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First published online July 9, 2004; 10.1104/pp.104.042820 Plant Physiology 135:1457-1470 (2004) © 2004 American Society of Plant Biologists Role of Hsp17.4-CII as Coregulator and Cytoplasmic Retention Factor of Tomato Heat Stress Transcription Factor HsfA21Biocenter of the Goethe University, D–60439 Frankfurt am Main, Germany
HsfA2 is a heat stress (hs)-induced Hsf in peruvian tomato (Lycopersicon peruvianum) and the cultivated form Lycopersicon esculentum. Due to the high activator potential and the continued accumulation during repeated cycles of heat stress and recovery, HsfA2 becomes a dominant Hsf in thermotolerant cells. The formation of heterooligomeric complexes with HsfA1 leads to nuclear retention and enhanced transcriptional activity of HsfA2. This effect seems to represent one part of potential molecular mechanisms involved in its activity control. As shown in this paper, the activity of HsfA2 is also controlled by a network of nucleocytoplasmic small Hsps influencing its solubility, intracellular localization and activator function. By yeast two-hybrid interaction and transient coexpression studies in tobacco (Nicotiana plumbaginifolia) mesophyll protoplasts, we found that tomato (Lycopersicon esculentum) Hsp17.4-CII acts as corepressor of HsfA2. Given appropriate conditions, both proteins together formed large cytosolic aggregates which could be solubilized in presence of class CI sHsps. However, independent of the formation of aggregates or of the nucleocytoplasmic distribution of HsfA2, its transcriptional activity was specifically repressed by interaction of Hsp17.4-CII with the C-terminal activator domain. Although not identical in all aspects, the situation with the highly expressed, heat stress-inducible Arabidopsis HsfA2 was found to be principally similar. In corresponding reporter assays its activity was repressed in presence of AtHsp17.7-CII but not of AtHsp17.6-CII or LpHsp17.4-CII.
Stress-induced gene expression leads to the rapid accumulation of heat stress proteins (Hsps). Many of them act as molecular chaperones with important functions not only for the protection of proteins against stress damage but also for their folding, intracellular distribution and degradation (Ellis, 2000  ; Dougan et al., 2002; Hartl and Hayer-Hartl, 2002; Haslbeck, 2002; Picard, 2002; Walter and Buchner, 2002; Young et al., 2003). Eukaryotic heat stress (hs)-inducible genes share conserved promoter elements with the palindromic consensus motif (AGAAnnTTCT; Pelham, 1982; Pelham and Bienz, 1982; Nover, 1987). They represent the recognition sites of the heat stress transcription factors (Hsf), which are the key regulators of the response conserved throughout the eukaryotic kingdom (Wu, 1995; Morimoto, 1998; Nover et al., 2001).
In plants the Hsf system is more complex than in any other organism investigated so far (for review, see Nover et al., 2001
The network of protein interactions influencing the function and intracellular distribution of HsfA2 has a second aspect. In the course of a heat stress response, the ongoing accumulation of HsfA2 and other hs-inducible proteins results in a unique storage form of the transcription factor in cytoplasmic chaperone complexes composed of the 40-nm heat stress granules (HSG; Nover et al., 1989
From the point of view of its abundance as well as its high activator potential, HsfA2 becomes the dominant Hsf in tomato after hs treatment (Mishra et al., 2002
Identification of Hsp17.4-CII as Specific Interaction Partner of Tomato HsfA2 in Yeast Two-Hybrid System
Yeast two-hybrid screening was used to identify tomato proteins interacting with heat stress transcription factor HsfA2 as bait. To this aim, the two previously identified activator motifs AHA1 and AHA2 in the C-terminal domain of HsfA2 had to be mutated (Döring et al., 2000
By directly testing different cytosolic sHsps as preys in combination with the C-terminal part of HsfA2 as bait, it was confirmed that the selectivity of interaction is very high (Fig. 2). Only Hsp17.4-CII interacts with HsfA2 (no. 2), but not Hsp17.3-CII (no. 3) nor any other sHsp of classes CI and CII from tomato or pea (Pisum sativum; nos. 10–12). To analyze the striking difference between the two types of Hsp17-CII, we created a number of mutant proteins of Hsp17.4-CII with single amino acid exchanges as indicated in Figure 1. The two-hybrid test clearly showed that only the mutant protein with exchange of V48>A in immediate vicinity of the  -crystalline domain lost the capability for interaction with HsfA2, whereas other amino acid exchanges discriminating the two different forms, i.e. K35>I, D56>N, V96>A, and Q151>E were unaffected in this respect (nos. 4 and 6–8). The V48>A exchange in Hsp17.4-CII abolished interaction, i.e. this loss-of-function mutation corresponds to the Hsp17.3-CII type of sHsps. We also tested the reverse exchange of A48>V in the Hsp17.3-CII background (no. 9). Although negative in the yeast two-hybrid test, this mutant form was shown to interact with HsfA2 in other test systems (see below).
We used tobacco (Nicotiana plumbaginifolia) protoplasts to confirm and complement the results of the yeast two-hybrid system. In two important aspects the situation in these plant cells is more complex and fundamentally different from the yeast two-hybrid system: (1) expression of the full length proteins results in formation of their native oligomeric structures, i.e. any interaction observed under these conditions reflects the specific recognition between the HsfA2 as trimeric or multimeric complex and Hsp17-CII which is usually a dodecamer; and (2) The function of HsfA2 as activator of hs-inducible genes may contribute to the complexity of protein interactions because of the synthesis of the endogenous chaperones. Whenever necessary, this problem was minimized by using HsfA2 mutants exhibiting identical properties with respect to the intracellular localization and interaction with HsfA1 and Hsp17.4-CII, but with strongly reduced or lacking activator function. On the other hand, we tried to mimic the complex natural situation of HsfA2 as component of the chaperone and Hsf networks by coexpression of defined mixtures of the proteins in tobacco protoplasts. One intriguing aspect of the interaction of HsfA2 with Hsp17.4-CII is the formation of insoluble aggregates. To investigate this in more detail, we used differential centrifugation to characterize the high Mr, salt- and detergent-resistant aggregates of HsfA2. To avoid the complex situation of protein recruitment into HSG or HSG-like complexes, samples were incubated at room temperature. Whole cell extracts of tobacco protoplasts expressing the indicated proteins were prepared in buffer containing high-salt (500 mM NaCl) and detergent (0.5% Nonidet and 0.2% Sarcosyl). After centrifugation for 1 h at 100,000g (see "Materials and Methods"), the distribution of HsfA2 between the soluble form in the supernatant (S100) and the sedimentable form in the pellet (P100) fractions was determined by immunoblot analysis (Fig. 3). If expressed alone, only 5% of the total HsfA2 was detected in the pellet fraction (Fig. 3A, lane 1). However, a considerable portion (48%) of HsfA2 was sedimentable in the presence of Hsp17.4-CII but not of Hsp17.3-CII (lanes 2 and 3). The numbers at the bottom of the immunoblots of the P100-fraction give an estimate of the relative amount of HsfA2 in the pellet fractions (S100 + P100 = 100%). Similar results were obtained with the two proteins from the culture tomato, i.e. LeHsp17.4-CII as the interacting type and LeHsp17.6-CII as the noninteracting type (lanes 4 and 5; for sequences see Fig. 1). Interestingly, the oligomerization state of the two class CII sHsps was not crucial for the interaction with HsfA2. The N-terminally Myc-tagged forms of both proteins did not form the usual dodecamers of about 220 kD (data not shown), but the specific influence on the sedimentation behavior of HsfA2 was not altered (Fig. 3, lanes 6 and 7). None of the other sHsps tested, i.e. LpHsp17.7-CI (lane 8) nor Hsp17-CII proteins from Arabidopsis or pea (lanes 9 and 10), influenced the aggregation of HsfA2. For control, expression levels of the proteins were analyzed by immunoblots (Fig. 3B).
We used the sedimentation assay also to test mutant forms of LpHsp17.4-CII and of the closely related LpHsp17.3-CII with exchanges of the amino acid residues discriminating the two forms (see sequences in Fig. 1). Similar to the results with the yeast two-hybrid test, only the Hsp17.4-CII mutant protein with V48>A (lane 14) but not the mutant forms with K35>I, D55>N, or Q151>E (Fig. 3, lanes 11–13) were disturbed in the interaction with HsfA2. Interestingly, in contrast to the results with the yeast two-hybrid system (Fig. 2, no. 9) the mutant of Hsp17.3-CII with A48>V showed a clearly detectable interaction with HsfA2 (Fig. 3, lane 15). These results confirmed the crucial role of the amino acid residue V48 for discriminating the interacting type (Hsp17.4-CII) from the noninteracting type (Hsp17.3-CII with A48).
The sedimentation assay offered also the opportunity for a simple check of different mutant forms of HsfA2 with respect to their interaction capacity (Fig. 3C). Although different to a certain extent, all forms of HsfA2 tested so far were affected in their sedimentation behavior in the presence of LpHsp17.4-CII (Fig. 3C, nos. 1, 2, and 4). The only exception was the C-terminally truncated form HsfA2
Particularly interesting are the results with two fusion proteins harboring the indicated parts of HsfA1 and HsfA2 respectively (Mishra et al., 2002
Based on the results from the two-hybrid screening, HsfA2 interacts with HsfA1 (Scharf et al., 1998a To elaborate more details of this network of protein interactions and mutual influences of the partners, we used tobacco protoplasts for coexpression of different combinations of Hsfs and members of the tomato sHsp family and tested the sedimentation behavior under control and hs conditions (Fig. 4). The results enlarge our perspectives of the influence of protein interactions on the stability and/or oligomerization state of the proteins involved. The major difference between the control (Fig. 4A) and hs samples (Fig. 4B) was the general tendency for structural binding of HsfA2 and Hsp17 classes CI and CII in the hs samples, irrespective of the mixture of proteins expressed. Selectivity was only observed in samples incubated under control temperature conditions, which are reflecting more likely the situation of tomato cells during recovery from a heat stress. We therefore concentrate our further discussion mainly on the results obtained with the control temperature samples (Fig. 4A).
Similar to previous experiments, the sedimentation of a considerable part of HsfA2 in high salt-resistant form was only observed in the presence of Hsp17.4-CII (lane 1 and 5) but not of Hsp17.3-CII (lane 2 and 6). Interestingly, nuclear retention of HsfA2 in the presence of HsfA1 (Scharf et al., 1998a ; Heerklotz et al., 2001) does not change the amount of HsfA2 detected in the sedimentable form (Fig. 4A, lane 5). However, in samples including Hsp17.4-CII and Hsp17.7-CI (lanes 3 and 7), the formation of sedimentable HsfA2 was drastically reduced or abolished. This solubilizing effect of Hsp17.7-CI was also observed with the Myc-tagged as well as untagged form of Hsp17.7-CI or with other representatives of tomato class CI small Hsps, e.g. LeHsp17.8-CI and LeHsp17.6-CI (Fig. 5, A and B) as well as with corresponding proteins from Arabidopsis and pea (AtHsp18.1-CI, AtHsp17.6-CI, PsHsp18.1-CI, data not shown). Evidently, in contrast to the exclusivity of the interaction between HsfA2 and Hsp17.4-CII, the solubilizing effect of class CI sHsps is more general and, probably, essential for maintaining sHsps in a soluble state at normal growth temperatures.
Two points are important to notice. First, in contrast to the situation under heat stress conditions where a considerable portion of both Hsp17-CI and Hsp17-CII together with HsfA2 are found in the high salt and detergent-resistant sedimentable fraction (Fig. 4B), this is not the case for samples maintained under control temperature conditions. Due to the specific buffer conditions used in the cosedimentation assays, the HsfA2 aggregates (Fig. 4A, lanes 1–5) contain only very small amounts of Hsp17.4-CII. This indicates that major structural differences might exist between HsfA2/Hsp17.4-CII complexes formed under control and hs conditions, respectively. Second, for these experiments we used the 3-HA-tagged form of HsfA2 and a C-terminally truncated form of HsfA1. Both have very low or no transcriptional activity. This special test situation avoids the synthesis of endogenous Hsp17-CI that evidently would influence the outcome of the experiment.
The results on the decisive role of class CI chaperones for the solubilization of aggregated HsfA2 and Hsp17 class CII complexes were confirmed by size exclusion chromatography of whole protein extracts from tobacco protoplasts expressing the indicated proteins or mixtures of proteins (Fig. 5). Expression of HsfA2 alone (sample 1) gave a broad elution peak with a maximum in fraction number 5 which corresponds to an apparent molecular size of about 350 kD for soluble oligomeric HsfA2 complexes. Coexpression with Hsp17.4-CII (sample 2) but not with Hsp17.3-CII (sample 3) shifted a considerable part of HsfA2 to fraction number 2, i.e. close to the exclusion volume. This fraction evidently represents the sedimentable aggregates of HsfA2 (see Fig. 5A) and contains also a significant portion of Hsp17.4-CII. As estimated on the basis of densitometer scans of the corresponding signals of protein imunoblots shown in Fig. 5A, the portions of HsfA2 and Hsp17.4-CII shifted to the high-molecular size fraction (Fig. 5A, sample 2, fraction no.2) correspond to approximately 27% and 22%, respectively, based on the total amounts of these proteins eluted from the size exclusion column. In contrast to the high salt and detergent conditions used for the sedimentation assay (Figs. 1–4 When we tested three different isoforms of Hsp17-CI from the cultivated tomato L. esculentum for their capacity to solubilize aggregated HsfA2 complexes (Fig. 5B), we were surprised to find that only two of them, LeHsp17.6-CI and LeHsp17.8-CI, were comparable to LpHsp17.7-CI. In contrast to this, LeHsp18.1-CI was much less efficient in solubilizing the HsfA2 aggregates formed in presence of Hsp17.4-CII (Fig. 5B, lane 5). The peculiarities of the three isoforms of tomato Hsp17-CI in this respect seem to be correlated with their capacity for interaction with Hsp17.4-CII, as detected by native gel electrophoresis (Fig. 5C). Hsp17.6-CI expressed alone formed small oligomers in the range of 100 kD, Hsp17.8-CI formed dodecamers of about 220 kD, whereas Hsp18.1-CI gave a complex of >700 kD. Upon coexpression with Hsp17.4-CII part of the former two class CI proteins were found in new complexes of about 235 kD containing all of the Hsp17.4-CII (bands marked with asterisks in lanes 6 and 7). However, coexpression of Hsp18.1-CI and Hsp17.4-CII gave no comparable shift of the Hsp17.4-CII signal.
It is worth repeating once more that all effects observed in tobacco protoplasts mimic at least parts of the natural situation in tomato cells in the recovery period. As reported earlier (Kirschner et al., 2000 An interesting common aspect of the protein interactions documented in Figure 4 is the stabilization of HsfA2, both in the presence of HsfA1 and Hsp17.4-CII. The relative numbers given on top for the signal density of HsfA2 in whole protoplast extracts (WPE) indicate a 2- to 3-fold increased level of HsfA2 in all combinations with HsfA1 and/or Hsp17.4-CII (lanes 1, 5–8, and 10) but not in combinations with Hsp17.3-CII or Hsp17.7-CI (lanes 2, 4, and 9). The same was true for the samples used for the size exclusion chromatography (compare Fig. 5A, lanes for the input controls for samples 3 versus 2 and 4). Stabilization of HsfA2 in the insoluble cytoplasmic aggregates seems plausible, but this cannot explain the stabilization by interaction with HsfA1.
The different types of protein interactions characterized in Figures 3 to 5 may also have marked influences on the intracellular localization of HsfA2. To investigate this in detail, we used tobacco protoplasts expressing the indicated proteins under control temperature conditions (Fig. 6). In cells with Hsp17.4-CII alone (Fig. 6A, sample 1), the small Hsp was found mainly distributed throughout the cytoplasm, but upon coexpression with HsfA2 (sample 2) large cytoplasmic aggregates of both proteins were observed. In the triple combination of Hsp17.4-CII and HsfA2 with HsfA1 (sample 3), the situation was very similar. However, as a result of the interaction with HsfA1, part of the HsfA2 was now found in the nucleus. The observed solubilizing effect of Hsp17.7-CI was also visible at the cellular level (samples 4 and 5 of Fig. 6A). In both cases, the cytoplasmic aggregates of HsfA2 completely disappeared, and the nuclear portion of HsfA2 in the presence of HsfA1 clearly increased (compare samples 3 and 5). Interestingly, a similar increase in the nuclear localization was also visible for Hsp17.4-CII in the corresponding immunofluorescence images. Neither HsfA2 nor sHsp aggregates were observed in the presence of Hsp17.3-CII (Fig. 6B), and changes in the nucleocytoplasmic localization of HsfA2 in these samples were only influenced by coexpression of HsfA1.
One obvious difference between the results in Figure 6A and those shown before in Figure 4A needs further explanations. Although the predominant portion of Hsp17.4-CII was clearly associated with the HsfA2 aggregates in situ (Fig. 6A, samples 2 and 3), this association is evidently not high salt/detergent-resistant and hence, it was not preserved under the high stringency conditions used for the sedimentation procedure (Fig. 4A, samples 1 and 5).
The functional significance of the different states of HsfA2 is particularly striking from the results shown in Figure 6C. Our earlier observations indicated that HsfA2 has nucleocytoplasmic localization but because of its strong NES the steady-state localization is more cytoplasmic (Heerklotz et al., 2001
Is the specific interaction between HsfA2 and Hsp17.4-CII limited to the cytoplasmic part of the transcription factor and, if not, does binding of the small Hsp also influence the activator potential of HsfA2? To answer these questions, we used reporter assays in tobacco protoplasts and coexpressed HsfA2 in presence of the C-terminally deleted (inactive) form of HsfA1 to provide the basis for nuclear retention of the former (see Fig. 6C, sample 2). The high activator potential of HsfA2 (Fig. 7A, sample 1) was markedly reduced in the presence of increasing amounts of Hsp17.4-CII (samples 3–5). Interestingly, the repressor effect of Hsp17.4-CII on HsfA2 were not altered in the presence of Hsp17.7-CI (sample 6), i.e. they reflect the interaction of HsfA2 and Hsp17.4-CII in the nucleus, and they are largely independent of the aggregation state of cytoplasmic HsfA2 and Hsp17.4-CII (see immunofluorescence data in Fig. 6). As expected, the noninteracting type (Hsp17.3-CII) had only weak effects on the reporter activity (samples 7–10). Samples 11 to 16 (Fig. 7A) complement the picture by showing the results obtained with the two mutant sHsps (Hsp17.4-CII V48A and Hsp17.3-CII A48V) tested earlier for their influence on the interaction and aggregation of HsfA2 (Figs. 2 and 3). In support of the earlier results on the decisive role of the V48 residue for the interaction with HsfA2, the former mutant sHsp lost its capability to interfere with HsfA2 function (samples 11–13), whereas the latter gained this capability (samples 14–16). In all cases, the signals from immunoblot analyses at the bottom serve as expression controls for the interacting proteins.
To confirm the role of the C-terminal part with the transcriptional activator modules of HsfA2 for the specific interaction with Hsp17.4-CII, we used a Gal4p-dependent  -glucuronidase (GUS) reporter construct (Döring et al., 2000) and expression constructs encoding chimeric activator proteins consisting of the yeast Gal4p DNA binding domain and the C-terminal activator domains of tomato HsfA1 and HsfA2 (for details, see block diagrams at the top of Fig. 7B). The activity of the two chimeric activators with the activator domain of HsfA1 or the C-terminally truncated form of HsfA2 were not affected by the presence of Hsp17.4-CII (samples 1–6), whereas the activator with the complete C-terminal domain of HsfA2 (amino acids 263–351) was strongly repressed in its activity by increasing amounts of Hsp17.4-CII (samples 7–9) but not by Hsp17.3-CII (sample 10). The results extend our earlier conclusions. Interaction between HsfA2 and Hsp17.4-CII is not restricted to the aggregated state but is also observed with the soluble nuclear HsfA2. Hsp17.4-CII acts as corepressor of HsfA2 irrespective of the presence or absence of Hsp17.7-CI, and for this effect only the C-terminal activator domain of HsfA2 is required and sufficient. To prove whether the specific repressor function of Hsp17.4-CII on the activity of HsfA2 is not only a peculiarity of the tomato system but rather presents a more general regulatory principle, we tested the activities of the orthologous proteins from Arabidopsis in the Hsf-dependent reporter assay. The results in Figure 7C confirm that the activity of AtHsfA2 was markedly repressed by coexpression with increasing amounts of AtHsp17.7-CII (Fig. 7C, lanes 1–3), but not in presence of AtHsp17.6-CII (lanes 4, 5). As observed for the tomato proteins, the functional interaction of AtHsfA2 with one of the two closely related members of class CII Hsps in Arabidopsis seems to be species-specific. Coexpression of AtHsfA2 with tomato Hsp17.4-CII had no effect on the level of GUS activity (lanes 6 and 7).
Although our knowledge is still rather fragmentary, there is experimental evidence that the maintenance of the inactive state of Hsfs requires additional proteins functioning as corepressors. On the one hand, a small Hsf-binding protein (HSBP1) in mammals and nematodes was reported to mask the HR-A/B region by coiled-coil interactions, and thus to stabilize the inactive, monomeric form of Hsfs (Satyal et al., 1998 ; Tai et al., 2002). Recently, a HSBP ortholog was identified in maize. The lack of HSBP in the mutant empty pericarp 2 (emp2) leads to an uncontrolled expression of hs genes and embryo lethality (Fu et al., 2002). On the other hand, Hsp70 and Hsp90 are evidently involved in the control of Hsf activity in yeast and vertebrates (Baler et al., 1996; Nair et al., 1996; Ali et al., 1998; Shi et al., 1998; Bharadwaj et al., 1999; Bonner et al., 2000; Guo et al., 2001), and the function of Hsp70 as coregulator of Hsf activity may be similar in plants as well (Lee and Schöffl, 1996; Kim and Schöffl, 2002). To our knowledge, a role of a sHsp for control of Hsf activity was never shown before.
The tomato Hsf system is characterized by an intriguing cooperation between two Hsfs, i.e. HsfA1 and HsfA2. As master regulator of the heat stress response, HsfA1 is responsible for the hs-induced synthesis of HsfA2 (Mishra et al., 2002
Results in this manuscript demonstrate that the three states of HsfA2 not only depend on its interaction with HsfA1 mediated by the similarity of their oligomerization domains, but also with Hsp17.4-CII binding to the C-terminal domain of HsfA2. The latter interaction has two effects. On the one hand, Hsp17.4-CII acts as corepressor of HsfA2. On the other hand, it promotes formation of high Mr aggregates of HsfA2 and Hsp17.4-CII even under control temperature conditions (Figs. 3, 4, and 6). This aggregation represents an experimental artifact valuable to investigate details of the protein network responsible for the intracellular localization and function of HsfA2. In the reality of tomato cells in the recovery period following a short hs treatment, no aggregates of HsfA2 are observed because two proteins counteract the structural binding of HsfA2 and Hsp17.4-CII. First, interaction with HsfA1 not only prevents aggregation of HsfA2, but it also promotes nuclear accumulation and keeps HsfA2 in a transcriptionally competent state (Scharf et al., 1998a
This solubilizing effect of Hsp17-CI under control temperature conditions is a class-specific but organism-independent character. It reflects the interaction between Hsp17.4-CII and Hsp17-CI in their oligomeric states as found upon coexpression in plant cells (Siddique et al., 2003 In contrast to the broad specificity of the class CI proteins, the interaction between HsfA2 and Hsp17.4-CII is highly selective. It was not observed with any other class CII protein tested so far. This is even true for the closely related tomato Hsp17.3-CII. A single amino acid exchange (V48>A) makes the crucial difference between the two proteins. Remarkably, this interaction can be detected in all test systems, e.g. in yeast two-hybrid tests (Fig. 2), in glutathione S-transferase (GST)-pull-down assays (data not shown) as well as in situ in protoplasts (Fig. 6) and in reporter assays (Fig. 7). Evidently, it is independent of the oligomeric state of the two proteins.
Analysis of the Arabidopsis orthologs, i.e. of HsfA2 and the two class CII sHsps, indicates that a comparable functional relationship might exist in this plant as well. Similar to tomato, the Arabidopsis HsfA2 is also strictly hs-inducible and, together with the small Hsps and other chaperones, it accumulates to high levels in cell cultures and leaves (Kotak et al., 2004
Our results show for the first time important elements of a protein network controlling the intracellular distribution, stability, and activator function of tomato HsfA2. Previously, we showed that HsfA1 as master regulator of the hs response in tomato is responsible for hs-induced new synthesis of HsfA2 (Mishra et al., 2002 Although we have probably identified the four major components of the HsfA2 network, we cannot exclude that in the reality of an hs-induced cell, additional proteins might be important for the control of the activity state and stability of HsfA2 as well. Our analysis was mainly based on the coexpression of the partner proteins in tobacco protoplasts. Although probably very similar, this cannot completely simulate the situation in native tomato cells. Further experiments are required, e.g. an RNAi approach in tomato protoplasts that would allow selectively knocking-down expression of these and other components with putative functional interactions to the HsfA2 network.
General Materials and Methods
The use of tobacco (Nicotiana plumbaginifolia) mesophyll protoplasts for polyethylene glycol-mediated transformation and transient gene expression was published (Treuter et al., 1993
LMB was kindly provided by Minoru Yoshida, Tokyo, and used as specific repressor of the nuclear export receptor exportin 1 as described before (Kudo et al., 1999
For indirect immunofluorescence of protoplasts we followed the procedures described by Scharf et al. (1998a) For microscopic analysis a Zeiss Axiophot microscope (Zeiss, Oberkochen, Germany) combined with a Color View F12 System (Olympus, Hamburg, Germany) was used. Captured images were resized and combined using Photoshop 5.5 Software (Adobe Systems, La Jolla, CA). Confocal laser scan micrographs were captured using a Leica CLSM (Leica, Bensheim, Germany) and Imaris Software (Bitplane, Zurich).
Standard procedures were used for cloning (Ausubel et al., 1993
To minimize possible interferences of the endogenous set of sHsps in tobacco protoplasts, two inactive HsfA2 variants were used in those experiments where the intracellular distribution or the aggregation behavior of HsfA2 and coexpressed sHsps was analyzed. The corresponding expression plasmids are pRTHsfA2M2 encoding a mutant form of HsfA2 impaired in DNA-binding (Lyck et al., 1997
For Hsf-dependent GUS reporter assays, we used the phsp17gus vector containing the promoter region of the soybean hsp17.3-B gene upstream of the TATA box (Treuter et al., 1993
The yeast two-hybrid screening of a tomato cDNA library derived from heat stressed Lycopersicon peruvianum cell cultures and protein interaction tests were done with the pBDGal4 bait and pADGal4 prey vector system (Stratagene, Amsterdam) as described before (Scharf et al., 1998a
The prey vectors pADGal4xLpHsp17.4-CII and pADGal4xLpHsp17.3-CII encoding fusion proteins of the Gal4 activator domain with the two formes of cytosolic Hsp17 class CII proteins known in tomato. The exchange of individual amino acid residues that are distinct in both sequences (see also Fig. 1) was done by PCR using the Taq Plus Precision system (Stratagene) and forward and reverse primer complementary to plasmid sequences flanking the Hsp17 cDNA inserts in combinations with the following mutagenesis primers:
Amplified DNA fragments were cut at the appropriate restriction sites and ligated into EcoRI-XbaI linearized pADGal4. The mutant Hsp17.4-CII Q151E was generated by subcloning a PagI-XbaI cDNA fragment derived from pADGal4xLpHsp17.3-CII into PagI-XbaI linearized pADGal4xLpHsp17.4-CII. The corresponding plant expression constructs were generated by ligation of the mutagenized cDNA fragments into pRT104 linearized with EcoRI and XbaI.
Prey constructs and plant expression vectors encoding class CI sHsps of tomato (LpHsp17.7-CI) or pea (PsHsp18.1-CI) and class CII sHsps of pea (Pisum sativum; PsHsp17.1-CII) or Arabidopsis (AtHsp17.6-CII) were described before (Kirschner et al., 2000
Genomic DNA was used as template for PCR amplification of DNA fragments encoding class CI and class CII sHsps from the tomato cv L. esculentum var Moneymaker (Mishra et al., 2002
For protein extraction harvested protoplasts were lysed by three cycles of freeze-thawing in NEB500 buffer containing 25 mM HEPES, pH 7.5, 500 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 10 mM NaF, 0.2% (w/v) NP40, and 10% (w/v) glycerol. For all protein extraction buffers used in this study, Complete protease inhibitor cocktail tablets were added as recommended by the manufacturer (Roche Diagnostics, Mannheim, Germany). Cellular debris was removed by centrifugation for 5 min at 10,000 rpm at 4°C. Aliquots corresponding to approximately 15,000 protoplasts were heated with 1 vol. of 2x SDS sample buffer and separated on 14% SDS-polyacrylamide gels (SDS-PAGE).
Protein extracts for separation on native 3% to 20% polyacrylamide pore exclusion gels were prepared in nondenaturing buffer as described before (Kirschner et al., 2000 For immunoblot analysis, proteins were transferred to 45-µm nitrocellulose membranes (PROTRAN BA85, Schleicher and Schuell, Dassel, Germany) and processed for chemiluminescence detection following the manufacturer's protocol (Perkin Elmer Life Sciences, Rodgau-Jügesheim, Germany).
For separation of soluble and insoluble protein fractions, protoplasts were extracted with high salt and detergent buffer (50 mM Tris-HCl, pH 7.8, 500 mM NaCl, 25 mM KCl, 5 mM MgCl2, 30 mM EDTA, 0.5% [w/v] NP40, 0.2% [w/v] sarcosyl, 5% [w/v] saccharose, 5% [w/v] glycerol, 14.2 mM Corresponding chemiluminescence signals from the supernatant (S100) and pellet (P100) fractions were quantified by using the Image 1D software (Amersham Biosciences, Freiburg, Germany). Relative amounts of a given protein in the insoluble P100 fraction were calculated on the basis that the signal densities of both fractions (S100 + P100) correspond to 100%.
Protein extracts in NEB500 corresponding to approximately 500,000 protoplasts were injected on a Superdex 200 HR30/10 filtration column (Amersham Biosciences). Size exclusion chromatography (SEC) was performed at 4°C with elution buffer (25 mM HEPES, pH 7.5, 500 mM NaCl, 1 mM EDTA, 0.2% [w/v] NP40, and 1 µg mL–1 pefabloc) at a flow rate of 0.4 mL min–1. Fractions of 0.8 mL were collected and after acetone precipitation the distribution of indicated proteins was analyzed by SDS-PAGE and immunoblot detection. As molecular mass standards thyroglobulin (669 kD), ferritin (440 kD), catalase (232 kD), lactate dehydrogenase (140 kD), and bovine serum albumin (67 kD) were used. Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession number AY608694.
We thank Gisela English for the excellent technical assistance and Drs. Lutz Nover, Markus Fauth, and Pascal von Koskull-Döring for their helpful and critical discussions during preparation of this manuscript. Received March 16, 2004; returned for revision May 16, 2004; accepted May 16, 2004.
1 This work was supported by the Deutsche Forschungsgemeinschaft (grant no. SCHA 577/6 to K.-D.S.) and Fonds der Chemischen Industrie (to K.-D.S.).  Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.042820. * Corresponding author; e-mail scharf{at}cellbiology.uni-frankfurt.de; fax 49–69–798–29286.
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C323 (no. 3). Evidently, the interaction with HsfA2 and the resulting formation of high Mr forms require the C-terminal activation domain of HsfA2 but are not dependent on the oligomeric state. The HsfA2
