Chaperone activity of recombinant maize chloroplast protein synthesis elongation factor, EF-Tu Damodara Rao1, Ivana Momcilovic1, Satoru Kobayashi1, Eduardo Callegari2and Zoran Ristic1 1
Trang 1Chaperone activity of recombinant maize chloroplast protein synthesis elongation factor, EF-Tu
Damodara Rao1, Ivana Momcilovic1, Satoru Kobayashi1, Eduardo Callegari2and Zoran Ristic1
1
Department of Biology, University of South Dakota and2Department of Basic Biomedical Sciences, University of South Dakota School of Medicine, Vermillion, SD, USA
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
activity) and was stable at 45C, 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
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]
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
identity of this protein was not known until the report of Bhadula et al [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 inEscherichia coli
E coliexpressing 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 colicells 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
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)
Trang 2Isolation and purification of recombinant pre-EF-Tu
fromE coli
E colicells 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 mMphenylmethanesulfonyl 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 C until further use
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 mMacetate buffer (pH 4.8) consisting of 10 mMMgCl2,
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
0.0–0.5M 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
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
primary antibody raised against the myc epitope, which is included near the C-terminus of recombinant protein [9] Mass spectrometry
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 byMASSLYNX4.0 software (Micromass), and peptide matching and protein searches were performed automatically using thePROTEINLYNX1.1 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 mMTris/HCl, pH 7.4, 50 mMMgCl2,
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
37C 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
Heat stability of recombinant pre-EF-Tu 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 25C (control) or 45C (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
45C for 45 min in covered quartz cuvettes, and the heat stability of EF-Tu was assessed by monitoring light
Trang 3scattering 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 45C) by the [3H]GDP
exchange assay [14] as outlined above
Chaperone assays
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 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 mMEDTA, 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: 41C or 45 C; MDH: 45 C) for
45 min, and CS or MDH stability was estimated by
monitoring light scattering at 320 nm during incubation
[21]
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 lMof 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 mMNADH, 0.1 mM oxaloacetic acid
in 50 mMTris/HCl (pH 7.5)] were then heated at various
high temperatures (38C, 41 C, 43 C, and 45 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 43C 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
and MDH was then measured at various times of recovery
as described above
Results
One-dimensional SDS/PAGE, Western blot, and mass spectrometry analysis of purified recombinant pre-EF-Tu 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 than that of 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) 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 ( 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.
Trang 4purified 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)
Heat stability of recombinant maize pre-EF-Tu 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 45C, 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 45C, indicated by
an increase in relative light scattering (Fig 4A) The pre-EF-Tu also maintained its activity at high temperature (45C) As indicated by the [3H]GDP exchange assay, the binding of heated pre-EF-Tu with GDP was similar to the binding of nonheated (25C) pre-EF-Tu with GDP (Fig 3) Western blot analysis of the supernatant 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 45C, indicating its stability at this temperature (Fig 4B)
Recombinant maize pre-EF-Tu protected CS and MDH from thermal aggregation
The recombinant maize pre-EF-Tu protected CS from thermal aggregation When heated at either 41C or 45 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
41C (Fig 5A) and 6.7 at 45 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 45C, MDH began
to form insoluble aggregates, indicated by an increase in relative light scattering (Fig 5C) Addition of various
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.
Fig 3 Binding of recombinant maize pre-EF-Tu (EF-Tu) to [3H]GDP.
Purified pre-EF-Tu was incubated alone at 25 C or 45 C for 45 min.
Following incubation, the activity of the protein was assessed by the
[ 3 H]GDP exchange assay [14] Reaction mixture (total volume 200 lL)
contained binding buffer, 4.5 nmol of [ 3 H]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 [ 3 H]GDP suggests that this protein
(pre-EF-Tu) is probably in a physiologically active form [14] Similar results
were obtained in a duplicate experiment.
Trang 5aggregation 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
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 lMCS was heated at 43C alone or
in the presence of either 4 lMBSA or 4 lMovalbumin; less
than 20% of the original CS activity remained after 30 min
at 43C (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 lMCS was heated at 43C 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
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 C Aliquot of protein sample (1 mL) was incubated at 45 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 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 l M ) was incubated at 25 C (control) or 45 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 C, indicating pre-EF-Tu stability at
this temperature.
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 l M BSA was mixed with CS and 3 l M 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 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).
Trang 6CS activity were obtained when this enzyme was heated at
other high temperatures, 38C, 41 C, and 45 C (not
shown)
The recombinant pre-EF-Tu also protected MDH from
thermal inactivation When 0.5 lM MDH was heated at
43C 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 38C, 41 C, and 45 C
(not shown)
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 43C, 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
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]
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]
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]
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 °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 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 C (0 min of recovery) Similar results were obtained at 38 C,
41 C and 45 C (not shown).
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 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.
Trang 7In 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 45C (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 40C to 45 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
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
43C 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 45C) observed in our study
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)
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
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])
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
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
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 sativumL) HSP17.7 and HSP18.1, for example, minimally protected CS activity at 38C, but helped this enzyme regain 65–70% of its original activity after 60 min of recovery at 22C [20] The reactivation activity of HSP17.7 and HSP18.1, however, seemed to be limited to tempera-tures below 40C, as these two sHSPs had no effect on CS reactivation following CS exposure to 45C [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
Trang 8other 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 40C (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 vivothis 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
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.
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