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First published online March 21, 2008; 10.1104/pp.108.118208 Plant Physiology 147:381-390 (2008) © 2008 American Society of Plant Biologists OPEN ACCESS ARTICLE
Chaperone Activity of ERD10 and ERD14, Two Disordered Stress-Related Plant Proteins1,[OA]Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest, Hungary H–1113 (D.K., P.T.); Department of Medical Chemistry, Molecular Biology, and Pathobiochemistry, Semmelweis University, Budapest, Hungary H–1088 (E.K.); and Institute of Biochemistry, Biological Research Center, H–6701 Szeged, Hungary (Z.T.)
ERD10 and ERD14 (for early response to dehydration) proteins are members of the dehydrin family that accumulate in response to abiotic environmental stresses, such as high salinity, drought, and low temperature, in Arabidopsis (Arabidopsis thaliana). Whereas these proteins protect cells against the consequences of dehydration, the exact mode(s) of their action remains poorly understood. Here, detailed evidence is provided that ERD10 and ERD14 belong to the family of intrinsically disordered proteins, and it is shown in various assays that they act as chaperones in vitro. ERD10 and ERD14 are able to prevent the heat-induced aggregation and/or inactivation of various substrates, such as lysozyme, alcohol dehydrogenase, firefly luciferase, and citrate synthase. It is also demonstrated that ERD10 and ERD14 bind to acidic phospholipid vesicles without significantly affecting membrane fluidity. Membrane binding is strongly influenced by ionic strength. Our results show that these intrinsically disordered proteins have chaperone activity of rather wide substrate specificity and that they interact with phospholipid vesicles through electrostatic forces. We suggest that these findings provide the rationale for the mechanism of how these proteins avert the adverse effects of dehydration stresses.
Late embryogenesis-abundant (LEA) proteins are expressed in late stages of seed maturation and/or upon water stress conditions in plants (Rorat, 2006  ; Tunnacliffe and Wise, 2007). They constitute a highly divergent group of proteins that can be classified into three loosely defined groups (or superfamilies) by the presence of distinct, short sequence motifs (Wise and Tunnacliffe, 2004). ERD10 and ERD14 (for early response to dehydration) belong to the dehydrin (DHN) family, also known as group 2 LEA proteins (Kiyosue et al., 1994b), isolated from Arabidopsis (Arabidopsis thaliana). In unstressed plants, these proteins are expressed in late phases of seed development (Kiyosue et al., 1994a) and also in some actively dividing tissues, such as the tips of roots and leaves (Welin et al., 1994; Nylander et al., 2001). Their expression increases in response to low temperature (Nylander et al., 2001) and other dehydration stress conditions, such as high salinity and drought, through the abscisic acid cascade (Kiyosue et al., 1994a; Welin et al., 1994; Shinozaki and Yamaguchi-Shinozaki, 1996; Nylander et al., 2001; Rorat, 2006). Recent results have shown that some of the DHNs also accumulate during high-light stress (Kimura et al., 2003). Whereas these proteins confer the plant desiccation stress tolerance, their exact mechanism of action is not known in detail. Earlier studies have suggested various possible biochemical activities for DHNs, such as buffering water, sequestering ions, stabilizing membranes, or acting as chaperones (Rorat, 2006; Tunnacliffe and Wise, 2007). Pertinent to the latter function is that most of these proteins are extremely flexible or completely lack a well-defined structure (i.e. they belong to the family of intrinsically disordered/unstructured proteins [IDPs/IUPs], as suggested for Glycine max rGmDHN1 [Soulages et al., 2003], DHN1 [Koag et al., 2003], and ERD10 [Mouillon et al., 2006]).
IDPs are widely distributed in prokaryotic and eukaryotic proteomes (Tompa et al., 2006
Recently, some DHNs were characterized in detail, and the results provide the rationale for their function. Their amino acid compositions provide an overall hydrophilic character, which potentially confers a high potential hydration capacity (McCubbin et al., 1985
Perhaps most intriguing with respect to dehydration stress function is the potential chaperone activity of LEA proteins, for which only limited evidence has been presented to date. For two LEA proteins (one of group 1 and another of group 3), protection of citrate synthase from heat-induced aggregation and of lactate dehydrogenase from cold-induced aggregation have been described (Goyal et al., 2005
IDP Character of ERD10 and ERD14
Based on observations by circular dichroism (CD) spectroscopy (Mouillon et al., 2006
The polypeptide chain of IDPs is more accessible to proteases than that of globular proteins, which results in much higher protease sensitivity, and this is diagnostic for their open structures. This was tested with proteases of broad substrate specificity (subtilisin and proteinase K) and proteases of narrow substrate specificity (trypsin and chymotrypsin); the pattern of the digestion was similar for all proteases, so we show only the results of proteinase K (Fig. 2A). ERD10 and ERD14 do not show any ordered motifs, which would show resistance against proteolysis. The 1H-NMR spectra of ERD10 and ERD14 (Fig. 3A ; only ERD10 is shown) also suggest their disordered nature, because the chemical shift dispersion is narrower than is typical for globular proteins. For globular proteins, 1H chemical shifts usually spread out in the region 9.5 to 6.0 ppm, whereas for IDPs, they are usually confined in the region 8.0 to 8.5 ppm (Dyson and Wright, 2004 ). The CD spectra of ERD10 and ERD14 with a minimum close to 200 nm (Fig. 3B) are also typical of disordered proteins, because regular secondary structural elements, such as the  -helix and the β-sheet, in globular proteins give characteristic spectral peaks at 208, 222, and 214 nm, whereas a large negative peak at approximately 200 nm is characteristic of the coil conformation that dominates in IDPs (Receveur-Brechot et al., 2005). It should be noted that the small negative peak at approximately 224 nm suggests some residual helical content in both proteins. When ERD10 and ERD14 were titrated with trifluoro acetic acid (data not shown), a transition from the disordered state to the helix was observed within the range of 15% to 30% trifluoro acetic acid, which suggests a significant local preference for the helical conformation and a possible induced folding mechanism during interaction with partners (Johansson et al., 2003; Meszaros et al., 2007). In all, these observations unequivocally suggest that ERD10 and ERD14 are highly disordered proteins.
ERD10 and ERD14 as Chaperones
Thermal Inactivation of Alcohol Dehydrogenase
Thermal Aggregation of Citrate Synthase Citrate synthase is a thermally unstable protein that makes it suitable for analyzing chaperone activities. Here, citrate synthase was incubated at an elevated temperature and aggregation was followed by an increase in absorbance. ERD10 and ERD14 at an equimolar concentration showed significant protective activity (Fig. 5 ), slowing the rate of aggregation to about half of its control value; this effect was significantly higher than that of BSA but fell short of that of HSP90. Upon increasing ERD10 and ERD14 concentrations further, the protective effect did not change much and did not reach the efficiency of HSP90 (Table I).
Thermal Aggregation of Firefly Luciferase
Chemically Induced Inactivation of Lysozyme
Membrane Binding of ERD10 and ERD14
We also analyzed the CD spectra of ERD10 and ERD14 in the presence of PLVs. Contrary to other proteins (Koag et al., 2003 ), ERD10 and ERD14 did not show any detectable structural change (data not shown). Furthermore, we also checked the effect of 100 µM CaCl2 and high ionic strength on the interaction; we found that the latter (800 mM NaCl) reduced the interaction dramatically, which suggests an electrostatic interaction. Calcium reduced the amount of ERD10 and ERD14 (Fig. 8, B and C; Table II
) bound to the vesicles (P = 0.003 and 0.056 for ERD10 and ERD14, respectively, compared with the interaction with PLVs), but it did not change the amount of ERD10 and ERD14 found in the flow through (P = 0.24 and 0.20 for ERD10 and ERD14, respectively, compared with the flow through). Accordingly, calcium only affects the interaction between ERD10, ERD14, and PLVs (Fig. 8, B and C, lanes 2 and 3; Table II). These results suggest an interaction with calcium in the nonphosphorylated state of the proteins, in accordance with previous observations (Svensson et al., 2000; Alsheikh et al., 2003). Liposome binding suggested a possible effect of ERD10 and ERD14 on membrane fluidity. This was analyzed by fluorescence anisotropy measurements, which showed no effect of ERD10 or ERD14 on phosphatidylcholine (PC):PS (1:1) vesicles (Fig. 9
).
Previous and recent results with ERD10 and ERD14 provide an intriguing picture of the biochemical behavior of these proteins. Prior subcellular localization data have shown that DHNs are localized in various cell compartments, such as cytosol, nucleus, mitochondria, and vacuole (Houde et al., 1992 ; Leung and Giraudat, 1998; Li et al., 1998; Ismail et al., 1999b; Richard et al., 2000; Tamminen et al., 2001). ERD10 and ERD14 are expressed ubiquitously during environmental stress and under normal environmental conditions, including in the tips of roots and leaves (Welin et al., 1994; Nylander et al., 2001). DHNs are capable of interacting with membranes (Danyluk et al., 1998; Ismail et al., 1999a; Nylander et al., 2001; Koag et al., 2003), and some of them have a preference for membranes composed of acidic phospholipids (Koag et al., 2003). They can be phosphorylated, which usually results in lower SDS-PAGE mobility. Furthermore, phosphorylation makes these proteins capable of binding bivalent metal ions (Alsheikh et al., 2003, 2005).
For some DHNs, significant flexibility has been suggested, and limited evidence indicated that they might belong to the family of IDPs (Mouillon et al., 2006
Of particular relevance to their stress-related functions is that for several IDPs and/or DHNs, chaperone or molecular shield function has already been demonstrated (Wisniewskia et al., 1999
To investigate these functions of ERD10 and ERD14, we studied their protective effects on the heat-induced loss of enzyme activity and/or aggregation of four different substrates. The selected assays represent different modes and mechanisms of possible chaperone action and included thermal inactivation of ADH, thermal aggregation of firefly luciferase and citrate synthase, and the chemically induced inactivation of lysozyme. In the analyses shown here, ERD10 and ERD14 demonstrated marked effects on the prevention of aggregation/deactivation of protein substrates. A comparison of related data published in the literature, such as for periplasmic disulfide isomerase of Gram-negative bacteria (DsbC; Chen et al., 1999
Another putative mechanism of the protective function of ERD10 and ERD14 is membrane binding and stabilization. We could address this cryoprotective effect by analyzing their binding of PLVs and their effects on membrane fluidity. Generally, the fluorescence anisotropy is characteristic of the movement rate of the fluorescent dye in the phospholipid layer, which showed no difference in the presence or absence of ERD10 and ERD14. Similarly, melittin (Ohki et al., 1994
ERD10 and ERD14 were previously identified in Arabidopsis as cDNA clones that encode two members of the DHN family. Although the expression pattern and transcriptional regulation of these DHNs are well characterized, the specific biochemical functions of these proteins and their physiological roles in plants, especially during stress conditions, are not fully understood. Here, we provide evidence that the nonphosphorylated form of ERD10 and ERD14 act as chaperones in vitro and that they bind to phospholipid layers via electrostatic forces. Further studies will be needed to address the functionality of these proteins in their phosphorylated form under normal and stress conditions.
All chemicals were obtained from Sigma-Aldrich or Fluka. HSP90 expression vector (HSP90-pMAL) was obtained from Dr. Csaba S  ti.
For the lack of a stress-treated Arabidopsis (Arabidopsis thaliana) cDNA library, genomic segments corresponding to ERD10 (National Center for Biotechnology Information [NCBI] accession no. NP_564114) and ERD14 (NCBI accession no. NP_177745) were isolated from Arabidopsis genomial DNA. Genomial DNA was extracted and purified as described previously (Sambrook et al., 1989 Both ERD10 and ERD14 have only one intron in their genes. The genes were isolated from the genomial DNA with terminal primers. To isolate the corresponding exons, internal and terminal primers were designed for both exons. The internal primers were designed to anneal to the end of the corresponding exon and provide a flanking region that overlaps with the other exon. The corresponding cDNAs were isolated from cross-annealing of the exons, and the missing parts were filled up with DNA polymerase. After this step, the terminal primers were added to the reaction solution to amplify the full-length cDNAs. The cDNAs of ERD10 and ERD14 were then ligated into pET22b expression plasmids (Novagen), with a stop codon before the His tag to obtain the nontagged version of the proteins.
The expression of the recombinant proteins (ERD10 and ERD14) was induced in Escherichia coli strain BL21(DE) at 30°C by 0.5 mM isopropyl-β-D-thiogalactopyranoside. The cells were harvested by centrifugation and resuspended in 1/100th volume of lysis buffer (50 mM Tris, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 2 mM benzamidine, 5 mM mercaptoethanol, and 2 mM EDTA, pH 7.5). The cells were disrupted by sonication six times for 15 s each in ice with 30-s breaks, and crude cell debris were removed by centrifugation (10,000g for 10 min at 4°C). The supernatant was placed in a boiling-water bath for 5 min for heat fractionation, and aggregated proteins were removed by centrifugation (100,000g for 30 min at 4°C). The supernatant was stored at –20°C until further purification steps. The supernatant was dialyzed into buffer A (50 mM Tris and 2 mM EDTA, pH 7.5) and loaded onto an equilibrated 7-mL DEAE cellulose (Pharmacia) column. The column was washed with buffer A with 50 mM NaCl, and the protein was eluted with a linear gradient of 50 to 500 mM NaCl in buffer A. The protein fractions detected by SDS-PAGE were pooled and dialyzed into buffer B (100 mM acetic acid and 1 mM EDTA, pH 6). The protein sample was loaded onto an equilibrated CM-Sepharose column (Pharmacia) and washed with buffer B, in which ERD10 and ERD14 appeared in the flow through. The protein fractions were dialyzed into Millipore water for lyophilization. The final yield was approximately 15 to 17 mg L–1 medium, with purity typically exceeding 90%. The expression of HSP90 was induced with 0.5 mM isopropyl-β-D-thiogalactopyranoside in BL21 (DE) cells at 30°C. The cells were harvested by centrifugation and resuspended in 1/50th volume of lysis buffer (20 mM Tris and 200 mM NaCl, pH 7.5). The cells were disrupted by sonication six times for 10 s each in ice with 30-s breaks, and crude cell debris were removed by centrifugation (10,000g for 10 min at 4°C). The supernatant was then immediately loaded onto 3 mL of amylose resin (New England Biolabs) and washed three times with lysis buffer. The elution was carried out via 10 mM maltose in lysis buffer, and the fractions were analyzed with SDS-PAGE. The protein fractions were then dialyzed against buffer A (50 mM Tris and 1 mM EDTA, pH 7.5) and cleaved with 10 µg mL–1 factor Xa (New England Biolabs) for 24 h at room temperature. The sample was loaded onto a 5-mL DEAE cellulose (Pharmacia) column, washed two times with 7 mL of buffer A with 50 mM NaCl, and eluted with a linear gradient of 50 to 500 mM NaCl in buffer A. The fractions were analyzed with SDS-PAGE, and the protein samples were dialyzed against 15 mM NaH2PO4, 50 mM NaCl, 1 mM EDTA, 0.5 mM benzamidine, and 0.1 mM phenylmethylsulfonyl fluoride, pH 7.5.
1H-NMR spectra were recorded on a Bruker DRX 500-MHz spectrometer. Samples were dissolved at 1 mM concentration in H2O:D2O (9:1, v/v) and centrifuged prior to use. The spectra were recorded at 25°C. For CD spectroscopy, ERD10 and ERD14 were dissolved in 10 mM NaH2PO4 and 100 mM NaCl, pH 7.5, at a protein concentration of 0.5 mg mL–1, and the spectra were recorded with a JASCO J-720 spectropolarimeter in a 1-mm cuvette at 25°C. The disordered characteristics were also predicted by the in silico prediction methods IUPred (http://www.enzim.hu/IUPred) and PONDR (http://www.pondr.com). Protease sensitivity was tested with four proteases: subtilisin and proteinase K (proteases of broad specificity) and trypsin and chymotrypsin (proteases of narrow specificity). Typically, 0.5 mg mL–1 ERD10 or ERD14 was treated with the appropriate protease at ratios of 1:50, 1:250, 1:600, 1:900, and 1:1,500 in separate reactions for 30 s. The reactions were stopped by adding SDS-PAGE loading buffer and heated at 100°C for 3 min. The samples were analyzed with 12.5% SDS-PAGE. The resistance to heat-induced aggregation was analyzed with SDS-PAGE. ERD10 and ERD14 (0.3 mg mL–1 of either) and 0.2 mg mL–1 BSA as a control were treated at 100°C for 5 min and centrifuged at 12,000g for 15 min at room temperature. The supernatants were applied to a 12.5% SDS-PAGE gel.
Thermal Inactivation of ADH
Thermal Aggregation of Citrate Synthase
Thermal Aggregation of Luciferase
Chemical Aggregation of Lysozyme
The interaction between lipid vesicles and ERD10 and ERD14 was analyzed by a mini-gel filtration assay as described previously (Tompa et al., 2001 PLVs were prepared with the following procedure. PC and PS were dissolved at a 1:1 ratio in 50 mM Tris, pH 7.5, sonicated at 16 µ (four times for 10 s each, with 30-s breaks, on ice), and centrifuged at 14,000g for 2 min. ERD10 and ERD14 (0.5 mg mL–1, 17 µM ERD10 and 24 µM ERD14) was incubated with 200 µg mL–1 PLVs for 5 min in the same buffer, and 30 µL was applied onto an equilibrated 5- x 5-mm Sephadex G200 column. The column was spun down at 1,000 rpm for 1 min, and the flow through was analyzed by 12.5% SDS-PAGE and evaluated with the software Quantity One (Bio-Rad). The effects of calcium (100 µM CaCl2) and high ionic strength (0–1.2 M NaCl) on the interaction were analyzed under the same conditions.
The effects of ERD10 and ERD14 on membrane fluidity were tested with fluorescence anisotropy measurements as described before (Torok et al., 1997
The temperature dependence of anisotropy was determined with and without added protein, which enables quantitation of the effect, on a T-format fluorescence spectrometer (Quanta Master QM-1; Photon Technology International) as described (Schlame et al., 1990 Sequence data from this article can be found in the GenBank data libraries under accession numbers D17714 (ERD10) and D17715 (ERD14).
We are indebted to Prof. András Perczel (Department of Organic Chemistry, Eötvös Loránd University) for measuring the 1H-NMR spectra and to Dr. Csaba S ti (Department of Medical Biochemistry, Semmelweis University) for his help in performing the luciferase assay and providing the HSP90 expression vector. Received February 21, 2008; accepted March 10, 2008; published March 21, 2008.
1 This work was supported by the Hungarian Scientific Research Fund (grant no. K60694), the Ministry of Health of Hungary (grant no. ETT 245/2006), and an International Senior Research Fellowship from the Wellcome Trust (grant no. ISRF 067595).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Peter Tompa (tompa{at}enzim.hu).
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.108.118208 * Corresponding author; e-mail tompa{at}enzim.hu.
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ABSTRACT
RESULTS
segments), of which the Lys-rich K segment represents a lipid-binding A2 amphipathic 
) or in the presence of 1 µM BSA (0.066 mg mL–1; 
), 1 µM HSP90 (0.084 mg mL–1; 
), 1 µM ERD10 (0.03 mg mL–1; 
), or 1 µM ERD14 (0.02 mg mL–1; 
). Activity is given as percentage of the zero-time value. Data points represent averages ± SD for five separate experiments.
). Results from a typical experiment in which aggregation was followed by measuring A400 are shown.
