-A/B region (clone 8).

View larger version (42K): [in this window] [in a new window]   FIG. 9.   Yeast two-hybrid test for interaction between HsfA1 and HsfA2. (Top) Basic structure of the two tomato Hsfs. AHA1 and AHA2, activator modules (for details, see reviews by Nover et al. [30] and Nover and Scharf [27]). Sequence details for the HR-A/B regions with the conserved heptad repeat pattern of hydrophobic amino acid residues are given. For the two-hybrid constructs, we used the usual N-terminal (aa 1 to 147) and C-terminal (aa 768 to 881) parts of the yeast Gal4 activator protein (see Materials and Methods). (Bottom) For the two-hybrid test, the baits were composed of the DBD or HR region of Gal4p (aa 1 to 147) alone (bait A) or fused to the indicated parts of the tomato Hsfs, i.e., HsfA1 (aa 23 to 448 [bait B]) and HsfA2 (aa 122 to 209 [bait C]). The activators combined with them were Hsf fragments fused to the C-terminal Gal4p activator domain (Gal4p-AD) (samples 2 to 8). Interaction is indicated by growth (+) or nongrowth () of the cultures on His-free synthetic dextrose minimal (SD) medium and by the activity measured in the lacZ assay (RLU per second and milligram of protein [see Materials and Methods]). -Gal, -galactosidase; prot., protein.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

On the basis of structural peculiarities of their oligomerization domains (HR-A/B region), the plant Hsfs are assigned to classes A and B. In contrast to class B and all non-plant Hsfs so far investigated, the HR-A/B region of the class A Hsfs is extended by an insertion of 21 aa residues creating a structure of the putative coiled-coil region with two overlapping heptad hydrophobic repeats (see sequences given in Fig. 9 and the summary by Nover et al. [30]). The consequences for the structural organization of this domain and the functional significance of the differences between class A and class B Hsfs are unclear. This reflects also our limited knowledge about the detailed structure of this domain, which is important for oligomerization and control of activity of Hsfs (33, 35, 50, 55).

Here, we present experimental evidence that the tomato Hsf system is characterized by an intriguing interaction between two Hsfs (HsfA1 and HsfA2) which is essential for the nuclear import and probably physical stabilization of HsfA2. The latter is synthesized only in HS cells and accumulates to fairly high levels (Fig. 3). Following the preinduction protocol, we can distinguish three major states of HsfA2 in tomato cell cultures. They are evident from the immunofluorescence (Fig. 1 and 4) and cell fractionation (Fig. 6) data. (i) Shortly after the temperature upshift from 25 to 40°C, most of the HsfA1 and HsfA2 is found in the nucleus (Fig. 4C and H). This corresponds to the behavior of HsfA2 if coexpressed with HsfA1 in tobacco protoplasts (Fig. 1D). (ii) After 2 h of HS with ongoing synthesis, a considerable proportion of the HsfA2, together with Hsp17, can be detected in large cytoplasmic aggregates representing the well-known HSG (Fig. 5). The structural binding of HsfA2 is specific; HsfA1 and other cytosolic proteins (tubulin, Hsp90) are not bound to the HSG fraction (Fig. 6). (iii) The HS-specific, high-salt-resistant structural binding of HsfA2 in the nuclear and HSG fractions is reversible. At the end of a 2-h recovery period, most of the HsfA2 is found in soluble form in the cytoplasm (Fig. 4G and sample 6 in Fig. 6).

Formation of HSG is a general phenomenon in all plant tissues with sufficiently high levels of Hsp synthesis (25). They can readily be detected as large aggregates of electron-dense material composed mainly of 40-nm-diameter particles (Fig. 5A and B). There is experimental evidence that they may function as sites for transient storage and protection of housekeeping mRNP (29). Incorporation of HsfA2 into HSG and its rapid release in the recovery period are new aspects which may indicate an additional function as storage sites for excess Hsf and probably other HS-induced proteins. Our knowledge about the fine structure of the HSG complexes is still very limited. We previously reported on the electron microscopic and biochemical characterization of 10-nm-diameter hollow-core particles (precursor HSG) with a sedimentation coefficient of about 15S (29). We are currently investigating whether HsfA2 is already incorporated into the pre-HSG particles or, more likely, associates with the HSG complexes only during the HS-induced aggregation of the pre-HSG. Transient-expression assays with individual components (Hsp17, HsfA2) in tobacco protoplasts, which are capable of forming HSG from the endogenous precursor particles, are valuable tools in this context.

The HsfA1-assisted nuclear import of HsfA2 (Fig. 1 and 4) depends on a direct physical interaction between the two proteins. This is documented by the coimmunoprecipitation experiments (Fig. 7 and 8) and by the yeast two-hybrid tests (Fig. 9). As expected, the interaction is mediated by the A-type-specific HR-A/B region only (Fig. 8 and 9). HsfB1 does not function as a mediator of nuclear import of HsfA2 or as bait in the two-hybrid test (data not shown). A series of partial clones resulting from the two-hybrid screening allowed definition of the part of the oligomerization domain which is indispensable for the interaction (sequence data in Fig. 9). The strongest interaction is actually found for a HsfA2 fragment lacking the whole N-terminal half of the HR-A/B region. Thus, structural prerequisites for the HsfA1-mediated nuclear import of HsfA2 are the C-terminal half of the HR-A/B region and a functional NLS (Fig. 1E and F). On the other hand, HsfA1 as a carrier does not need its activator domain. Truncated versions, inactive as transcription factors (e.g., HsfA1C396), are equally effective and represent valuable tools with which to demonstrate the increased activator potential of HsfA2 if the cytoplasmic retention is released in coexpression experiments (Fig. 2). It is worth mentioning that the two-hybrid test indicates an unexpected peculiarity. The heterologous combinations of HsfA1 with HsfA2 are more stable (efficient) than the homologous combinations. In fact, the A1-A1 combination cannot be detected at all. However, this matter requires further investigation, since (i) only Hsf fragments with unknown conformational abnormalities were tested in the two-hybrid system and (ii) a positive two-hybrid effect probably requires dimer formation, whereas the natural interaction of Hsfs by their oligomerization domains leads preferentially to trimers.

The pronounced negative effect of the C-terminal HR-C region on the activator function of HsfA2 can be released by deletion of the last 8 aa (15). HsfA2C343 is transported to the nucleus without need for interaction with HsfA1. Though not directly comparable with the particular situation of HsfA2, the role of the C-terminal HR-C region for maintenance of the inactive state and the cytoplasmic localization was also demonstrated for the Drosophila melanogaster, chicken, and human Hsfs (1, 19, 20, 35, 47). The model of an intramolecular interaction between the HR-A/B and HR-C regions in the monomeric Hsf proposed by Rabindran et al. (35) is very suggestive but, unfortunately, not supported by direct experimental evidence, e.g., a two-hybrid test. Probably, the generation and/or maintenance of the inactive state requires not only the HR-C region but additional factors, e.g., corepressors of the chaperone type interacting with it (18, 31, 36, 39, 55, 56).

The time course of formation and the stability of the putative hetero-oligomers of HsfA1 and HsfA2 as well as their composition are unknown. They are evidently not formed readily upon mixing protoplast lysates containing the two proteins separately (Fig. 8, lane 3). However, the procedure of coimmunoprecipitation can be used to search for factors or conditions allowing postsynthetic formation of the HsfA1-HsfA2 hetero-oligomers. Unfortunately, we were never able to demonstrate the existence of monomeric Hsf forms in tomato cells or in tobacco protoplasts during transient expression, nor is there any real evidence for HS-dependent changes of the oligomeric state of plant Hsfs (12, 15, 41a). The HS-induced transition between monomeric, inactive Hsf and the active trimeric form as an inherent part of the activation-deactivation cycle in Drosophila and vertebrates (10, 35, 53, 55, 56) appears to be lacking in plants. It is tempting to speculate that the HsfA2 form in the cytoplasm with a shielded NLS motif represents a homo-oligomeric form, whereas the transport-competent form may represent hetero-oligomers with one or two subunits of HsfA1. Oligomerization itself is evidently not sufficient to open the shielded conformation of HsfA2.

Our findings about a physical interaction of HsfA1 and HsfA2 as prerequisite for efficient nuclear import of the latter help to understand the dynamic changes of HsfA2 distribution in tomato cells. The accumulation and stability of this transcription factor during a prolonged stress period (Fig. 4) are connected with its aggregation in the cytoplasmic HSG together with other HS-induced proteins. Monitoring the Hsf levels by Western blotting, though valuable, may have limited value for the evaluation of data about Hsf-mediated reporter gene expression. In particular, the more-than-additive increase of Gus levels in protoplast samples coexpressing HsfA2 with HsfA1 (Fig. 2) may reflect the increased availability of HsfA2 homo-oligomers in the nucleus. Alternatively, the HsfA1-HsfA2 hetero-oligomers represent not only the transport-competent form but also the more stable and transcriptionally active form. In vivo footprinting data with a defined set of different HS reporter constructs can help to clarify this problem.

One point of concern in the evaluation of our results obtained with tobacco protoplasts is the ill-defined role and expression of the endogenous Hsfs. Because of the evident conservation of Hsf types between distantly related plant species such as maize, Arabidopsis thaliana, soybean, and tomato (30), we can assume that N. plumbaginifolia also contains Hsfs of the A1, A2, and B1 types. Fortunately for the application of our antisera used for immunofluorescence (Fig. 1), Western blotting (Fig. 8 and reference 15), and coimmunoprecipitation (Fig. 8), there is essentially no detectable cross-reactivity of Hsfs or cross-reacting Hsf material in the tobacco protoplasts. This situation probably reflects the low level of endogenous Hsfs and the well-known high degree of sequence variability in the C-terminal parts of plant Hsfs. However, even a low level of the putative tobacco HsfA1 might be sufficient to cause some nuclear import and thus transcriptional activity of the tomato HsfA2 if expressed in the absence of the homologous HsfA1 (Fig. 2, sample 4). Formally, we cannot exclude this possibility at present. It may affect the sensitivity of the detection system by reducing the synergistic effects by coexpression (Fig. 2), but it would not influence the general conclusions.