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Zoran Ristic - One of the best experts on this subject based on the ideXlab platform.
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Heat tolerance and expression of protein synthesis elongation factors, EF-Tu and EF-1α, in spring wheat.
Functional plant biology : FPB, 2009Co-Authors: Urska Bukovnik, P. V. Vara Prasad, Miranda Bennett, Zoran RisticAbstract:Protein elongation factors, EF-Tu and EF-1α, have been implicated in cell response to heat stress. We investigated the expression (accumulation) of EF-Tu and EF-1α in mature plants of spring wheat cultivars Kukri and Excalibur, and tested the hypothesis that cultivars with contrasting tolerance to heat stress differ in the accumulation of these elongation factors under prolonged exposure to high temperature (16 days at 36/30°C). In addition, we investigated the expression of EF-Tu and EF-1α in young plants experiencing a 24-h heat shock (43°C). Excalibur showed better tolerance to heat stress than Kukri. Heat stress induced accumulation of EF-Tu and EF-1α in mature plants of both cultivars, but to a greater extent in Excalibur. Young plants did not show appreciable accumulation of EF-Tu in response to heat shock. However, these plants showed increased accumulation of EF-1α and the accumulation appeared greater in Excalibur than in Kukri. The results support the hypothesis that EF-Tu plays a role in heat tolerance in spring wheat. The results also suggest that EF-1α may be of importance to wheat response to heat stress.
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Chloroplast protein synthesis elongation factor, EF-Tu, reduces thermal aggregation of rubisco activase.
Journal of plant physiology, 2007Co-Authors: Zoran Ristic, Ivana Momčilović, Eduardo Callegari, Benjamin P. DeridderAbstract:Chloroplast protein synthesis elongation factor, EF-Tu, has been implicated in heat tolerance in maize. The recombinant precursor of this protein, pre-EF-Tu, has been found to exhibit chaperone activity and protect heat-labile proteins, such as citrate synthase and malate dehydrogenase, from thermal aggregation. Chloroplast EF-Tu is highly conserved and it is possible that the chaperone activity of this protein is not species-specific. In this study, we investigated the effect of native wheat pre-EF-Tu on thermal aggregation of rubisco activase. Additionally, we investigated the effect of native and recombinant maize pre-EF-Tu on activase aggregation. Activase was chosen because it displays an exceptional sensitivity to thermal aggregation and constrains photosynthesis at high temperature. The native precursors of both wheat and maize EF-Tu displayed chaperone activity, as shown by the capacity of both proteins to reduce thermal aggregation of rubisco activase in vitro. Similarly, the recombinant maize pre-EF-Tu protected activase from thermal aggregation. This is the first report on chaperone activity of native pre-EF-Tu and the first evidence for thermal protection of a photosynthetic enzyme by this putative chaperone. The results are consistent with the hypothesis that chloroplast EF-Tu plays a functional role in heat tolerance by acting as a molecular chaperone.
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Heat-induced accumulation of chloroplast protein synthesis elongation factor, EF-Tu, in winter wheat
Journal of plant physiology, 2007Co-Authors: Zoran Ristic, Ivana Momčilović, Urska Bukovnik, P. V. Vara PrasadAbstract:Chloroplast protein synthesis elongation factor, EF-Tu, has been implicated in heat tolerance in maize (Zea mays). Chloroplast EF-Tu is highly conserved, and it is possible that this protein may be of importance to heat tolerance in other species including wheat (Triticum aestivum). In this study, we assessed heat tolerance and determined the relative levels of EF-Tu in mature plants (at flowering stage) of 12 cultivars of winter wheat experiencing a 16-d-long heat treatment (36/30 degrees C, day/night temperature). In addition, we also investigated the expression of EF-Tu in young plants experiencing a short-term heat shock (4h at 43 degrees C). Heat tolerance was assessed by examining the stability of thylakoid membranes, measuring chlorophyll content, and assessing plant growth traits (shoot dry mass, plant height, tiller number, and ear number). In mature plants, relative levels of EF-Tu were determined after 7 d of heat stress. High temperature-induced accumulation of EF-Tu in mature plants of all cultivars, and a group of cultivars that showed greater accumulation of EF-Tu displayed better tolerance to heat stress. Young plants of all cultivars but one did not show significant increases in the relative levels of EF-Tu. The results of the study suggest that EF-Tu protein may play a role in heat tolerance in winter wheat.
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Chaperone activity of recombinant maize chloroplast protein synthesis elongation factor, EF-Tu.
European journal of biochemistry, 2004Co-Authors: Damodara Rao, Ivana Momčilović, Eduardo Callegari, Satoru Kobayashi, Zoran RisticAbstract: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 tolerance, 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 inactivation. In this study we investigated the effect of the recombinant precursor of maize EF-Tu (pre-EF-Tu) on thermal aggregation and inactivation of the heat-labile proteins, citrate 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 45 °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.
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Localization and abundance of chloroplast protein synthesis elongation factor (EF-Tu) and heat stability of chloroplast stromal proteins in maize
Plant Science, 2004Co-Authors: Ivana Momčilović, Zoran RisticAbstract:Chloroplasts from a line of maize with high levels of chloroplast protein synthesis elongation factor (EF-Tu), ZPBL 1304, display greater heat stability than chloroplasts from a line with lower levels of EF-Tu, ZPL 389. We hypothesize that the greater heat stability of chloroplasts from ZPBL 1304 line may partly be attributed to EF-Tu, which may be protecting chloroplasts from heat injury. In this study, we investigated the subcellular distribution of EF-Tu and the heat stability of chloroplast stromal proteins in ZPBL 1304 and ZPL 389. Immunogold labeling and transmission electron microscopy (TEM) revealed that the chloroplast EF-Tu is localized mostly in the stroma and that under normal conditions agranal chloroplasts have a higher relative level of this protein than granal chloroplasts. Light scattering experiments with chloroplast stromal extracts showed that stromal proteins from the maize line with higher levels of EF-Tu, ZPBL 1304, display greater heat stability than stromal proteins from the line with lower levels of EF-Tu, ZPL 389. The results support the hypothesis that maize EF-Tu plays a role in the development of heat tolerance, possibly by acting as a molecular chaperone and protecting chloroplast stromal proteins from thermal aggregation.
Linda L. Spremulli - One of the best experts on this subject based on the ideXlab platform.
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Effects of mutagenesis of residue 221 on the properties of bacterial and mitochondrial elongation factor EF-Tu.
Biochimica et biophysica acta, 2004Co-Authors: Senyene Eyo Hunter, Linda L. SpremulliAbstract:During protein biosynthesis, elongation factor Tu (EF-Tu) delivers aminoacyl-tRNA (aa-tRNA) to the A-site of ribosomes. This factor is highly conserved throughout evolution. However, several key residues differ between bacterial and mammalian mitochondrial EF-Tu (EF-Tu(mt)). One such residue is Ser221 (Escherichia coli numbering). This residue is conserved as a Ser or Thr in the bacterial factors but is present as Pro269 in EF-Tu(mt). Pro269 reorients the loop containing this residue and shifts the adjoining beta-strand in EF-Tu(mt) compared to that of E. coli EF-Tu potentially altering the binding pocket for the acceptor stem of the aa-tRNA. Pro269 was mutated to a serine residue (P269S) in EF-Tu(mt). For comparison, the complementary mutation was created at Ser221 in E. coli EF-Tu (S221P). The E. coli EF-Tu S221P variant is poorly expressed in E. coli and the majority of the molecules fail to fold into an active conformation. In contrast, EF-Tu(mt) P269S is expressed to a high level in E. coli. When corrected for the percentage of active molecules, both variants function as effectively as their respective wild-type factors in ternary complex formation using E. coli Phe-tRNA(Phe) and Cys-tRNA(Cys). They are also active in A-site binding and in vitro translation assays with E. coli Phe-tRNA(Phe). In addition, both variants are as active as their respective wild-type factors in ternary complex formation, A-site binding and in vitro translation assays using mitochondrial Phe-tRNA(Phe).
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Effects of mutagenesis of residue 221 on the properties of bacterial and mitochondrial elongation factor EF-Tu.
Biochimica et Biophysica Acta, 2004Co-Authors: Senyene Eyo Hunter, Linda L. SpremulliAbstract:Abstract During protein biosynthesis, elongation factor Tu (EF-Tu) delivers aminoacyl-tRNA (aa-tRNA) to the A-site of ribosomes. This factor is highly conserved throughout evolution. However, several key residues differ between bacterial and mammalian mitochondrial EF-Tu (EF-Tu mt ). One such residue is Ser221 ( Escherichia coli numbering). This residue is conserved as a Ser or Thr in the bacterial factors but is present as Pro269 in EF-Tu mt . Pro269 reorients the loop containing this residue and shifts the adjoining β-strand in EF-Tu mt compared to that of E. coli EF-Tu potentially altering the binding pocket for the acceptor stem of the aa-tRNA. Pro269 was mutated to a serine residue (P269S) in EF-Tu mt . For comparison, the complementary mutation was created at Ser221 in E. coli EF-Tu (S221P). The E. coli EF-Tu S221P variant is poorly expressed in E. coli and the majority of the molecules fail to fold into an active conformation. In contrast, EF-Tu mt P269S is expressed to a high level in E. coli . When corrected for the percentage of active molecules, both variants function as effectively as their respective wild-type factors in ternary complex formation using E. coli Phe-tRNA Phe and Cys-tRNA Cys . They are also active in A-site binding and in vitro translation assays with E. coli Phe-tRNA Phe . In addition, both variants are as active as their respective wild-type factors in ternary complex formation, A-site binding and in vitro translation assays using mitochondrial Phe-tRNA Phe .
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Tomato EF-Tsmt, a functional mitochondrial translation elongation factor from higher plants
Plant Molecular Biology, 2003Co-Authors: Mohamed Benichou, Li Zhengguo, Barthélémy Tournier, Ana Chavez, Hicham Zegzouti, Alain Jauneau, Corinne Audran-delalande, Alain Latché, Mondher Bouzayen, Linda L. SpremulliAbstract:Ethylene-induced ripening in tomato (Lycopersicon esculentum) resulted in the accumulation of a transcript designated LeEF-Ts(mt) that encodes a protein with significant homology to bacterial Ts translational elongation factor (EF-Ts). Transient expression in tobacco and sunflower protoplasts of full-length and truncated LeEF-Ts(mt)-GFP fusion constructs and confocal microscopy observations clearly demonstrated the targeting of LeEF-Ts(mt) to mitochondria and not to chloroplasts and the requirement for a signal peptide for the proper sorting of the protein. Escherichia coli recombinant LeEF-Ts(mt) co-eluted from Ni-NTA resins with a protein corresponding to the molecular weight of the elongation factor EF-Tu of E. coli, indicating an interaction with bacterial EF-Tu. Increasing the GDP concentration in the extraction buffer reduced the amount of EF-Tu in the purified LeEF-Ts(mt) fraction. The purified LeEF-Ts(mt) stimulated the poly(U)-directed polymerization of phenylalanine 10-fold in the presence of EF-Tu. Furthermore, LeEF-Ts(mt) was capable of catalysing the nucleotide exchange reaction with E. coli EF-Tu. Altogether, these data demonstrate that LeEF-Ts(mt) encodes a functional mitochondrial EF-Ts. LeEF-Ts(mt) represents the first mitochondrial elongation factor to be isolated and functionally characterized in higher plants.
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High resolution crystal structure of bovine mitochondrial EF-Tu in complex with GDP.
Journal of molecular biology, 2000Co-Authors: Gregers R. Andersen, Linda L. Spremulli, Søren Thirup, Jens NyborgAbstract:The crystal structure of bovine mitochondrial elongation factor Tu (EF-Tu) in complex with GDP has been determined at a resolution of 1.94 A. The structure is similar to that of EF-Tu:GDP from Escherichia coli and Thermus aquaticus, but the orientation of the GDP-binding domain 1 is changed relative to domains 2 and 3. Sixteen conserved water molecules common to EF-Tu and other G-proteins in the GDP-binding site are described. These water molecules create a network linking separated parts of the binding pocket. Mitochondrial EF-Tu binds nucleotides less tightly than prokaryotic EF-Tu possibly due to an increased mobility in regions close to the GDP-binding site. The C-terminal extension of mitochondrial EF-Tu has structural similarities with DNA recognising zinc fingers suggesting that the extension may be involved in recognition of RNA.
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Interaction of mammalian mitochondrial elongation factor EF-Tu with guanine nucleotides
Protein science : a publication of the Protein Society, 2000Co-Authors: Ying Chun Cai, James M. Bullard, Nancy L. Thompson, Linda L. SpremulliAbstract:Elongation factor Tu (EF-Tu) promotes the binding of aminoacyl-tRNA (aa-tRNA) to the acceptor site of the ribosome. During the elongation cycle, EF-Tu interacts with guanine nucleotides, aa-tRNA and its nucleotide exchange factor (EF-Ts). Quantitative determination of the equilibrium dissociation constants that govern the interactions of mammalian mitochondrial EF-Tu (EF-Tu(mt)) with guanine nucleotides was the focus of the work reported here. Equilibrium dialysis with [3H]GDP was used to measure the equilibrium dissociation constant of the EF-Tu(mt) x GDP complex (K(GDP) = 1.0 +/- 0.1 microM). Competition of GTP with a fluorescent derivative of GDP (mantGDP) for binding to EF-Tu(mt) was used to measure the dissociation constant of the EF-Tu(mt) x GTP complex (K(GTP) = 18 +/- 9 microM). The analysis of these data required information on the dissociation constant of the EF-Tu(mt) x mantGDP complex (K(mGDP) = 2.0 +/- 0.5 microM), which was measured by equilibrium dialysis. Both K(GDP) and K(GTP) for EF-Tu(mt) are quite different (about two orders of magnitude higher) than the dissociation constants of the corresponding complexes formed by Escherichia coli EF-Tu. The forward and reverse rate constants for the association and dissociation of the EF-Tu(mt) x GDP complex were determined using the change in the fluorescence of mantGDP upon interaction with EF-Tu(mt). These values are in agreement with a simple equilibrium binding interaction between EF-Tu(mt) and GDP. The results obtained are discussed in terms of the recently described crystal structure of the EF-Tu(mt) x GDP complex.
Jens Nyborg - One of the best experts on this subject based on the ideXlab platform.
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Ternary Complex of EF-Tu and Its Action on the Ribosome
The Ribosome, 2014Co-Authors: Gregers R. Andersen, Victor G. Stepanov, Søren Thirup, Morten Kjeldgaard, Jens NyborgAbstract:This chapter describes the advances made in the structural studies of elongation factor EF-Tu during the last decade, and shows that the structural transition between the active, aa-tRNA binding form and the inactive form of EF-Tu is surprisingly large. Most of the various functional states of EF-Tu have been illustrated by structural results over the last few years. Very recently the authors had finished the refinement of the structure of bovine mitochondrial EF-Tu·GDP at a resolution of 1.94 A in a collaboration with Linda Spremulli, University of North Carolina. The modes by which EF-G and the ternary complex of EF-Tu interact with the ribosome have been elegantly demonstrated by cryo-electron microscopy (EM) reconstructions. The structure of the ternary complex on the ribosome is blocked with kirromycin. The mechanism of the GTPase reaction of EF-Tu has been exceedingly difficult to pin down. The nucleotide exchange mechanism of EF-Ts is not well understood in structural terms. We know the structure of the nucleotide-free complex of EF-Tu·EF-Ts, but we do not know the structure of the intact free form of EF-Ts, although the structure of a fragment of EF-Ts from Thermus thermophilus has been determined. The GTP hydrolysis reactions of the G proteins of translation are all highly stimulated during interaction with the ribosome.
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Efficient Separation of Thermus aquaticus EF-Tu Functional Complexes
Biochemical and biophysical research communications, 2001Co-Authors: Victor G. Stepanov, Jens NyborgAbstract:A new method for fast separation of the main functional complexes of the elongation factor Tu from Thermus aquaticus has been developed. Binary complexes EF-Tu ∗ GDP and EF-Tu ∗ GDPNP as well as the ternary complex EF-Tu ∗ GDPNP ∗ Leu ∼ tRNA were separated from each other by means of HPLC on a hydrophobic sorbent TSK-Gel Phenyl 5PW in a reverse gradient of ammonium sulfate. This technique is suitable for monitoring EF-Tu activity, characterisation of the ratio between different EF-Tu forms in cell extracts, and isolation of individual EF–Tu complexes for structural and functional investigations. In order to illustrate the potentials of the method, we used HPLC on a TSK-Gel Phenyl 5PW matrix to determine the ratio between affinities of GDP and GDPNP for EF-Tu. We found that Ka(GDP) is about 27 times higher than Ka(GDPNP) at 37°C, the value being close to the one reported for Thermus thermophilus EF-Tu.
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High resolution crystal structure of bovine mitochondrial EF-Tu in complex with GDP.
Journal of molecular biology, 2000Co-Authors: Gregers R. Andersen, Linda L. Spremulli, Søren Thirup, Jens NyborgAbstract:The crystal structure of bovine mitochondrial elongation factor Tu (EF-Tu) in complex with GDP has been determined at a resolution of 1.94 A. The structure is similar to that of EF-Tu:GDP from Escherichia coli and Thermus aquaticus, but the orientation of the GDP-binding domain 1 is changed relative to domains 2 and 3. Sixteen conserved water molecules common to EF-Tu and other G-proteins in the GDP-binding site are described. These water molecules create a network linking separated parts of the binding pocket. Mitochondrial EF-Tu binds nucleotides less tightly than prokaryotic EF-Tu possibly due to an increased mobility in regions close to the GDP-binding site. The C-terminal extension of mitochondrial EF-Tu has structural similarities with DNA recognising zinc fingers suggesting that the extension may be involved in recognition of RNA.
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Helix unwinding in the effector region of elongation factor EF-Tu-GDP.
Structure (London England : 1993), 1996Co-Authors: Galina Polekhina, Søren Thirup, Morten Kjeldgaard, Poul Nissen, Corinna Lippmann, Jens NyborgAbstract:Abstract Background Elongation factor Tu (EF-Tu) in its GTP conformation is a carrier of aminoacylated tRNAs (aa-tRNAs) to the ribosomal A site during protein biosynthesis. The ribosome triggers GTP hydrolysis, resulting in the dissociation of EF-Tu–GDP from the ribosome. The affinity of EF-Tu for other molecules involved in this process, some of which are unknown, is regulated by two regions (Switch I and Switch II) that have different conformations in the GTP and GDP forms. The structure of the GDP form of EF-Tu is known only as a trypsin-modified fragment, which lacks the Switch I, or effector, domain. The aim of this work was to establish the overall structure of intact EF-Tu–GDP, in particular the structure of the effector domain. Results The crystal structures of intact EF-Tu–GDP from Thermus aquaticus and Escherichia coli have been determined at resolutions of 2.7 a and 3.8 a, respectively. The structures confirm the domain orientation previously found in the structure of partially trypsin-digested EF-Tu–GDP. The structures of the effector region in T. aquaticus and E. coli EF-Tu–GDP are very similar. The C-terminal part of the effector region of EF-Tu–GDP is a β hairpin; in EF-Tu–GTP, this region forms an α helix. This conformational change is not a consequence of crystal packing. Conclusions EF-Tu undergoes major conformational changes upon GTP hydrolysis. Unlike other GTP-binding proteins, EF-Tu exhibits a dramatic conformational change in the effector region, involving an unwinding of a small helix and the formation of a β hairpin structure. This change is presumably involved in triggering the release of tRNA, and EF-Tu, from the ribosome.
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The ternary complex of aminoacylated tRNA and EF-Tu-GTP. Recognition of a bond and a fold
Biochimie, 1996Co-Authors: Poul Nissen, Morten Kjeldgaard, Søren Thirup, Brian F.c. Clark, Jens NyborgAbstract:Abstract The refined crystal structure of the ternary complex of yeast Phe-tRNA Phe , Thermus aquaticus elongation factor EF-Tu and the non-hydrolyzable GTP analog, GDPNP, revelas many details of the EF-Tu recognition of aminoacylated tRNA (aa-tRNA). EF-Tu-GTP recognizes the aminoacyl bond and one side of the backbone fold of the acceptor helix and has a high affinity for all ordinary elongator aa-tRNAs by binding to this aa-tRNA motif. Yet, the binding of deacylated tRNA, initiator tRNA, and selenocysteine-specific tRNA (tRNA Sec ) is effectively discriminated against. Subtle rearrangements of the binding pocket may occur to optimize the fit to any side chain of the aminoacyl group and interactions with EF-Tu stabilize the 3′-aminoacyl isomer of aa-tRNA. A general complementarity is observed in the location of the binding sites in tRNA for synthetases and for EF-Tu. The complex formation is highly specific for the GTP-bound conformation of EF-Tu, which can explain the effects of various mutants.
Gilbert Richarme - One of the best experts on this subject based on the ideXlab platform.
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Specificity of elongation factor EF-Tu for hydrophobic peptides.
Biochemical and biophysical research communications, 2002Co-Authors: Abdelharim Malki, Teresa Caldas, Andrea Parmeggiani, Masamichi Kohiyama, Gilbert RicharmeAbstract:The elongation factor EF-Tu carries aminoacyl-tRNAs to the A-site of the ribosome during the elongation process of protein biosynthesis. We, and others, have recently reported that the Escherichia coli EF-Tu interacts with unfolded and denatured proteins and behaves like a chaperone in protein folding and protection against protein thermal denaturation. In this study, we have identified EF-Tu binding sites in protein substrates by screening cellulose-bound peptides scanning the sequences of several proteins. The binding motifs recognized by EF-Tu in protein substrates are also recognized by the chaperone DnaK and mainly consist of hydrophobic clusters. EF-Tu interacts as efficiently as DnaK with the membrane spanning sequence of the membrane protein phospholemman and with the signal sequence of alkaline phosphatase. It interacts less efficiently with several other hydrophobic clusters of lysozyme and alkaline phosphatase, which are also DnaK substrates and fails to bind to several DnaK binding sites. Our results suggest that EF-Tu, like DnaK, interacts albeit more weakly with the hydrophobic regions of substrate protein and are consistent with the hypothesis that it possesses chaperone properties.
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Protein-disulfide isomerase activity of elongation factor EF-Tu.
Biochemical and biophysical research communications, 1998Co-Authors: Gilbert RicharmeAbstract:EF-Tu is involved in the binding and transport of the appropriate codon-specified aminoacyl-tRNA to the aminoacyl site of the ribosome. We and others have recently shown that the Escherichia coli EF-Tu, in additon to its acknowledged role in translation elongation, displays chaperone-like properties. We report here that EF-Tu, like thioredoxin, protein disulfide isomerase, and DsbA, catalyzes protein disulfide formation (oxidative renaturation of reduced RNase), reduction (reduction of insulin disulfides), and isomerization (refolding of randomly oxidized RNase). In contrast with most protein disulfide isomerases which possess vicinal cysteines and form an intramolecular disulfide upon oxidation, EF-Tu, which does not possess vicinal cysteines, forms intermolecular disulfides upon oxidation, resulting in the appearance of multimeric forms.
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Chaperone Properties of Bacterial Elongation Factor EF-Tu
The Journal of biological chemistry, 1998Co-Authors: Teresa Caldas, Abdelhamid El Yaagoubi, Gilbert RicharmeAbstract:Abstract Elongation factor Tu (EF-Tu) is involved in the binding and transport of the appropriate codon-specified aminoacyl-tRNA to the aminoacyl site of the ribosome. We report herewith that theEscherichia coli EF-Tu interacts with unfolded and denatured proteins as do molecular chaperones that are involved in protein folding and protein renaturation after stress. EF-Tu promotes the functional folding of citrate synthase and α-glucosidase after urea denaturation. It prevents the aggregation of citrate synthase under heat shock conditions, and it forms stable complexes with several unfolded proteins such as reduced carboxymethyl α-lactalbumin and unfolded bovine pancreatic trypsin inhibitor. The EF-Tu·GDP complex is much more active than EF-Tu·GTP in stimulating protein renaturation. These chaperone-like functions of EF-Tu occur at concentrations that are at least 20-fold lower than the cellular concentration of this factor. These results suggest that EF-Tu, in addition to its function in translation elongation, might be implicated in protein folding and protection from stress.
Ivana Momčilović - One of the best experts on this subject based on the ideXlab platform.
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Roles of Protein Synthesis Elongation Factor EF-Tu in Heat Tolerance in Plants
Journal of Botany, 2012Co-Authors: Ivana Momčilović, P. V. Vara PrasadAbstract:EF-Tu proteins of plastids, mitochondria, and the cytosolic counterpart EF-1α in plants, as well as EF-Tu proteins of bacteria, are highly conserved and multifunctional. The functions of EF-Tu include transporting the aminoacyl-tRNA complex to the A site of the ribosome during protein biosynthesis; chaperone activity in protecting other proteins from aggregation caused by environmental stresses, facilitating renaturation of proteins when conditions return to normal; displaying a protein disulfide isomerase activity; participating in the degradation of N-terminally blocked proteins by the proteasome; eliciting innate immunity and triggering resistance to pathogenic bacteria in plants; participating in transcription when an E. coli host is infected with phages. EF-Tu genes are upregulated by abiotic stresses in plants, and EF-Tu plays important role in stress responses. Expression of a plant EF-Tu gene confers heat tolerance in E. coli, maize knock-out EF-Tu null mutants are heat susceptible, and over-expression of an EF-Tu gene improves heat tolerance in crop plants. This review paper summarizes the current knowledge of EF-Tu proteins in stress responses in plants and progress on application of EF-Tu for developing crop varieties tolerant to abiotic stresses, such as high temperatures.
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Chloroplast protein synthesis elongation factor, EF-Tu, reduces thermal aggregation of rubisco activase.
Journal of plant physiology, 2007Co-Authors: Zoran Ristic, Ivana Momčilović, Eduardo Callegari, Benjamin P. DeridderAbstract:Chloroplast protein synthesis elongation factor, EF-Tu, has been implicated in heat tolerance in maize. The recombinant precursor of this protein, pre-EF-Tu, has been found to exhibit chaperone activity and protect heat-labile proteins, such as citrate synthase and malate dehydrogenase, from thermal aggregation. Chloroplast EF-Tu is highly conserved and it is possible that the chaperone activity of this protein is not species-specific. In this study, we investigated the effect of native wheat pre-EF-Tu on thermal aggregation of rubisco activase. Additionally, we investigated the effect of native and recombinant maize pre-EF-Tu on activase aggregation. Activase was chosen because it displays an exceptional sensitivity to thermal aggregation and constrains photosynthesis at high temperature. The native precursors of both wheat and maize EF-Tu displayed chaperone activity, as shown by the capacity of both proteins to reduce thermal aggregation of rubisco activase in vitro. Similarly, the recombinant maize pre-EF-Tu protected activase from thermal aggregation. This is the first report on chaperone activity of native pre-EF-Tu and the first evidence for thermal protection of a photosynthetic enzyme by this putative chaperone. The results are consistent with the hypothesis that chloroplast EF-Tu plays a functional role in heat tolerance by acting as a molecular chaperone.
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Heat-induced accumulation of chloroplast protein synthesis elongation factor, EF-Tu, in winter wheat
Journal of plant physiology, 2007Co-Authors: Zoran Ristic, Ivana Momčilović, Urska Bukovnik, P. V. Vara PrasadAbstract:Chloroplast protein synthesis elongation factor, EF-Tu, has been implicated in heat tolerance in maize (Zea mays). Chloroplast EF-Tu is highly conserved, and it is possible that this protein may be of importance to heat tolerance in other species including wheat (Triticum aestivum). In this study, we assessed heat tolerance and determined the relative levels of EF-Tu in mature plants (at flowering stage) of 12 cultivars of winter wheat experiencing a 16-d-long heat treatment (36/30 degrees C, day/night temperature). In addition, we also investigated the expression of EF-Tu in young plants experiencing a short-term heat shock (4h at 43 degrees C). Heat tolerance was assessed by examining the stability of thylakoid membranes, measuring chlorophyll content, and assessing plant growth traits (shoot dry mass, plant height, tiller number, and ear number). In mature plants, relative levels of EF-Tu were determined after 7 d of heat stress. High temperature-induced accumulation of EF-Tu in mature plants of all cultivars, and a group of cultivars that showed greater accumulation of EF-Tu displayed better tolerance to heat stress. Young plants of all cultivars but one did not show significant increases in the relative levels of EF-Tu. The results of the study suggest that EF-Tu protein may play a role in heat tolerance in winter wheat.
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Chaperone activity of recombinant maize chloroplast protein synthesis elongation factor, EF-Tu.
European journal of biochemistry, 2004Co-Authors: Damodara Rao, Ivana Momčilović, Eduardo Callegari, Satoru Kobayashi, Zoran RisticAbstract: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 tolerance, 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 inactivation. In this study we investigated the effect of the recombinant precursor of maize EF-Tu (pre-EF-Tu) on thermal aggregation and inactivation of the heat-labile proteins, citrate 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 45 °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.
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Localization and abundance of chloroplast protein synthesis elongation factor (EF-Tu) and heat stability of chloroplast stromal proteins in maize
Plant Science, 2004Co-Authors: Ivana Momčilović, Zoran RisticAbstract:Chloroplasts from a line of maize with high levels of chloroplast protein synthesis elongation factor (EF-Tu), ZPBL 1304, display greater heat stability than chloroplasts from a line with lower levels of EF-Tu, ZPL 389. We hypothesize that the greater heat stability of chloroplasts from ZPBL 1304 line may partly be attributed to EF-Tu, which may be protecting chloroplasts from heat injury. In this study, we investigated the subcellular distribution of EF-Tu and the heat stability of chloroplast stromal proteins in ZPBL 1304 and ZPL 389. Immunogold labeling and transmission electron microscopy (TEM) revealed that the chloroplast EF-Tu is localized mostly in the stroma and that under normal conditions agranal chloroplasts have a higher relative level of this protein than granal chloroplasts. Light scattering experiments with chloroplast stromal extracts showed that stromal proteins from the maize line with higher levels of EF-Tu, ZPBL 1304, display greater heat stability than stromal proteins from the line with lower levels of EF-Tu, ZPL 389. The results support the hypothesis that maize EF-Tu plays a role in the development of heat tolerance, possibly by acting as a molecular chaperone and protecting chloroplast stromal proteins from thermal aggregation.