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| Plant Physiol. 2008 May; 147(1): 381–390. doi: 10.1104/pp.108.118208. | PMCID: PMC2330285 |
Copyright © 2008, American Society of Plant Biologists Chaperone Activity of ERD10 and ERD14, Two Disordered Stress-Related Plant Proteins 1[OA]Denes Kovacs, Eva Kalmar, Zsolt Torok, and Peter Tompa* 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.) Received February 21, 2008; Accepted March 10, 2008. |
Abstract 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), and they have fundamental functions in the cell, primarily in regulating signal transduction or gene expression ( Tompa, 2002; Dyson and Wright, 2005). These functions are usually intimately related to the structural characteristics of these proteins, such as high flexibility, structural adaptability, and extended conformational states. IDPs have been shown to have high ion-binding capacity ( Heyen et al., 2002; Alsheikh et al., 2003, 2005; Saavedra et al., 2006; Tompa et al., 2006), to be able to bind membranes ( Danyluk et al., 1998; Ismail et al., 1999a; Koag et al., 2003), and to have both RNA and protein chaperone activity ( Tompa and Csermely, 2004). In Arabidopsis, approximately 23% of proteins are predicted to be fully disordered ( Oldfield et al., 2005). The lack of a stable structure for IDPs enables them to contribute functions relevant to the dehydration stress conditions encountered by plants. 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; Goyal et al., 2003), also demonstrated recently experimentally ( Bokor et al., 2005). It was also reported that both ERD10 and ERD14 can be phosphorylated at various sites, which promotes the binding of bivalent metal ions, and this might be related to their ion-sequestering activity ( Rorat, 2006; Tunnacliffe and Wise, 2007). Of the various ions, primary importance has been ascribed to calcium, which suggests a calcium activation route for the protein ( Welin et al., 1994; Shinozaki and Yamaguchi-Shinozaki, 1996; Nylander et al., 2001). DHNs also have the capacity to bind to membranes, which results from their diverse combinations of short sequence motif domains (K, S, Y, and ![[var phi]](corehtml/pmc/pmcents/x03C6.gif) segments), of which the Lys-rich K segment represents a lipid-binding A2 amphipathic α-helix ( Close, 1996). In maize ( Zea mays) scutellar parenchyma cells, DHN1 is associated with membrane-rich areas surrounding lipid and protein bodies ( Asghar et al., 1994; Egerton-Warburton et al., 1997). DHN1 preferentially binds to small unilamellar vesicles composed of acidic phospholipids through the A2 amphipathic α-helical structure formed by the K segment ( Koag et al., 2003). In wheat ( Triticum aestivum), members of the wcor410 protein family accumulate near the plasma membrane under cold stress ( Danyluk et al., 1998). An effect on membrane stability has been suggested to contribute to the function of DHNs under stress conditions ( Danyluk et al., 1998; Rorat, 2006). 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). These protective effects, however, manifest only in a trehalose-dependent manner, with low protective efficiency. A broad protein stabilization function and antiaggregation activity have been shown for a group 3 LEA protein from Aphelenchus avenae ( Chakrabortee et al., 2007). Cryoprotective activity on lactate dehydrogenase has been demonstrated for two DHN-type proteins ( Momma et al., 2003), and a similar effect was also shown for PCA60, a 60-kD protein from winter bark tissues of peach ( Prunus persica; Wisniewskia et al., 1999). All of these points might be taken to suggest that ERD10 and ERD14 exploit their structural disorder in fulfilling protective functions by chaperone activity, membrane binding, or both. We decided to investigate these possibilities. We report here that they do act as potent chaperones of broad substrate specificity and that they also have membrane-binding capacity. The implications of these findings are discussed in the context of the dehydration stress functions of LEA proteins in general and DHNs in particular. |
RESULTS IDP Character of ERD10 and ERD14 Based on observations by circular dichroism (CD) spectroscopy ( Mouillon et al., 2006) and relaxation 1H-NMR measurements ( Bokor et al., 2005), the IDP character of ERD10 and ERD14 has already been suggested. It should not be overlooked, however, that for a definitive conclusion on the disordered status of a protein a combination of various techniques is required ( Tompa, 2002). Thus, we present here further evidence for the structural disorder of these proteins. In silico predictions by IUPred ( Dosztanyi et al., 2005) and PONDR ( Romero et al., 2004) predicted both ERD10 and ERD14 as highly disordered proteins ( Fig. 1), as the scores of the particular amino acids were mostly above 0.5, which is the threshold separating ordered from disordered regions in proteins. Because the score is above 0.5 for most of the sequence, these predictions suggest that ERD10 and ERD14 are mostly disordered proteins. In SDS-PAGE, they show a characteristic low mobility: the real molecular mass of ERD10 is 29 kD but it runs at 45 kD, whereas the real molecular mass of ERD14 is 20 kD but it runs at 37 kD ( Fig. 2A). This high apparent molecular mass caused by a highly hydrophilic character is often diagnostic of IDPs ( Tompa, 2002). In addition, both proteins are heat stable ( Fig. 2B) and are very sensitive to proteases ( Fig. 2A).  | Figure 1.In silico prediction of protein disorder. Predictions were performed with the amino acid sequences taken from the NCBI (ERD10, NP_564114; ERD14, NP_177745), which are identical to the amino acid sequences translated from the cDNA used for ERD10 (dashed (more ...) |
 | Figure 2.IDP character of ERDs. A, Limited proteolysis of BSA, ERD10, and ERD14 with proteinase K. The ratio of proteinase concentration (mg mL−1) was varied from 1:1,500 to 1:50 relative to substrate concentration at 0.5 mg mL−1. Incubation was (more ...) |
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 α-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.  | Figure 3.NMR and CD spectra of ERD10 and ERD14. A, The NMR spectrum of ERD10 was recorded on a 500-MHz spectrometer at 1 mm protein concentration in Millipore water at 25°C with water suppression (inset shows the range 7.0–9.0 ppm blown up). B, (more ...) |
 | Figure 4.Protection of ADH from heat-induced inactivation by ERDs. ADH (2 μm, 0.3 mg mL−1) was treated at 43°C alone (![[filled square]](corehtml/pmc/pmcents/x25AA.gif) ) or in the presence of 1 μm BSA (0.066 mg mL−1; •), 1 μm HSP90 (0.084 mg mL (more ...) |
ERD10 and ERD14 as Chaperones Thermal Inactivation of Alcohol Dehydrogenase ERD10 and ERD14 show potent protective effects on the thermally induced inactivation of alcohol dehydrogenase (ADH). The chaperone effect is apparent under various conditions and ADH:ERD ratios, and here we show the chaperone activity of ERD10 and ERD14 at molar ratios of 2:1 ( Fig. 1; Table I) and 1:5 ( Table I). Chaperone activity is characterized by the effect on ADH activity remaining after 60 min of incubation, when at a molar ratio of 2:1 ERD10 and ERD14 have somewhat weaker protective effects than heat shock protein 90 (HSP90), whereas at a molar ratio 1:5 almost complete protection is seen ( Table I). The significance of this effect is underscored by the fact that it exceeds that of bovine serum albumin (BSA) and is commensurate with that of HSP90. The molar excesses of ERD10 and ERD14 under the conditions of dehydration stress are relevant, because they may represent approximately 4% of the soluble cellular protein ( Wise and Tunnacliffe, 2004). 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).  | Figure 5.Effects of ERDs on the heat-induced aggregation of citrate synthase. Citrate synthase at 0.5 μm was incubated at 45°C alone () or in the presence of 0.5 μm BSA (•), 0.5 μm HSP90 (![[filled triangle]](corehtml/pmc/pmcents/utrif.gif) ), 0.5 μ (more ...) |
Thermal Aggregation of Firefly Luciferase Both ERD10 and ERD14 showed significant protective effects on the thermal aggregation of firefly luciferase at a 2× molar excess ( Fig. 6). Under these conditions, aggregation was almost completely inhibited, similar to the effect of HSP90 ( Table I). BSA was practically ineffective in this assay.  | Figure 6.Effects of ERDs on the heat-induced aggregation of luciferase. Results from a typical experiment on the thermal aggregation of luciferase are shown. The denaturation of 1.1 μm luciferase was induced at 45°C alone () or in the (more ...) |
Chemically Induced Inactivation of Lysozyme ERD10, ERD14, and HSP90 had similar protective effects on the enzyme activity of lysozyme at substoichiometric ratios of lysozyme:chaperone (7:1; Fig. 7; Table I). Although the time course of the loss of enzyme activity was somewhat different in the presence of ERD10, ERD14, and HSP90, their protective effects, as expressed by residual enzyme activity after 25 min of incubation, were commensurate. BSA, again, had a negligible effect.  | Figure 7.Effects of ERDs on the chemically induced inactivation of lysozyme. Lysozyme at 34 μm was treated with DTT either without additions () or in the presence of 5 μm BSA (•), 5 μm HSP90 (), 5 μm ERD10 (more ...) |
Membrane Binding of ERD10 and ERD14 The protective effects under dehydration stress conditions of ERD10 and ERD14 may also have a component of membrane stabilization ( Rorat, 2006; Tunnacliffe and Wise, 2007). To check this possibility, we analyzed the liposome binding of ERD10 and ERD14. From previous results, it was already known that DHNs preferably bind to small unilamellar vesicles composed of acidic phospholipids, such as phosphatidylserine (PS; Koag et al., 2003). The interaction of ERD10 and ERD14 with phospholipid vesicles (PLVs) was analyzed with mini gel filtration columns, as described in “Materials and Methods.” The analysis and comparison with the behavior of a positive control, calpain domain III, which shows calcium-dependent phospholipid binding, and a negative control, BSA, which does not bind to vesicles either in the absence or the presence of calcium ( Tompa et al., 2001), confirm that ERD10 and ERD14 bind to PLVs. The interaction was statistically significant, as suggested by Student's t test ( P = 0.003 and 0.056 for ERD10 and ERD14, respectively). Under the given conditions, approximately 2 μg of ERD10 and 2.5 μg of ERD14 were bound to 25 μg of PLVs, out of 15 μg total ( Fig. 8A).  | Figure 8.Binding of ERDs to PLVs. A, The binding analysis between PLVs and ERD10 or ERD14 was carried out using a mini gel filtration assay (Tompa et al., 2001). Protein samples were applied to a gel filtration column either in the absence (ØPLV) or presence (more ...) |
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 CaCl 2 and high ionic strength on the interaction; we found that the latter (800 m m 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).  | Figure 9.Fluidity of phospholipid membranes. The membrane fluidity was determined via fluorescence anisotropy measurement. The PC:PS (1:1) phospholipids were suspended at 40 mg mL−1 concentration with 1 mm 1,6-diphenyl-1,3,5-hexatriene dye (DPH). Large (more ...) |
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DISCUSSION 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). Here, the combination of various techniques provides a detailed picture of the structural state of ERD10 and ERD14. CD showed their lack of significant secondary structure. SDS-PAGE mobility suggested their unusual amino acid composition related to other IDPs with similar behavior ( Tompa, 2002). Extreme susceptibility to proteases suggested the extended nature and ensuing exposure of their polypeptide chain. NMR spectra suggested that their chain is of a highly flexible state, reminiscent in behavior of short peptides. Bioinformatics predictions suggested a fully disordered state along the entire length of the chain. In all, these and previous data confirm that these proteins exist in a highly flexible, largely unfolded conformational state with a highly charged polypeptide chain. According to Bokor et al. (2005) and to the resistance of these proteins to heat-induced aggregation, they have high hydration potential and are highly soluble without any propensity to aggregate. 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; Kim et al., 2000; Hara et al., 2001; Momma et al., 2003; Tompa and Csermely, 2004; Goyal et al., 2005). However, the distinction between the two functions is not entirely apparent. The term chaperone is used for a specific interaction with the client protein and/or a protective effect in a heat-induced aggregation or inactivation assay, whereas molecular shield is used in a situation in which protection against dehydration/freezing-induced aggregation is offered by the protein aspecifically filling the space between client molecules ( Goyal et al., 2005; Chakrabortee et al., 2007; Tunnacliffe and Wise, 2007). However, direct evidence that the latter mode of entropic effect is not utilized by bona fide chaperones is lacking. The entropy transfer model incorporates both mechanistic elements (i.e. transient interaction with the client and entropic filling of space between client molecules; Tompa and Csermely, 2004). In the case of group 1 and group 3 LEA proteins, the evidence mostly points to their involvement in aggregation caused by freezing ( Wisniewskia et al., 1999; Hara et al., 2001; Momma et al., 2003; Goyal et al., 2005; Chakrabortee et al., 2007), but the possible chaperone activity of group 2 LEA proteins has never been tested. 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), α-synuclein ( Kim et al., 2000; Souza et al., 2000; Ahn et al., 2006), HSP90 ( Minami et al., 2001), or tubulin ( Guha et al., 1998), makes it safe to conclude that ERD10 and ERD14 are chaperones of commensurate potency to previously described representatives of this functional class. The significance of the effect is underlined by the fact that it far exceeds that of BSA and in several cases is commensurate with, or even exceeds, that exerted by HSP90. These activities in various and unrelated assays point to the possibility of a broad and rather nonspecific mechanism of action. The ensuing possible general and nondiscriminate effects of ERD10 and ERD14 provide the rationale for their general protective effects under stress conditions, as also noted in the case of LEA group 1 and group 3 proteins ( Goyal et al., 2005). In general mechanistic terms, such a loose specificity is in agreement with the elements of the rather nonspecific nature of interaction and entropic exclusion of the entropy transfer mechanism of IDP chaperones ( Tompa and Csermely, 2004), which originate from the highly flexible and charged nature of these proteins. There is also a strong correlation between this phenomenon and the ability of some IDPs to carry out multiple functions, termed moonlighting ( Tompa et al., 2005). As a final note, its should be stressed that by these novel results with ERD10 and ERD14, the strict distinction between classical chaperone action and cryoprotective molecular shield activity ( Chakrabortee et al., 2007; Tunnacliffe and Wise, 2007) may not be tenable. In our view, these seemingly contrasting behaviors can be unified in a detailed mechanistic picture based on a description originating from the intrinsic disorder of these proteins. 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) adsorbs to the surface of the negatively charged PS membrane due to electrostatic binding and also does not change the properties of PLVs. This phenomenon, that an electrostatic adsorption does not contribute appreciably to the change of the biophysical properties of the vesicles, was also postulated before by Arnold et al. (1992). This result suggests that the interaction is electrostatic, affecting membranes only peripherally, via phospholipid head groups. We have verified this presumption by analyzing the ionic strength dependence of the interaction and found that the amounts of bound ERD10 and ERD14 decrease significantly above 500 m m NaCl. Furthermore, the membrane-binding function of DHNs was ascribed to the K segment, for which it was shown that it is disordered in the free form, even in α-helix-promoting environments ( Mouillon et al., 2006). All of these results suggest that ERD10 and ERD14 interact with membranes through electrostatic means. |
CONCLUSION 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. |
MATERIALS AND METHODS All chemicals were obtained from Sigma-Aldrich or Fluka. HSP90 expression vector (HSP90-pMAL) was obtained from Dr. Csaba Sőti. Preparation of Expression Plasmids 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), with the modification that imbibed seeds (12 h in water) were disrupted by mechanical fracturing in lysis buffer (10 m m Tris, 0.1 m m EDTA, 20 μg mL −1 ribonuclease, and 0.5 mass percent SDS). 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. Protein Expression and Purification 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. Characterization of IDP Nature 1H-NMR spectra were recorded on a Bruker DRX 500-MHz spectrometer. Samples were dissolved at 1 m m concentration in H 2O:D 2O (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 m m NaH 2PO 4 and 100 m m 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 12,000 g for 15 min at room temperature. The supernatants were applied to a 12.5% SDS-PAGE gel. Chaperone Assays Thermal Inactivation of ADH The heat-induced inactivation of yeast ADH (Sigma-Aldrich) was carried out at 43°C for 1 h. ADH (2 μm, 0.3 mg mL−1) was mixed with either ERD10 and ERD14 from 1 to 10 μm (ERD10, 0.03–0.3 mg mL−1; ERD14, 0.02–0.2 mg mL−1) or with 1 μm (0.084 mg mL−1) HSP90 or 1 μm (0.066 mg mL−1) BSA as a control (in 100 mm NaH2PO4 and 0–250 mm NaCl, pH 8.8). The samples were incubated on ice for 5 min prior to the assay. The reaction solution was then placed into a 43°C water bath, and enzyme activity was determined every 10 min. The enzyme activity was measured with 1.25 mm ethanol and 2 mm NAD+ (100 mm NaH2PO4, pH 7.5) by adding 20 μL (to 600 μL) of reaction solution. The increase in NADH A340 was followed at room temperature with a JASCO UV-550 spectrophotometer. Thermal Aggregation of Citrate Synthase The aggregation of citrate synthase (from porcine heart; Sigma) was induced at 45°C in 50 mm Tris, 100 mm NaCl, pH 7.5, with 0.5 μm citrate synthase (0.043 mg mL−1) without additions or in the presence of 0.5 μm BSA (0.033 mg mL−1), 0.5 μm HSP90 (0.045 mg mL−1), 0.5 to 2 μm ERD10 (0.015–0.06 mg mL−1), or 0.5 to 2 μm ERD14 (0.010–0.041 mg mL−1). The samples were preincubated for 5 min on ice and then placed into the thermostatted cell holder of a JASCO UV-550 spectrophotometer, and aggregation was followed for 40 min by measuring the increase in A400. Thermal Aggregation of Luciferase The aggregation of firefly luciferase (Sigma) was induced at 45°C in 50 mm Tris, pH 7.5. Luciferase at 1.1 μm (0.064 mg mL−1) was incubated without additions or in the presence of 2 μm BSA (0.130 mg mL−1), 2 μm HSP90 (0.18 mg mL−1), 2 μm ERD10 (0.06 mg mL−1), or 2 μm ERD14 (0.045 mg mL−1). The samples were incubated for 5 min on ice prior to use and then placed into the thermostatted cell holder. The aggregation was followed for 40 min with a JASCO UV-550 spectrophotometer by measuring the increase in A400. Chemical Aggregation of Lysozyme The reduction of disulfide bonds was carried out with dithiothreitol (DTT; Sigma), which causes unfolding of the enzyme with a concomitant loss of activity. Inactivation of lysozyme was studied in 50 mm Tris, 150 mm NaCl, 2 mm EDTA, and 20 mm DTT, pH 7.5. Lysozyme (34 μm, 0.5 mg mL−1) was incubated without additions or in the presence of 5 μm BSA (0.33 mg mL−1), 5 μm HSP90 (0.16 mg mL−1), 5 μM ERD10 (0.15 mg mL−1), or 5 μm ERD14 (0.1 mg mL−1) at 25°C. Aliquots (20 μL) were withdrawn every 5 min to determine enzyme activity by following the decomposition of a 0.1 mg mL−1 Micrococcus lysodecticus cell wall preparation (Sigma) suspended in 20 mm NaH2PO4 buffer, pH 7.0, at 25°C. The activity was measured for 1 min in a JASCO FP-6300 spectrofluorimeter at 450-nm extinction and emission wavelengths. Vesicle-Binding Assay The interaction between lipid vesicles and ERD10 and ERD14 was analyzed by a mini-gel filtration assay as described previously ( Tompa et al., 2001). Protein samples were prepared in the presence and absence of PLVs and passed through the mini gel filtration column. The amount of the protein in the flow through is characteristic of the strength of the interaction. 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- × 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. Effects on Membrane Fluidity The effects of ERD10 and ERD14 on membrane fluidity were tested with fluorescence anisotropy measurements as described before ( Torok et al., 1997). This technique is based on the analysis of the mobility of a fluorescent dye (1,6-diphenyl-1,3,5-hexatriene) inside the phospholipid layer, which is linked directly to membrane fluidity. The dye and phospholipids were mixed in chloroform and dried under nitrogen gas. The phospholipid film was dissolved in 50 m m Tris, pH 7.5 (at a final concentration of 40 μg mL −1), with vortexing until it dissolved fully. Large unilamellar vesicles were prepared by freezing the suspension 10 times, followed by extrusion using a Liposofast extruder (Avestin), with two stacked polycarbonate filters of 100-nm pore size, as described previously ( Mayer et al., 1986). Fluorescence anisotropy was determined according to the following equation: 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). The analysis was carried out at different ERD10 and ERD14 concentrations ranging from 10 to 200 μg mL −1. Sequence data from this article can be found in the GenBank data libraries under accession numbers D17714 (ERD10) and D17715 (ERD14). |
Acknowledgments We are indebted to Prof. András Perczel (Department of Organic Chemistry, Eötvös Loránd University) for measuring the 1H-NMR spectra and to Dr. Csaba Sőti (Department of Medical Biochemistry, Semmelweis University) for his help in performing the luciferase assay and providing the HSP90 expression vector. |
Notes |
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See the addendum "Disordered plant LEA proteins as molecular chaperones" in Plant Signal Behav, volume 3 on page 710.
