Báo cáo khoa học: Chaperone activity of recombinant maize chloroplast protein synthesis elongation factor, EF-Tu

doi:10.1111/j.1432-1033.2004.04309.x

Eur. J. Biochem. 271, 3684–3692 (2004) (cid:2) FEBS 2004

Chaperone activity of recombinant maize chloroplast protein synthesis elongation factor, EF-Tu

Damodara Rao1, Ivana Momcilovic1, Satoru Kobayashi1, Eduardo Callegari2 and Zoran Ristic1 1Department of Biology, University of South Dakota and 2Department of Basic Biomedical Sciences, University of South Dakota School of Medicine, Vermillion, SD, USA

activity) and was stable at 45 (cid:1)C, the highest temperature used in this study to test this protein for possible chaperone activity. Importantly, the recombinant maize pre-EF-Tu displayed chaperone activity. It protected citrate synthase and malate dehydrogenase from thermal aggregation and inactivation. To our knowledge, this is the first observation of chaperone activity by a plant/eukaryotic pre-EF-Tu protein. The results of this study support the hypothesis that maize EF-Tu plays a role in heat tolerance by acting as a molecular chaperone and protecting chloroplast proteins from thermal aggregation and inactivation.

Keywords: chloroplast protein synthesis elongation factor (EF-Tu); chaperones; heat stress; heat tolerance; Zea mays.

The protein synthesis elongation factor, EF-Tu, is a protein that carries aminoacyl-tRNA to the A-site of the ribosome during the elongation phase of protein synthesis. In maize (Zea mays L) this protein has been implicated in heat tol- erance, and it has been hypothesized that EF-Tu confers heat tolerance by acting as a molecular chaperone and protecting heat-labile proteins from thermal aggregation and inactiva- tion. In this study we investigated the effect of the recom- binant precursor of maize EF-Tu (pre-EF-Tu) on thermal aggregation and inactivation of the heat-labile proteins, cit- rate synthase and malate dehydrogenase. The recombinant pre-EF-Tu was purified from Escherichia coli expressing this protein, and mass spectrometry confirmed that the isolated protein was indeed maize EF-Tu. The purified protein was capable of binding GDP (indicative of protein

identity of this protein was not known until the report of Bhadula et al. [5].)

Chloroplast protein synthesis elongation factor, EF-Tu, is a protein (45–46 kDa) that plays a key role in the elongation phase of protein synthesis [1–3]. This protein catalyzes the GTP-dependent binding of aminoacyl-tRNA to the A-site of the ribosome [3]. In land plants, EF-Tu is encoded by the nuclear genome and synthesized in the cytosol [4]. Chloro- plast EF-Tu is highly conserved, and it shows a high sequence similarity to prokaryotic EF-Tu [3,5].

A hypothesis has been developed that maize EF-Tu may confer heat tolerance by protecting other proteins from heat-induced aggregation and inactivation (thermal dam- age), thus acting as a molecular chaperone [10,11]. In this study we investigated the effect of the recombinant precur- sor of maize EF-Tu (pre-EF-Tu, which has a 58 amino acid long chloroplast targeting sequence at the N-terminal end [5]) on thermal aggregation and inactivation of the heat- labile proteins, citrate synthase (CS) and malate dehydro- genase (MDH). Here we report, for the first time, that the recombinant maize pre-EF-Tu displays chaperone proper- ties, as it protected heat-labile proteins from thermal damage.

Materials and methods

Expression of maize pre-EF-Tu in Escherichiacoli

Studies from our laboratory have suggested that in maize (Zea mays L) chloroplast EF-Tu may play a role in the development of heat tolerance. The evidence for this conclusion includes: (a) positive correlation between the heat-induced accumulation of EF-Tu and plant ability to tolerate heat stress in several genotypes of maize [5–7], (b) association between the heat-induced synthesis of EF-Tu and the maize heat tolerance phenotype [8], (c) increased tolerance to heat stress in Escherichia coli expressing maize EF-Tu [9], (d) decreased tolerance to heat stress in a maize mutant with reduced capacity to accumulate EF-Tu [10], and (e) increased thermal stability of chloroplast proteins in maize with higher levels of EF-Tu [10,11]. (It should be noted that in the previous studies [6–8] maize EF-Tu was referred to as a 45–46 kDa heat shock protein because the

Correspondence to Z. Ristic, Department of Biology, University of South Dakota, Vermillion, SD 57069, USA. Fax: +1 605 677 6557, E-mail: zristic@usd.edu Abbreviations: CS, citrate synthase; MDH, malate dehydrogenase; HSPs, heat shock proteins; sHSPs, small heat shock proteins. (Received 5 May 2004, revised 14 July 2004, accepted 27 July 2004)

E. coli expressing maize pre-EF-Tu was previously trans- formed [9] using a cDNA for maize (Z. mays L) EF-Tu, designated as Zmeftu1 [5]. Zmeftu1 was subcloned into the expression vector pTrcHis2A, which adds C-terminal c-myc and polyhistidine tags to the protein, and the pTrcHis2A vector carrying Zmeftu1 was used to transform competent E. coli cells of the strain DH5a [9].

In the current study, the induction of expression of maize pre-EF-Tu in E. coli was carried out according to Moriarty et al. [9]. Following induction, the recombinant protein was isolated and purified from the E. coli culture.

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primary antibody raised against the myc epitope, which is included near the C-terminus of recombinant protein [9]. Isolation and purification of recombinant pre-EF-Tu from E.coli

Mass spectrometry

E. coli cells expressing maize pre-EF-Tu were collected by centrifugation (50 mL cell culture; 10 000 g, 30 min), washed, and suspended in isolation buffer [20 mM Tris/ HCl pH 8.0 containing 20 mM NH4Cl, 10 mM MgCl2, 2 mM dithiothreitol, 0.1 mM EDTA, 10% (v/v) glycerol, 1 mM phenylmethanesulfonyl fluoride] according to Stanzel et al. [12]. Crude protein extract from harvested cells was then prepared by the lysozyme/EDTA method [13]. Cells were sonicated at a medium intensity setting, holding the suspension on ice. After sonication, insoluble debris was removed by centrifugation at 5000 g for 15 min. The supernatant (lysate) was then passed through a 0.8 lm syringe filter and stored at )70 (cid:1)C until further use.

Mass spectrometry analysis was performed according to Koc et al. [17]. Purified protein was separated on a 10% (w/v) polyacrylamide gel with SDS and stained by Coomassie Brilliant Blue R250. The stained protein band was then excised from the gel, and the protein spot was digested in-gel with Trypsin (Promega, Madison, WI) [18]. The peptides produced were injected into a Capillary LC (Waters Corporation, Milford, MA) to be desalted and separated using a C18 RP PepMap, 75 lm (internal diameter) column (LC Packings, Dionex, San Francisco, CA). The standard gradient used was as follows: 0–2 min, 3% B isocratic; 2–40 min, 3–80% B linear. Mobile phase A was water/ acetonitrile/formic acid (98.9 : 1 : 0.1, v/v/v), and phase B was acetonitrile/water/formic acid (99 : 0.9 : 0.1, v/v/v). The total solvent flow was 8 lLÆmin)1. Samples were analyzed under nano-ESI/MS and nano-ESI-MS/MS using a Q-TOF micro mass spectrometer (Micromass, Manchester, UK). The spectrum data were acquired by MASSLYNX 4.0 software (Micromass), and peptide matching and protein searches were performed automatically using the PROTEINLYNX 1.1 Global Server (Micromass). The peptide masses and sequence tags were searched against the NCBI nonredundant protein database.

[3H]GDP exchange assay

The activity of recombinant pre-EF-Tu was assessed by the [3H]GDP exchange assay [14]. Various amounts of purified protein were added to scintillation vials containing 40 lL of binding buffer (250 mM Tris/HCl, pH 7.4, 50 mM MgCl2, 250 mM NH4Cl, 25 mM dithiothreitol) and 4.5 nCi of [3H]GDP (specific activity 27.7 mCiÆmg)1 or 12.3 CiÆ mmol)1; total volume of reaction mixture, 200 lL). As controls, BSA and ovalbumin (Sigma) were used. The reaction mixtures were allowed to equilibrate for 10 min at 37 (cid:1)C then were diluted with 2 mL of wash buffer (10 mM Tris/HCl pH 7.4, 10 mM MgCl2, 10 mM NH4Cl), filtered using Millipore discs (pore size, 0.2 lm; diameter, 50 mm), and washed three times with 3 mL of the same buffer. The filters were dissolved in 5 mL of scintillation fluid, and the radioactivity was monitored using a Beckman LS 6500 scintillation counter.

Purification of recombinant pre-EF-Tu was carried out according to Stanzel et al. [12]. Recombinant protein was purified by SP-Sepharose, Q-Sepharose and gel filtration chromatography. SP-Sepharose was packed in a column (25 cm · 1 cm), equilibrated with eight column volumes of 20 mM acetate buffer (pH 4.8) consisting of 10 mM MgCl2, 2 mM dithiothreitol, 0.1 mM EDTA, 10% (v/v) glycerol, and 1 mM phenylmethanesulfonyl fluoride. The fractions from the SP-Sepharose column were analyzed by 1D SDS/ PAGE, and fractions having prominent bands between 45 kDa and 55 kDa were pooled [typically, pooled fractions had a total of four to five bands with molecular masses ranging from 20 to 100 kDa but the prominent bands (1–2) were between 45 and 55 kDa range] and dialyzed against isolation buffer overnight. After dialysis, the dialysate was applied to Q-Sepharose column (30 cm · 1 cm), followed by washing the column with the same buffer. The bound recombinant pre-EF-Tu was eluted with a linear gradient of 0.0–0.5 M NaCl in the isolation buffer. Fractions (2 mL) were collected and analyzed by 1D SDS/PAGE. The fractions containing purified pre-EF-Tu were pooled and concentrated using a centrifuge filter device Amicon )50 (Millipore, Bedford, MA). The concentrated protein was then applied to Sephacryl SS-100 (50 cm · 2.5 cm), eluted with the isolation buffer, and protein concentration was determined using the Bradford Assay (Bio-Rad, CA). The purity of recombinant pre-EF-Tu preparation was checked using 1D SDS/PAGE and Western blotting [5], the identity of the purified protein was verified using mass spectrometry, and the ability of the protein to bind GDP (indicative of EF-Tu activity [14]) was assessed using the [3H]GDP exchange assay [14,15]. In addition, the heat stability of purified pre-EF-Tu was also assessed as described below. Heat stability of recombinant pre-EF-Tu

One-dimensional SDS/PAGE and Western blotting

One-dimensional SDS/PAGE of purified recombinant pre- EF-Tu was carried out according to Laemmli [16]. In separate trials, 1D SDS/PAGE gels were stained using Coomassie Brilliant Blue R250 and Silver stain (Amersham). Western blot analysis was performed as described by Moriarty et al. [9]. The purified protein was resolved on 10% (w/v) polyacrylamide gel with SDS, and then trans- ferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA). The blot was probed for recombinant protein using the ECL chemiluminescent development method with The heat stability of recombinant pre-EF-Tu was assessed using two approaches. In the first approach, 1 mL samples of purified protein (0.5 lM) were incubated at 25 (cid:1)C (control) or 45 (cid:1)C (heated) for 45 min. After incubation, samples were centrifuged and the supernatant of each sample (control and heated) was analyzed for the presence of pre-EF-Tu using Western blotting and anti-myc Ig as described above. Equal volumes of protein samples were loaded in each gel lane. In the second approach, 1 mL aliquot of recombinant protein (0.5 lM) was incubated at 45 (cid:1)C for 45 min in covered quartz cuvettes, and the heat stability of EF-Tu was assessed by monitoring light

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and MDH was then measured at various times of recovery as described above.

Results

scattering at 320 nm during incubation [11,19]. As a control, a heat-labile protein, citrate synthase, [20] was used. In addition, the activity of recombinant pre-EF-Tu was also measured after heating (45 min at 45 (cid:1)C) by the [3H]GDP exchange assay [14] as outlined above.

One-dimensional SDS/PAGE, Western blot, and mass spectrometry analysis of purified recombinant pre-EF-Tu Chaperone assays

than that of

The recombinant pre-EF-Tu was tested for possible chap- erone activity using two approaches: (a) by monitoring thermal aggregation of heat-labile CS or MDH in the presence or absence of pre-EF-Tu, and (b) by measuring residual activity of CS or MDH after heating in the presence or absence of pre-EF-Tu. CS and MDH were chosen as model substrates because they are known to be relatively heat-labile and have been used in chaperone studies [20–23]. CS and MDH were obtained from Boehringer Mannheim. Both enzymes are homodimers, and in the text and figures the concentrations of CS and MDH refer to the 98 kDa homodimer and 70 kDa homodimer, respectively.

The recombinant maize pre-EF-Tu was purified to homo- geneity from E. coli expressing this protein [9]. A previous study has shown that the expressed maize pre-EF-Tu appears in E. coli in a highly soluble form [9]. Both silver stained (Fig. 1, lane 1) and Coomassie Blue stained (Fig. 1, lane 2) 1D SDS/PAGE gels showed a single band indicating purified protein. The molecular mass of the purified protein was approximately 50–51 kDa; this molecular mass was greater the native chloroplast EF-Tu (45–46 kDa) [5] because of the presence of a chloroplast targeting sequence at the N-terminal end and the c-myc and polyhistidine tags at the C-terminus of the polypeptide [9]. Western blot probed with anti-myc Ig, which is specific to recombinant pre-EF-Tu carrying the c-myc tag [9], also showed a single band with the molecular mass of 50–51 kDa (Fig. 1, lane 3). The identity of the purified protein was further confirmed by mass spectrometry, which showed that the purified protein was indeed chloroplast pre-EF-Tu (Fig. 2). The recombinant protein amino acid sequence obtained by mass spectrometry (Fig. 2B,C) matched the sequence of maize chloroplast EF-Tu [5] and chloroplast EF-Tu from Oryza sativa L., Glycine max (L) Merr, Pisum sativum L., and Nicotiana silvestris Speg. (NCBI nonredundant protein database; data not shown).

The thermal aggregation of CS and MDH was tested according to Lee et al. [21]. In separate trials, CS (0.15 lM) and MDH (0.3 lM) were mixed with various amounts of purified recombinant pre-EF-Tu (as indicated in Fig. 6) in 20 mM Tris/HCl buffer [7 mM MgCl2, 60 mM NH4Cl, 0.2 mM EDTA, and 10% (v/v) glycerol; pH 8; total volume 1 mL] in covered quartz cuvettes. Three controls were used: CS or MDH alone, CS or MDH mixed with BSA, and CS or MDH mixed with ovalbumin (molar concentrations are indicated in Fig. 6). Samples were incubated at indicated high temperatures (CS: 41 (cid:1)C or 45 (cid:1)C; MDH: 45 (cid:1)C) for 45 min, and CS or MDH stability was estimated by monitoring light scattering at 320 nm during incubation [21]. GDP binding activity of purified recombinant pre-EF-Tu

We assessed the activity of purified pre-EF-Tu using the [3H]GDP exchange assay [14]. The assay showed that the

Fig. 1. One-dimensional SDS/PAGE gels and Western blot of purified recombinant maize pre-EF-Tu. The recombinant protein was purified from E. coli expressing this protein. Lane 1, gel stained with silver stain; lane 2, gel stained with Coomassie Brilliant Blue R250; lane 3, Western blot probed with anti-myc Ig. Arrow indicates recombinant pre-EF-Tu ((cid:1) 50–51 kDa). Note: E. coli EF-Tu has a mass of 43 kDa [22], and the protein band of this mass is not seen in lanes 1 and 2. Hence, the purified protein seen in lanes 1 and 2 is most probably maize pre-EF-Tu.

The residual activity of CS and MDH was determined using the experimental design of Lee et al. [21]. In separate trials, CS (2 lM) and MDH (0.5 lM) were mixed with 4 lM and 2 lM of purified recombinant pre-EF-Tu, respectively. Aliquots (1 mL) of the mixtures [CS mixture: 0.2 mM acetyl-CoA, 0.5 mM oxaloacetic acid, 0.1 mM 5,5¢-dithio- bis(2-nitrobenzoic acid) in 100 mM Tris/HCl (pH 7.5); MDH mixture: 0.1 mM NADH, 0.1 mM oxaloacetic acid in 50 mM Tris/HCl (pH 7.5)] were then heated at various high temperatures (38 (cid:1)C, 41 (cid:1)C, 43 (cid:1)C, and 45 (cid:1)C) for 30 min. As controls, CS or MDH alone and CS or MDH mixed with BSA or ovalbumin were used (molar concen- trations for BSA and ovalbumin are indicated in Fig. 7). After heating, aliquots were quickly cooled to room temperature and then kept on ice for up to 75 min (75 min recovery). The residual activity of CS and MDH was measured at room temperature immediately after heating and at various times of recovery, according to Srere [24] and Banaszak & Bradshaw [25], respectively.

We also investigated the possible effect of recombinant pre-EF-Tu on reactivation of heat-inactivated CS and MDH. In separate trials, CS (2 lM) and MDH (0.5 lM) were incubated at 43 (cid:1)C for 30 min, without the presence of pre-EF-Tu. Immediately after incubation, pre-EF-Tu was added to the heated protein samples (molar concentrations for pre-EF-Tu are indicated in Fig. 7) and the reaction mixtures were allowed to recover for 45 min. The mixtures were kept on ice during recovery. The residual activity of CS

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A

B

C

Fig. 2. Mass spectrometry analysis of recom- binant maize pre-EF-Tu (EF-Tu) isolated and purified from E. coli expressing this protein. (A) Score, number of matches, molecular mass, and pI (isoelectric point) of purified protein identified by nano-ESI-MS/MS. The score was determined by the PROTEINLYNX 1.1 Global Server (Micromass) and is an indicator of search result quality. (B) Matching peptides and amino acid sequences of peptide ion spectra obtained from the trypsin digestion of purified maize pre-EF-Tu. (C) Matching sites of peptide products in the complete sequence of maize chloroplast EF-Tu protein. The peptide products [from (B)] are shown in red, blue, and green. The complete sequence was obtained from the database using PROTEIN- LYNX 1.1 Global Server.

Heat stability of recombinant maize pre-EF-Tu

purified pre-EF-Tu binds [3H]GDP (Fig. 3) suggesting that this protein was probably in a physiologically active form. As indicated by an increase in radioactivity (disintegration per minute), the binding of pre-EF-Tu with GDP increased with an increase in the concentration of recombinant protein (Fig. 3). No significant radioactivity, however, was detected when [3H]GDP was mixed with control proteins, BSA or ovalbumin (Fig. 3).

The heat stability of recombinant pre-EF-Tu was assessed as its ability to remain soluble and maintain activity at high temperature [20]. Light scattering experiments with heated aliquots of purified pre-EF-Tu showed that this protein was heat stable (remained soluble) at 45 (cid:1)C, as no increase in relative light scattering was observed when the protein was heated at this temperature (Fig. 4A). The control protein (CS), in contrast, showed no stability at 45 (cid:1)C, indicated by an increase in relative light scattering (Fig. 4A). The pre- EF-Tu also maintained its activity at high temperature (45 (cid:1)C). As indicated by the [3H]GDP exchange assay, the binding of heated pre-EF-Tu with GDP was similar to the binding of nonheated (25 (cid:1)C) pre-EF-Tu with GDP the supernatant of (Fig. 3). Western blot analysis of centrifuged heated samples of purified pre-EF-Tu corro- borated the results of light the scattering experiments. A Western blot revealed that the recombinant pre-EF-Tu was present in the soluble fraction (supernatant) at 45 (cid:1)C, indicating its stability at this temperature (Fig. 4B).

Recombinant maize pre-EF-Tu protected CS and MDH from thermal aggregation

Fig. 3. Binding of recombinant maize pre-EF-Tu (EF-Tu) to [3H]GDP. Purified pre-EF-Tu was incubated alone at 25 (cid:1)C or 45 (cid:1)C for 45 min. Following incubation, the activity of the protein was assessed by the [3H]GDP exchange assay [14]. Reaction mixture (total volume 200 lL) contained binding buffer, 4.5 nmol of [3H]GDP (12.3 CiÆmmol)1), and various amounts of EF-Tu. As controls, BSA and ovalbumin were used. Reaction mixtures were allowed to equilibrate at room tem- perature for 10 min. Radioactivity was monitored using a Beckman LS 6500 scintillation counter. Increase in radioactivity (DPM, disin- tegration per minute) indicates binding of pre-EF-Tu to [3H]GDP [14]. Binding of pre-EF-Tu to [3H]GDP suggests that this protein (pre-EF- Tu) is probably in a physiologically active form [14]. Similar results were obtained in a duplicate experiment.

The recombinant maize pre-EF-Tu protected CS from thermal aggregation. When heated at either 41 (cid:1)C or 45 (cid:1)C, CS began to form insoluble aggregates, indicated by an increase in relative light scattering (Fig. 5A,B). The CS aggregation, however, was reduced in the presence of various amounts of pre-EF-Tu and was almost completely suppressed at an pre-EF-Tu : CS molar ratio of 3.3 at 41 (cid:1)C (Fig. 5A) and 6.7 at 45 (cid:1)C (Fig. 5B). Ovalbumin (0.5 lM, Fig. 5A,B) and BSA (not shown) added to CS had no protective effect on CS aggregation.

Recombinant pre-EF-Tu also protected MDH from thermal aggregation. When heated at 45 (cid:1)C, MDH began to form insoluble aggregates, indicated by an increase in relative light scattering (Fig. 5C). Addition of various reduced MDH amounts of pre-EF-Tu, however,

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A

B

Fig. 4. Heat stability of purified recombinant maize pre-EF-Tu (EF-Tu). (A) Relative light scattering of purified pre-EF-Tu during incubation at 45 (cid:1)C. Aliquot of protein sample (1 mL) was incubated at 45 (cid:1)C for 45 min, and light scattering was monitored at 320 nm. As a control, heat-labile CS was used. Data represent averages of two independent experiments. Bars indicate standard errors. Note that during incuba- tion at 45 (cid:1)C there is no increase in relative light scattering indicating that the purified maize pre-EF-Tu is heat stable at this temperature. (B) Western blot of purified pre-EF-Tu. A sample of purified protein (1 mL; 0.5 lM) was incubated at 25 (cid:1)C (control) or 45 (cid:1)C for 45 min. Following incubation, samples were centrifuged and the supernatant of each sample was analyzed for the presence of pre-EF-Tu using Western blotting and anti-myc Ig. Equal volumes of protein samples were loaded in each lane. Note that the pre-EF-Tu protein band is present in the sample heated at 45 (cid:1)C, indicating pre-EF-Tu stability at this temperature.

aggregation and almost completely suppressed it at a pre- EF-Tu : MDH molar ratio of 10 (Fig. 5C). Ovalbumin (3 lM, Fig. 5C) and BSA (not shown), in contrast, did not protect MDH from thermal aggregation.

Fig. 5. Effect of recombinant maize pre-EF-Tu (EF-Tu) on thermal aggregation of citrate synthase (CS; A and B) and malate dehydrogenase (MDH; C). In separate trials, CS and MDH were mixed with various amounts of pre-EF-Tu. Three controls were used: CS or MDH alone, CS or MDH mixed with ovalbumin, and CS or MDH mixed with BSA (0.5 lM BSA was mixed with CS and 3 lM BSA was mixed with MDH; not shown). Mixtures (total volume of each mixture, 1 mL) were incubated at indicated temperature for 45 min. During incuba- tion, samples were monitored for their absorbance at 320 nm, which is indicative of light scattering due to CS or MDH aggregation [20,21]. Data represent averages of two independent experiments. Bars indicate standard errors. Note that pre-EF-Tu protected CS and MDH from thermal aggregation. Note: BSA and ovalbumin were chosen as con- trol proteins because they are known to be relatively heat stable and have been used in chaperone (protein aggregation) studies [40]. In addition, our preliminary light scattering experiments showed that BSA and ovalbumin are stable at 45 (cid:1)C, as no increase in light scattering (indicative of protein aggregation [20]) was detected when BSA or ovalbumin were heated at this temperature for 45 min (not shown).

Recombinant maize pre-EF-Tu protected CS and MDH from thermal inactivation

The recombinant maize pre-EF-Tu protected CS from thermal inactivation. The enzymatic activity of CS was severely halted when 2 lM CS was heated at 43 (cid:1)C alone or in the presence of either 4 lM BSA or 4 lM ovalbumin; less than 20% of the original CS activity remained after 30 min at 43 (cid:1)C (Fig. 6A). Upon temperature shift of the samples to room temperature, the CS activity did not change significantly, as less than 20% of the original CS activity remained after 75 min of recovery (Fig. 6A). In contrast, when 2 lM CS was heated at 43 (cid:1)C in the presence of 4 lM pre-EF-Tu, 46% of CS activity remained after 30 min (Fig. 6A). During the recovery period, the activity of CS gradually increased, reaching a maximum of 68% of its original activity after 45 min (Fig. 6A). Similar results on

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Fig. 7. Effect of recombinant maize pre-EF-Tu (EF-Tu) on the activity of heat-inactivated CS and MDH. CS and MDH were incubated at 43 (cid:1)C for 30 min. Following incubation, indicated amounts of pre-EF- Tu were added to the heated protein samples, and the reaction mix- tures (total volume of each mixture, 1 mL) were allowed to recover on ice for 45 min. The residual activity of CS and MDH was measured at room temperature at various times of recovery. Data represent aver- ages of two independent experiments. Bars indicate standard errors.

The recombinant pre-EF-Tu did not show an effect on reactivation of heat-inactivated CS and MDH (Fig. 7). When CS and MDH were heated at 43 (cid:1)C, without pre-EF- Tu, the activity of CS and MDH was severely reduced, and the addition of pre-EF-Tu after heating did not change their activity during the recovery (Fig. 7).

Discussion

Fig. 6. Effect of recombinant maize pre-EF-Tu (EF-Tu) on the activity of citrate synthase (CS; A) and malate dehydrogenase (MDH; B) after incubation at 43 (cid:1)C. In separate trials, CS and MDH were mixed with indicated amounts of recombinant pre-EF-Tu. Reaction mixtures (total volume of each mixture, 1 mL) were incubated at 43 (cid:1)C for 30 min. After incubation, the mixtures were quickly cooled to room temperature and then kept on ice for up to 75 min (75 min recovery). Where indicated, a mixture of CS and BSA or ovalbumin (ovalb.) was used as control. CS and MDH activity was measured at various times of recovery. Data represent averages of three independent experiments (standard errors are plotted but they are often smaller than the sym- bols). Note that in the presence of recombinant pre-EF-Tu, CS and MDH showed a relatively high activity immediately after exposure to 43 (cid:1)C (0 min of recovery). Similar results were obtained at 38 (cid:1)C, 41 (cid:1)C and 45 (cid:1)C (not shown).

Elevation of ambient temperature (heat shock or heat stress) affects cell metabolism, causing changes in the rates of biochemical reactions and injuries to cellular membranes [26,27]. Moreover, increases in ambient temperature also cause denaturation and aggregation of most proteins [27], but protein denaturation due to heat shock is reversible unless followed by aggregation [28].

Plants generally respond to high temperatures with the induction of heat shock proteins (HSPs). HSPs are thought to play a role in heat tolerance by acting as molecular chaperones; that is, they bind and stabilize other proteins, protecting them from thermal aggregation and inactivation (thermal damage) [29–31].

CS activity were obtained when this enzyme was heated at other high temperatures, 38 (cid:1)C, 41 (cid:1)C, and 45 (cid:1)C (not shown).

Recent studies have suggested that some protein synthesis elongation factors may be involved in heat tolerance by acting as molecular chaperones. Prokaryotic elongation factors, EF-G [23] and EF-Tu [22], for example, interact with unfolded and denatured proteins, as do molecular chaperones, and protect them from thermal aggregation. Also, E. coli EF-Tu interacts preferentially with hydropho- bic regions of substrate proteins [32], a strategy used by molecular chaperones to prevent thermal aggregation of their substrate proteins [30].

The recombinant pre-EF-Tu also protected MDH from thermal inactivation. When 0.5 lM MDH was heated at 43 (cid:1)C alone or in the presence of either 2 lM BSA or 2 lM ovalbumin, the MDH activity was very low, less than 1% of its original activity after 30 min (Fig. 6B). However, when MDH was heated in the presence of 2 lM pre-EF-Tu, 50% of MDH activity remained immediately after heating (Fig. 6B). During the recovery period, the activity of MDH did not change significantly (Fig. 6B). A similar effect of pre-EF-Tu on MDH activity was seen when MDH was incubated at 38 (cid:1)C, 41 (cid:1)C, and 45 (cid:1)C (not shown). Studies from our laboratory have implicated maize EF-Tu in heat tolerance [5,9–11]. Maize EF-Tu exhibits > 80% amino acid identity with bacterial EF-Tu [5], and it has been hypothesized that, in maize, this protein may show chaperone activity similar to prokaryotic EF-Tu [5,9–11].

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genotypes with higher levels of EF-Tu display greater heat stability (lower thermal aggregation) than chloroplast stromal proteins from genotypes with lower levels of EF-Tu. The above hypothesis is also corroborated by studies which showed that whole chloroplasts from a high- level EF-Tu maize line are more heat stable than whole chloroplasts from a low-level EF-Tu line [34,35] (in these previous studies, maize EF-Tu was referred to as a 45–46 kDa HSP because the identity of this protein was not known until the report of Bhadula et al. [5]).

In this study, we isolated and purified the recombinant precursor of maize EF-Tu from E. coli and tested it for possible chaperone activity. Before the chaperone studies were conducted, the recombinant pre-EF-Tu was tested for its purity, identity, ability to bind GDP (indicative of EF-Tu activity [14]), and thermal stability. The results showed that the recombinant pre-EF-Tu was isolated in highly pure form (Fig. 1) capable of binding GDP (Fig. 3), and that the identity of the protein, as determined by mass spectrometry, was indeed maize EF-Tu (Fig. 2). In addition, the heat stability tests showed that the recombinant pre-EF-Tu was stable at 45 (cid:1)C (Figs 3 and 4), the highest temperature used in our study to test this protein for possible chaperone activity. The thermal stability of maize pre-EF-Tu observed in our study was similar to the thermal stability of bacterial EF-Tu, which has been shown to be stable at temperatures ranging from 40 (cid:1)C to 45 (cid:1)C [22].

Importantly, the recombinant maize pre-EF-Tu dis- played chaperone activity. It protected the heat-labile proteins, CS and MDH, from thermal aggregation and inactivation. The protective role of pre-EF-Tu against thermal aggregation was exhibited in a concentration dependent manner with the most effective protection seen when the molar ratio of pre-EF-Tu : substrate protein (CS or MDH) was 6.7 for CS and 10 for MDH.

One could argue that the chaperone activity observed in our study may be an attribute of pre-EF-Tu, because of the presence of chloroplast targeting sequence, and that the native EF-Tu may not have chaperone properties. We do not completely rule out this possibility, however, the evidence supports the hypothesis that the native maize EF-Tu displays chaperone activity. Like native chloroplast EF-Tu [12], the recombinant pre-EF-Tu shows the ability to bind GDP (Fig. 3), an indication that the targeting sequence does not significantly affect the activity of this protein. Furthermore, as stated earlier, the amino acid sequence of native maize EF-Tu is highly similar to that of bacterial EF-Tu [5], which is known to display chaperone activity [22]. In addition, a comparison of the predicted two- dimensional (SCRATCH servers; http://www.igb.uci.edu/ tools/scratch/) and three-dimensional [36] structure reveals a striking similarity between the native maize EF-Tu and its precursor (pre-EF-Tu) implying that the functional proper- ties of the native EF-Tu and pre-EF-Tu are similar. The hypothesis that the native maize EF-Tu acts as a chaperone and protects chloroplast proteins from thermal aggregation is consistent with the lower thermal aggregation of chloro- plast stromal proteins in maize genotypes with higher levels of EF-Tu as outlined above.

The results on the influence of maize pre-EF-Tu on thermal aggregation of CS were similar to those reported for bacterial EF-Tu [22]. Bacterial EF-Tu was also found to protect CS from thermal aggregation in a concentration dependent manner [22]. When 0.8 lM CS was heated at 43 (cid:1)C it formed insoluble aggregates [22]. However, the addition of 2 lM bacterial EF-Tu partially reduced, and 5 lM EF-Tu completely suppressed, the thermal aggrega- tion of CS [22]. Thus, the most effective bacterial EF- Tu : CS molar ratio that suppressed CS aggregation was 6.25 [22], and this is similar to the maize pre-EF-Tu : CS molar ratio (6.7 at 45 (cid:1)C) observed in our study.

Plant cells possess many structurally diverse chaperones [30,37,38], some of which, the small heat shock proteins (sHSPs), function in conjunction with other chaperones [21,31]. A model has been proposed for the activity of sHSPs [31,39]. During high temperature stress, sHSPs bind substrate proteins in an ATP-independent manner, pre- venting their aggregation and maintaining them in a state competent for subsequent ATP-dependent refolding, which is facilitated by other chaperones (e.g. HSP70 system) [21,31,39]. This model is supported by Lee & Vierling [31] who demonstrated that the HSP70 system is required for refolding of a sHSP18.1-bound firefly luciferase.

Bacterial EF-Tu has been found to facilitate refolding of denatured proteins [22,33]. Kudlicki et al. [33] have shown that Thermus thermophilus EF-Tu has chaperone-like capa- city to assist in the refolding of denatured rhodanese. Also, Caldas et al. [22] have observed refolding of urea-denatured CS in the presence of E. coli EF-Tu. In contrast to bacterial EF-Tu, however, recombinant maize pre-EF-Tu does not seem to have an effect on renaturation of denatured proteins. Rather, this protein appears to be important in protecting proteins from thermal damage during exposure to heat stress. As seen in our study, maize pre-EF-Tu helped CS and MDH maintain a relatively high activity during heat stress (Fig. 6) but it had no effect on reactivation of these enzymes following their almost complete thermal inactiva- tion (Fig. 7).

Some plant sHSPs, however, can facilitate reactivation of heat-inactivated proteins during recovery from stress with- out the presence of other chaperones and ATP. Pea (Pisum sativum L) HSP17.7 and HSP18.1, for example, minimally protected CS activity at 38 (cid:1)C, but helped this enzyme regain 65–70% of its original activity after 60 min of recovery at 22 (cid:1)C [20]. The reactivation activity of HSP17.7 and HSP18.1, however, seemed to be limited to tempera- tures below 40 (cid:1)C, as these two sHSPs had no effect on CS reactivation following CS exposure to 45 (cid:1)C [20].

Recombinant maize pre-EF-Tu does not seem to com- pletely fit the model proposed for the function of sHSPs, and it differs from pea HSP17.7 and HSP18.1 in some aspects of its chaperone activity. Maize pre-EF-Tu appears to be effective in protecting heat-labile proteins from thermal damage without a requirement for the presence of The ability of recombinant maize pre-EF-Tu to protect model substrates (CS and MDH) from thermal damage provides evidence for the possible role of native EF-Tu in heat tolerance. Native chloroplast EF-Tu is predominantly localized in the chloroplast stroma [11], and it is highly possible that during heat stress this protein may protect chloroplast stromal proteins from thermal damage by acting as a molecular chaperone. This possibility is suppor- ted by Momcilovic & Ristic [11] and Ristic et al. [10] who found that chloroplast stromal proteins from maize

Chaperone activity of recombinant maize EF-Tu (Eur. J. Biochem. 271) 3691

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shock proteins in maize hybrids from different climates. J. Plant Physiol. 149, 424–432.

8. Ristic, Z., Yang, G., Martin, B. & Fullerton, S. (1998) Evidence of association between specific heat-shock protein(s) and the drought and heat tolerance phenotype in maize. J. Plant Physiol. 153, 497–505.

9. Moriarty, T., West, R., Small, G., Rao, D. & Ristic, Z. (2002) Heterologous expression of maize chloroplast protein synthesis elongation factor (EF-Tu) enhances Escherichia coli viability under heat stress. Plant Sci. 163, 1075–1082.

10. Ristic, Z., Wilson, K., Nelsen, C., Momcilovic, I., Kobayashi, S., Meeley, R., Muszynski, M. & Habben, J. (2004) A maize mutant with decreased capacity to accumulate chloroplast protein synth- esis elongation factor (EF-Tu) displays reduced tolerance to heat stress. Plant Sci. 167, doi:10.1016/JPLANTSCI.2004.07.016. 11. Momcilovic, I. & Ristic, Z. (2004) Localization and abundance of chloroplast protein synthesis elongation factor (EF-Tu) and heat stability of chloroplast stromal proteins in maize. Plant Sci. 166, 81–88.

12. Stanzel, M., Schon, A. & Sprinzl, M. (1994) Discrimination against misacylated tRNA by chloroplast elongation factor Tu. Eur. J. Biochem. 219, 435–439.

13. Cull, M. & McHenry, C.S. (1990) Preparation of extracts from

prokaryotes. Methods Enzymol. 182, 147–153.

14. Zhang, Y.X., Shi, Y., Zhou, M. & Petsko, G.A. (1994) Cloning, sequencing, and expression in Escherichia coli of the gene encoding a 45-kilodalton protein, elongation factor Tu, from Chlamydia trachomatis serovar F. J. Bacteriol. 176, 1184–1187.

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other chaperones and ATP. As our in vitro experiments showed, maize pre-EF-Tu not only protected CS and MDH from thermal aggregation (Fig. 5) but also helped CS and MDH maintain a relatively high activity immediately after exposure to heat stress at temperature above 40 (cid:1)C (Fig. 6). We do not know, however, if and how maize pre-EF-Tu and/or native EF-Tu may function as molecular chaperones in vivo. Our observation that recombinant maize pre-EF-Tu acts independently in vitro, without a requirement for other chaperones and ATP, does not rule out the possibility that in vivo this protein and/or its native form may function in cooperation with other chaperones. Further studies are needed to investigate this possibility.

In conclusion, in this study we demonstrate that, in vitro, the recombinant maize pre-EF-Tu acts as a molecular chaperone and protects heat-labile proteins, CS and MDH, from thermal aggregation and inactivation. To our know- ledge, this is the first observation of chaperone activity by a plant/eukaryotic precursor of the EF-Tu protein. Previous studies have shown that whole chloroplasts [34,35] and chloroplast stromal proteins [10,11] from maize with higher levels of EF-Tu display greater heat stability (lower thermal aggregation) than whole chloroplasts and chloroplast stromal proteins from maize with lower levels of EF-Tu. Combined, our current and previous studies [10,11,34,35] strongly support the hypothesis that maize EF-Tu plays a role in heat tolerance by acting as a molecular chaperone and protecting chloroplast stromal proteins from thermal damage.

Acknowledgements

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17. Koc, E.C., Burkhart, W., Blackburn, K., Moyer, M.B., Schlatzer, D.M., Moseley, A. & Spremulli, L.L. (2001) The large subunit of the mammalian mitochondrial ribosome: analysis of the comple- ment of ribosomal proteins present. J. Biol. Chem. 276, 43958– 43969.

We acknowledge financial support for this research from the United States Department of Agriculture grant (Agreement no. 99-35100-8559) to Z. Ristic. The authors are thankful to Drs Karen L. Koster and Gary D. Small, The University of South Dakota, Dr Thomas E. Elthon, The University of Nebraska – Lincoln, and Dr David P. Horwath, the U.S. Department of Agriculture Experimental Research Laboratory, Fargo, ND for critical reading of the manuscript.

18. Kinter, M. & Sherman, N.E. (2000) The preparation of protein digests for mass spectrometric sequencing experiments. In Protein Sequencing and Identification Using Tandem Mass Spectrometry (Desiderio, D.M. & Nibbering, N.M.M., eds), pp. 147–164. Wiley- Interscience, New York.

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