Amino acids Thr56 and Thr58 are not essential for elongation factor function in yeast ˚ Galyna Bartish1,2, Hossein Moradi1,2 and Odd Nygard1 School of Life Sciences, Sodertorns hogskola, Huddinge, Sweden ă ă ă Department of Cell Biology, Arrhenius Laboratories, Stockholm University, Sweden Keywords elongation factor 2; functional complementation; osmostress; phosphorylation; yeast Correspondence ˚ O Nygard, School of Life Sciences, Sodertorns hogskola, S-141 89 Huddinge, ă ă ă Sweden Fax: +46 8608 4510 Tel: +46 8608 4701 E-mail: odd.nygard@sh.se (Received 10 January 2007, revised 27 June 2007, accepted 17 August 2007) doi:10.1111/j.1742-4658.2007.06054.x Yeast elongation factor is an essential protein that contains two highly conserved threonine residues, T56 and T58, that could potentially be phosphorylated by the Rck2 kinase in response to environmental stress The importance of residues T56 and T58 for elongation factor function in yeast was studied using site directed mutagenesis and functional complementation Mutations T56D, T56G, T56K, T56N and T56V resulted in nonfunctional elongation factor whereas mutated factor carrying point mutations T56M, T56C, T56S, T58S and T58V was functional Expression of mutants T56C, T56S and T58S was associated with reduced growth rate The double mutants T56M ⁄ T58W and T56M ⁄ T58V were also functional but the latter mutant caused increased cell death and considerably reduced growth rate The results suggest that the physiological role of T56 and T58 as phosphorylation targets is of little importance in yeast under standard growth conditions Yeast cells expressing mutants T56C and T56S were less able to cope with environmental stress induced by increased growth temperatures Similarly, cells expressing mutants T56M and T56M ⁄ T58W were less capable of adapting to increased osmolarity whereas cells expressing mutant T58V behaved normally All mutants tested were retained their ability to bind to ribosomes in vivo However, mutants T56D, T56G and T56K were under-represented on the ribosome, suggesting that these nonfunctional forms of elongation factor were less capable of competing with wild-type elongation factor in ribosome binding The presence of nonfunctional but ribosome binding forms of elongation factor did not affect the growth rate of yeast cells also expressing wild-type elongation factor Protein synthesis is one of the most complicated and energy consuming cellular processes Approximately 150 different proteins are required to facilitate the various processes involved in the translation process [1] Elongation factor (eEF2) is one of the key participants in the protein synthesis elongation cycle eEF2 is a 95 kDa GTP-binding protein that binds to pretranslocation ribosomes [2] The role of the factor, and its eubacterial homologue, elongation factor G (EFG), is to promote GTP-dependent translocation of the ribosome along the mRNA under simultaneous transfer of peptidyl-tRNA and deacylated tRNA to the ribosomal P- and E-sites, respectively This process is presumed to involve conformational changes in the ribosome as well as in the factor itself [2–4] Yeast eEF2 is a protein of 842 amino acids [5] The protein is evolutionary conserved and the amino acid sequence is 66% identical and 85% homologous to the sequence of human eEF2 [5] eEF2 is an essential protein coded for by two genes, EFT1 and EFT2 [5] The cellular level of eEF2 is strictly regulated [6] and cell viability requires that at least one of the two genes is functional Abbreviations CaMPKIII, Ca2+ and calmodulin-dependent protein kinase III; eEF2, eukaryotic elongation factor 2; EFG, elongation factor G; MAP, mitogenactivated protein; SC, synthetic complete; 5-FOA, 5-fluoroortic acid FEBS Journal 274 (2007) 5285–5297 ª 2007 The Authors Journal compilation ª 2007 FEBS 5285 Role of Thr56 and Thr58 for eEF2 function in yeast G Bartish et al eEF2 is subjected to post-translational modifications The C-terminal part of the protein contains a histidine residue (H699 in yeast) that is converted to diphthamide, a unique amino acid only found in eEF2 [7] The N-terminal part of eEF2 contains two highly conserved threonine residues (T56 and T58 in yeast) that can be phosphorylated The primary phosphorylation target is T56 but phosphorylation at the second threonine has also been observed [8,9] Phosphorylation decreases the affinity of eEF2 for pretranslocation ribosomes, thereby preventing the factor from stimulating translocation [10–12] The observation that threonines T56 and T58 are highly conserved in eEF2 [5,13] has led to the suggestion that threonine phosphorylation may play a general role in regulating the activity of eEF2 in eukaryotes In mammals, an altered phosphorylation status of eEF2 has been connected to different physiological situations and severe diseases [14] Mammalian eEF2 is phosphorylated by a specific Ca2+ and calmodulindependent protein kinase (CaMPKIII) [15,16] The activity of the eEF2 kinase is regulated by the mitogen-activated protein (MAP) kinase and mTOR-signalling pathways [17] These signalling pathways activate the eEF2 kinase in response to mitogens and other stimuli that increase the cellular energy demand [18– 21] Unicellular eukaryotes such as yeast appear to lack CaMPKIII [22] However, yeast eEF2 can serve as substrate for mammalian CaMPKIII [23] Donovan and Bodley [23] noted that yeast eEF2 was phosphorylated in vivo by an endogenous kinase present in the yeast cells Furthermore, peptide mapping suggested that both phosphorylation by the endogenous and the mammalian kinases occurred at the same site in yeast eEF2 [23] The endogenous yeast kinase was identified by Teige et al [24] as the Rck2 kinase, a Ser ⁄ Thr protein kinase homologous to mammalian calmodulindependent kinases Like the mammalian eEF2 kinase, Rck2 activity is regulated via phosphorylation Activation of the Rck2 kinase is mediated by the MAP kinase Hog1 in response to osmostress [24], an environmental stress condition known to reduce the rate of protein synthesis in fission yeast [25] Site directed mutagenesis has frequently been used to analyse the function of specific amino acids in bacterial EFG [26–29] To date, there are only a few reports in which this technique has been used to acquire information on the importance of specific amino acids and amino acid motifs for eEF2 function [6,13,30,31] In the present study, we have used site directed mutagenesis to analyse the importance of threonines T56 and T58 for cell viability in yeast 5286 Results Yeast eEF2 has two putative phosphorylation sites, threonines T56 and T58 We have used site directed mutagenesis to analyse the role of these two amino acids for viability of yeast cells A total of 13 eEF2 mutants were created All except three contained single amino acid substitutions The constructs were inserted in the expression vector pCBG1202 (Table 1) under the control of the GAL1 promoter The expression plasmid contains a 3¢-located sequence coding for an inframe V5 epitope that could be used for immunodetection of the plasmid-encoded protein All constructs were sequenced to confirm the presence of the introduced mutations and to assure that the correct reading frame was maintained To ascertain that the cloned constructs were expressed, cells from the haploid yeast strain YOR133w were transformed with the expression vector pCBG1202 containing the various constructs YOR133w cells retain one of the two EFT genes normally coding for the essential protein eEF2 Viability of the cells was therefore independent of the functional properties of the plasmid-encoded eEF2 Control cells were transformed with the identical plasmid containing the sequence coding for V5-tagged wild-type eEF2 (GA2 cells Table 1) As eEF2 exert its function on the ribosome, functional complementation studies require that the tag attached to the C-terminus of the plasmid-encoded eEF2 not interfere with the ribosomal binding properties of the factor As shown in Fig 1A, the tagged wild-type protein was able to bind to ribosomes Thus, the C-terminal tag did not prevent ribosomal binding Furthermore, all mutant forms of eEF2 used in the present study were also capable of binding to the ribosome (Fig 1A) A closer examination of the total expression levels of the mutant forms of eEF2 suggests that all mutants were expressed to the same level as tagged wild-type eEF2 with two exceptions (Fig 1B) The detectable levels of the double mutant T56V ⁄ T58V and the single mutant T56D was 75% and 50% of the wild-type levels, respectively Because all constructs are identical, except for the introduced point mutations, transcription levels should be equal It is therefore possible that the lower intracellular levels of these mutant forms of eEF2 reflect increased degradation The expression levels of tagged wild-type eEF2 from plasmid pCBG1202 in GB2 cells (Table 1) was used as a reference for maximum expression levels and ribosomal binding of tagged eEF2 analysed in the absence of competing eEF2 coded for by the yeast genome As shown in FEBS Journal 274 (2007) 5285–5297 ª 2007 The Authors Journal compilation ª 2007 FEBS G Bartish et al Role of Thr56 and Thr58 for eEF2 function in yeast Table Strains and plasmids used in the present study Euroscarf (Frankfurt, Germany) Strains and plasmids Source YOR133w (Mat a; his3D1; leu2D0 met15D0; ura3D0; yor133w::kanMX4) YDR385w (Mat a; his3D1; leu2D0; lys2D0; ura3D0; ydr385w::kanMX4) GA1 (YOR133w; pYES2.1 ⁄ URA3 ⁄ EF2) GA2 (YOR133w; pCBG1202 ⁄ HIS3 ⁄ EF2) GB1 (YOR133w; ydr385wDLEU2; pYES2.1 ⁄ URA3 ⁄ EF2) GB2 (YOR133w; ydr385wDLEU2; pCBG1202 ⁄ HIS3 ⁄ EF2) T56C as GB1 but (pCBG1202 ⁄ HIS3 ⁄ EF2T56C) T56M as GB1 but (pCBG1202 ⁄ HIS3 ⁄ EF2T56M) T56S as GB1 but (pCBG1202 ⁄ HIS3 ⁄ EF2T56S) T58S as GB1 but (pCBG1202 ⁄ HIS3 ⁄ EF2T58S) T58V as GB1 but (pCBG1202 ⁄ HIS3 ⁄ EF2T58 V) T56M ⁄ T58V as GB1 but (pCBG1202 ⁄ HIS3 ⁄ EF2T56M ⁄ T58V) T56M ⁄ T58W as GB1 but (pCBG1202 ⁄ HIS3 ⁄ EF2T56V ⁄ T58W) One Shot TOP10 cells (F- mcrA D(mrr-hsdRMS-mcrBC) /80lacZDM15 DlacX74 recA1 araD139 D(araleu) 7697 galU galK rpsL (StrR) endA1 nupG) DB3.1(F– gyrA462 endA1 D(sr1-recA) mcrB mrr hsdS20(rB-, mB-) supE44 ara-14 galK2 lacY1 proA2 rpsL20(SmR) xyl-5 Dleu mtl1) pYES2.1 (PGAL1, l, GAL1, URA3); pYES3 ⁄ CT (PGAL1, l, GAL1, TRP1) pDONR221 pCBG1202 (PGAL1, l, GAL1, HIS3, RFC) Euroscarf Euroscarf This study This study This study This study This study This study This study This study This study This study This study Invitrogen A Invitrogen Invitrogen Invitrogen Invitrogen This study B Fig Galactose induced expression levels and ribosome association of plasmid-encoded mutant and wild-type eEF2 Plasmid pCBG1202 containing mutant forms of eEF2 was inserted into Yor133w cells GA2 and GB2 cells expressing tagged wild-type eEF2 from the same plasmid was used as control (Table 1) Expression of the plasmid-encoded eEF2 was induced by incubating the transformed cells at 30 °C in the presence of galactose The induced cells were harvested and an aliquot of the total cell lysate was withdrawn before isolation of ribosomes The presence of plasmid-encoded eEF2 on isolated ribosomes was analysed by SDS gel electrophoresis and immunoblotting (A) Total expression and ribosome association of plasmid-encoded eEF2 was analysed by immunoblotting using a dot-blot technique The dot blots were quantified using computer-assisted densitometry Fig 1B, the expression level was almost twice that observed in cells also expressing genomic eEF2 The amount of plasmid-encoded eEF2 bound to ribosomes was also approximately double that seen in GA2 (Fig 1B) Most of the mutant forms of eEF2 were able to bind as efficient to ribosomes as wild-type eEF2 FEBS Journal 274 (2007) 5285–5297 ª 2007 The Authors Journal compilation ª 2007 FEBS 5287 Role of Thr56 and Thr58 for eEF2 function in yeast G Bartish et al The exceptions were mutants T56D, T56G and T56K These mutant forms of eEF2 were under-represented on the ribosome even after compensation for variation in total cellular levels of plasmid-encoded factor, suggesting that the mutation may have interfered with the ribosome-binding properties of eEF2 The low expression level of the double mutant T56V ⁄ T58V was not manifested in lower levels of ribosome-bound eEF2 (Fig 1B) Instead, this mutant appears to bind well to ribosomes This is in agreement with the lack of effect on ribosome binding seen with the single mutants T56V and T58V The ability of the eEF2 mutants to functionally complement wild-type eEF2 was analysed by transforming GB1 cells (Table 1) with expression vector pCBG1202 coding for mutant forms of eEF2 The GB1 strain lacks both genomic genes normally coding for eEF2 These cells are viable due to the presence of an URA3-plasmid, pYES2.1, containing the gene coding for wild-type eEF2 (Table 1) The transformed GB1 cells were allowed to grow on the appropriate selective medium Colonies from each transformation were isolated and plated onto solid media containing 5-fluoroortic acid (5-FOA) for counter selection As shown in Fig 2, seven of the mutant eEF2 constructs were able to support cell viability One colony from each functional construct was further characterized by growth on selective media The original GB1 strain was only able to grow on plates containing histidine (supplementary Fig S1) whereas the colonies in which the pYES2.1 plasmid was replaced by the HIS3-plasmid pCBG1202 containing the gene coding for wild-type (GB2-cells) or mutant but functional forms of eEF2 were only able to grow in the presence of uracil (supplementary Fig S1) Sequencing of plasmid pCBG1202 confirmed that the surviving eEF2 constructs contained the amino acid substitutions originally inserted in the eEF2 sequence by PCR The results from the functional complementation assay show that the threonine at position 56 could be replaced by cystein, methionine and serine (Fig 2) Mutants containing asparagine, aspartic acid, glycine, lysine or valine were nonfunctional (Fig 2) Clones expressing eEF2 in which the adjacent threonine T58 was replaced by amino acids serine or valine were viable (Fig 2) One possibility was that threonine T56 could be replaced by an amino acid that could not serve as phosphate acceptor (i.e cystein or methionine) as long as the second putative phosphorylation site T58 was left intact To investigate this possibility, we constructed double mutants in which both threonines were replaced by amino acids that could not be phosphory5288 Fig Ability of mutant forms of eEF2 to functionally complement yeast cells lacking genomic copies of the eEF2 genes Yeast GB1 cells were transformed with plasmid pCBG1202 carrying wild-type (wt, positive control) or mutant forms of the eEF2 gene Cells transformed with empty plasmid pCBG1202 were used as negative control The transformed cells were grown in SC ⁄ Gal-Ura-Leu-His medium until the D600 nm reached approximately Aliquots (5 lL) of the cell cultures (undiluted and diluted : 5) were spotted onto SC ⁄ Gal-Ura-Leu-His plates (left panel) or onto SC ⁄ Gal-Leu-His plates containing 5-FOA (right panel) The plates were incubated for days at 30 °C lated As shown in Fig 2, eEF2 containing the double mutants T56M ⁄ T58V and T56M ⁄ T58W could replace wild-type eEF2 in yeast whereas the construct T56V ⁄ T58V was nonfunctional During the experiment, we noted that some of the clones expressing functionally active but mutant forms of eEF2 appeared to grow slower than yeast expressing wild-type eEF2 from an otherwise identical plasmid The data presented in Fig 3A,D show that the doubling time for yeast cells expressing mutant T56M ⁄ T58V was increased by approximately 75% compared to that of yeast cells expressing the tagged wild-type protein For mutants T56C, T56S and T58S, the doubling time was increased by 15–25% whereas yeast cells expressing the double mutant T56M ⁄ T58W grew slightly faster than the control cells at 30 °C (Fig 3D) Expression of the double mutant T56M ⁄ T58V resulted in a marked reduction in the number of viable cells whereas mutants T56C, T56S and T58S only caused a slight increase in cell death (Table 2) FEBS Journal 274 (2007) 5285–5297 ª 2007 The Authors Journal compilation ª 2007 FEBS G Bartish et al Role of Thr56 and Thr58 for eEF2 function in yeast A D B E C F Fig Growth rate of yeast cells expressing plasmid born functional and nonfunctional eEF2 mutants Growth rate of yeast cells (GB) expressing wild-type or mutant functional forms of eEF2 from plasmid pCBG1202 under normal growth conditions (A,D) and under mild osmostress (C,D) Growth rate of yeast cells (YOR133w) expressing wild-type or mutant nonfunctional forms of eEF2 from plasmid pCBG1202 (B,E) Overnight cultures were diluted to approximately D600 nm ¼ 0.2 with SC ⁄ Gal-His medium (B,E) or with SC ⁄ Gal-His-Leu medium without (A,F) or with 0.4 M NaCl (C) The cells were allowed to grow at 30 °C under vigorous shaking The attenuance of the yeast cultures was measured at 600 nm at the intervals indicated (A–C) and the growth rates calculated (D,E) Temperature-dependent growth of yeast cell expressing mutant but functional eEF2 (F) Cells from overnight cultures were used for serial dilution (1 : 10) in SC-His-Leu medium Aliquots (5 lL) were spotted onto solid SC-His-Leu growth medium The plates were incubated for days at the temperatures indicated A comparison of the growth rate at various temperatures using solid medium showed that all mutants except T58V grew slightly slower than the control at 25 °C (Fig 3F) Cells expressing the T56M ⁄ T58V mutant failed to grow at this low temperature At 37 °C, cells expressing mutants T56M, T58S, T58V FEBS Journal 274 (2007) 5285–5297 ª 2007 The Authors Journal compilation ª 2007 FEBS 5289 Role of Thr56 and Thr58 for eEF2 function in yeast G Bartish et al Table Determination of the fraction of viable cells expressing functional but mutant forms of eEF2 Doubling time calculated after compensation for variations in the percentage of viable cells The originally observed doubling time was taken from Fig 3D Strain Viable cells (%) Estimated doubling time (min) GB2 T56C T56M T56S T58S T58V T56M ⁄ T58V T56M ⁄ T58W 89 80 89 83 85 81 65 88 287 319 301 342 323 284 370 275 and T56M ⁄ T58W grew at nearly the same rate as cells expressing wild-type eEF2 whereas the growth rate of cells expressing mutants T56C, T56S and T56M ⁄ T58V was more severely affected at 37 °C than at 30 °C (Fig 3F) For mutants T56C and T56M ⁄ T58V, the reduced growth was partly accounted for by a marked increase in the proportion of nonviable cells (not shown) The nonfunctional eEF2 constructs were able to bind to the ribosome in the presence of wild-type eEF2 coded for by the remaining eEF2 gene in the yeast strain YOR133w (Fig 1) It was therefore possible that these mutants could interfere with the function of wild-type eEF2 thereby reducing the rate of protein synthesis and the growth rate of the transformed cells As shown in Fig 3B, the doubling rate was only marginally affected by the presence of a nonfunctional eEF2 Thus, there was no negative effect on the growth rate caused by the presence of the nonfunctional ribosome-binding forms of eEF2 Phosphorylation of eEF2 in yeast cells can be triggered by osmostress [24] To investigate how yeast cells expressing eEF2 lacking the putative phosphorylation targets T56 and ⁄ or T58 responded to osmostress, control cells and cells containing the mutants T56M, T58V and T56M ⁄ T58W were grown in the presence of 0.4 m NaCl As shown in Fig 4C,D, mild osmostress had a slight negative effect on the growth rate of GB2 cells A limited effect was also seen on the growth rate of cells expressing the mutant T58V, suggesting that the mutation had little or no effect on the response to increased osmolarity By contrast, yeast cells expressing mutants T56M and T56M ⁄ T58W responded by a reduction in the growth rate by approximately 35% and 45%, respectively (Fig 3C,D) The fraction of dead cells was increased to approximately the same extent in cells expressing mutant eEF2 compared to 5290 that in cells expressing plasmid-encoded wild-type eEF2 (not shown) Discussion The ability of eEF2 to promote translocation in mammals is regulated by phosphorylation at T56 and ⁄ or T58 [8–10,12,32,33] Phosphorylation is catalysed by an eEF2-specific Ca2+ and calmodulin-dependent kinase Threonines Thr56 and Thr58 are highly conserved in eEF2 from several different organisms (Fig 4) [5,13] Phosphorylation at the homologous threonines has therefore been assumed to play a general role in the regulation of the rate of elongation in eukaryotes Yeast cells contain a Ser ⁄ Thr protein kinase called Rck2 that shows homology to the mammalian CaM-dependent eEF2-kinase [24] The Rck2 kinase phosphorylates eEF2 in vitro The activity of the kinase is increased under environmental stress conditions such as increased osmolarity [24] The activation is associated with elevated intracellular levels of phospho-eEF2 and reduced protein synthesis [24,25] The actual phosphorylation site(s) on eEF2 have not been identified but previous studies suggest that the target amino acids are identical to those phosphorylated by mammalian CaMPKIII in vitro (i.e T56 and ⁄ or T58) [23] Functional complementation under standard growth conditions and under environmental stress Our analysis of the role of the two threonines for eEF2 function in yeast cells showed that the threonine at position 56 could be replaced with serine as well as with cystein and methionine Cells expressing mutants T56C and T56S grew markedly slower than cells expressing tagged wild-type eEF2 while mutant T56M had less effect on the growth rate The decreased growth observed with T56C and T56S was partly accounted for by a slight increase in cell mortality Amino acid substitutions were also allowed at position 58 Cells expressing mutants T58S and T58V grew slower than control cells expressing the tagged wildtype eEF2 and showed a slight decrease in the number of viable cells Thus, T58 could be replaced by valine whereas the T56V mutation resulted in a nonfunctional eEF2 Consequently, double mutant T56V ⁄ T58V was also nonfunctional The observation that the functional properties of double mutant T56M ⁄ T58V was severely impaired was surprising because both mutants had little effect on eEF2 function, when occurring as single mutants Expression of the mutant had negative FEBS Journal 274 (2007) 5285–5297 ª 2007 The Authors Journal compilation ª 2007 FEBS G Bartish et al Role of Thr56 and Thr58 for eEF2 function in yeast Fig Comparison of the amino acid context surrounding the putative phosphorylation site in eEF2 from various fungi, plants and metazoans The position of threonines T56 and T58 (yeast numbering) are indicated by arrows Amino acid sequences from (accession numbers in parenthesis) Saccharomyces cerevisiae (NP_014776), Saccharomyces castellii (AAO32487), Saccharomyces kluyveri (AAO32562), Glugea plecoglossi (BAA11470), Ashbya gossypii (AAS53513), Candida albicans (CAA70857), Schizosaccharomyces pombe (CAB58373), Neurospora crassa (AAK49353), Gibberella zeae (XP_389750), Aspergillus nidulans (XP_663934), Aspergillus fumigatus (XP_755686), Cryptococcus neoformans (AAW43242), Entamoeba histolytica (BAA04800), Trypanosoma cruzi (BAA09433), Dictyostelium discoideum (EAL63212), Cyanidioschyzon merolae (BAC67668), Guillardia theta (AAK39722), Parachlorella kessleri (P28996), Chlorella pyrcnoidosa (BAE48222), Beta vulgaris (CAB09900), Arabidopsis thaliana (AAF02837), Oryza sativa (NP_001052057), Blastocystis hominis (BAA11469), Cryptosporidium parvum (AAC46607), Plasmodium falciparum (BAA97565), Tetrahymena thermophila (AAN04122), Drosophila pseudoobscura (EAL32818), Drosophila melanogaster (P13060), Aedes aegypti (AAK01430), Spodoptera exigua (AAL83698), Caenorhabditis elegans (AAD03339), Rattus norvegicus (NP_058941), Mus musculus (NP_031933), Cricetulus griseus (AAB60497), Pongo pygmaeus (CAH90954), Homo sapiens (AAH06547), Gallus gallus (NP_990699), Xenopus laevis (AAH44327), Xenopus tropicalis (NP_001015785), Danio rerio (AAH45488), Monosiga brevicollis (AAK27414), Pichia pastoralis (AAO39212) The arrows indicate the position of T56 and T58 effects on the proportion of viable cells and on the doubling time Double mutant T56M ⁄ T58W was fully functional and the growth rate of cells expressing this variant of eEF2 was almost indistinguishable from that of cells expressing the control factor In this mutant, the sequence TDT was replaced by the homologous motif MDW found in EFG from Escherichia coli Interestingly none of the organisms in the alignment shown in Fig have tryptophan at the position corresponding to T58 in yeast The results from the mutation experiments suggest that threonines T56 and T58 are not essential for the viability of yeast cells under normal growth conditions Both threonines could be replaced by amino acids that cannot be phosphorylated by serine ⁄ threonine protein kinases such as the Rck2 kinase [24] Phosphorylation of T56 and ⁄ or T58 therefore appears not to play an essential role in regulating the rate of protein synthesis in yeast under standard growth conditions Under conditions of increased osmolarity, the yeast cells rapidly reduce protein production [25] In Saccharomyces cerevisiae, the HOG MAP kinase pathway is activated under condition of increased extra cellular osmolarity [34] Activation of Hog1 is essential for survival of yeast cells at high osmolarity Hog1 activates Rck2, which in turn phosphorylates eEF2 [24] Mild osmostress reduced the growth rate of yeast cells expressing plasmid-encoded wild-type eEF2 (Fig 4) By contrast to what might have been expected, replacement of the two threonies with amino acids that could not serve as phosphorylation targets did not prevent the osmostress-dependent reduction in growth rate Cells expressing the T58V mutant behaved similar to cells expressing wild-type eEF2 whereas the growth rate of cells expressing mutants T56M and T56M ⁄ T58W was even more reduced than that observed in the presence of wild-type eEF2 The effect on the growth rate observed with the double mutant was probably caused by the amino acid replacement at position 56 because the effect on the growth rate was similar to that observed with the single mutation T56M The data suggest that phosphorylation at position 56 and ⁄ or 58 is not critical for the cellular response to increased extra cellular osmolarity Stress induced by increasing the growth temperature (37 °C) resulted in reduced growth rates for cells expressing wild-type as well as mutant forms of eEF2 FEBS Journal 274 (2007) 5285–5297 ª 2007 The Authors Journal compilation ª 2007 FEBS 5291 Role of Thr56 and Thr58 for eEF2 function in yeast G Bartish et al However, cells expressing mutants T56C, T56S and T56M ⁄ T58V were clearly more affected than cells expressing the other functional mutants The increased growth temperature also influenced the number of viable cells For most mutants, the effect was similar to that seen in cells expressing plasmid-encoded wild-type eEF2 The exception was cells expressing the double mutant T56M ⁄ T58V These cells showed markedly increased cell death The observation that mutants T56M, T58V and T56M ⁄ T58W did not alter the ability of the yeast cells to respond to temperature stress suggests that the ability to phosphorylate eEF2 at T56 and ⁄ or T58 is not crucial for regulating translation in response to temperature stress One possibility is that environmental stress induces phosphorylation at an alternative site in eEF2 A recent large-scale characterization of nuclear phosphoproteins in HeLa cells showed the presence of eEF2 phosphorylated at Ser485 (yeast numbering) located in the so-called hinge region of the factor [35] The presence of serine-phosphorylated eEF2 has also been demonstrated in vitro upon activation of a yeast kinase homologous to the type II Ca2+ and calmodulindependent kinases [36] It has been speculated that the general role of eEF2 phosphorylation may not be a massive shut-down of protein synthesis but rather a mechanism to promote translation of specific mRNAs that have difficulties in competing with more translation efficient mRNAs by slowing down the elongation rate [19,37] The situation would be analogous with that observed on translation after administration of limited concentrations of cycloheximide [38–42] Our results cannot rule out the possibility that a limited reduction of the elongation rate through phosphorylation at T56 is necessary to promote translation of mRNAs needed under specific stress situations An alignment of the amino acid sequences from a variety of eukaryotic organisms showed that Thr56 often is replaced by methionine in fungal eEF2 whereas Thr58 is much more conserved (Fig 4) It should be noted that none of the listed eEF2 sequences have amino acids S or V in position 58 and none of the sequences have C or S in position 56 The latter could be explained by the slower growth rate of yeast cells expressing eEF2 carrying these mutations The slower growth of the T58S mutant could also be an evolutionary disadvantage and hence explain the lack of serine at position 58, even if the resulting protein is functional However, the absence of valine at position 58 is notable because replacement of T58 with valine had limited effect on eEF2 function as determined by the effect of the mutation on the growth rate 5292 under both normal growth conditions and conditions of increased environmental stress The T56M mutation had little if any effect on the growth rate of yeast cells under standard laboratory growth conditions However, yeast cells expressing the T56M mutant (or the double mutant T56M ⁄ T58W) have considerable difficulties in coping with environmental stress situations as demonstrated by the effect of increased osmolarity Thus, the better ability to adapt to environmental stress may have constituted a strong evolutionary pressure in favour of threonine at position 56 in eEF2 Properties of the eEF2 mutants eEF2 is a GTP-binding protein that interacts with pretranslocation ribosomes and promotes ribosomal translocation along the mRNA under GTP-hydrolysis [2] All mutant forms of eEF2 described here (i.e even the mutants that were unable to functionally complement wild-type eEF2) were able to bind to ribosomes in cells also expressing wild-type eEF2 from one of the remaining eEF2 coding genes Because binding of eEF2 to the ribosome is dependent on the preformation of a guanosine nucleotide-factor complex [43], the observation suggests that the mutant forms of the factor were also able to interact with guanosine nucleotides Three of the nonfunctional mutants, T56D, T56G and T56K, were under-represented on the ribosome even after adjusting for variations in the intracellular concentrations of plasmid-encoded eEF2, indicating that these mutations had a negative effect on the ability of the factor to bind to ribosomes The other nonfunctional mutants had approximately the same ability to bind to ribosomes as the plasmidencoded wild-type eEF2, suggesting that the mutations interfere with the ability of the factor to sustain elongation rather than with the ability to associate with ribosomes The function of eEF2 in translocation requires reciprocation between two conformational states associated with the phosphorylation status of the bound guanosine nucleotide [2] The two putative phosphorylation sites are located in the so-called switch I region (also known as the effector-domain) [44,45], a flexible region known to be involved in the dynamic properties of elongation factors [46] Due to its flexible nature, the peptide sequence containing threonines T56 and T58 is missing in the crystal structure of yeast eEF2 [3] It is therefore difficult to estimate the structural effects caused by the introduced point mutations The observed phenotypic effects of the analysed eEF2 mutants, an inability to functionally complement wild-type eEF2 and the FEBS Journal 274 (2007) 5285–5297 ª 2007 The Authors Journal compilation ª 2007 FEBS G Bartish et al reduced growth rates obtained after expression of functional eEF2 mutants may be related to a loss of the dynamic properties of the factor Such a loss could lead to a reduced ability of the factor to participate in the elongation cycle, resulting in a reduced growth rate It has previously been shown that the level of eEF2 in yeast cells is tightly regulated [6] The regulation involves a post-transcriptional mechanism that keeps the cellular level of eEF2 constant Thus, over-expression of mutated eEF2 in cells also expressing plasmid encoded wild-type eEF2 result in decreased levels of the wild-type protein as the proportion of mutated factor increases As a consequence, nonfunctional mutants of eEF2 (e.g point mutations at V488 and H699) cause a dominant negative phenotype when expressed in cells also expressing wild-type eEF2 [6] The nonfunctional eEF2 mutants described in the present study were capable of binding to the ribosome and could therefore have been expected to interfere with the function of wild-type eEF2 even if expression of these mutants would have no effect on the intracellular level of wild-type eEF2 However, the nonfunctional mutants did not interfere with the growth rate of yeast cells also expressing wild-type eEF2 Only the expression of mutant T56V had a slight effect on the growth rate Thus, no dominant negative effect of the nonfunctional mutants could be observed In the present study, the nonfunctional mutants were expressed in yeast cells retaining one of the two genes normally coding for eEF2 in wild-type cells It is possible that these cells have a sub-optimal content of wild-type eEF2 If this is the case, the presence of nonfunctional eEF2 in the ribosomal fraction without noticeable effects on the growth rate may reflect an increased population of ‘hungry’ pretranslocation ribosomes waiting to interact with a functional eEF2 Experimental procedures Chemicals BP clonase enzyme mix, LR clonase enzymes mix, Reading frame cassette C, DNA polymerase (Klenow fragment) and anti-V5-HRP serum were obtained from Invitrogen (Carlsbad, CA, USA) Restriction nucleases AatII, ClaI, BamHI and XhoI were obtained from Roche (Mannheim, Germany) Alkaline phosphatase, Ready-To-Go ligation kit, ECL western blotting detection kit was obtained from Amersham Pharmacia Biotech Inc (Uppsala, Sweden) 5-FOA was provided by Larodan Fine Chemicals (Malmo, Sweden) Ampicillin, kanamycin, chloramphenicol and synthetic dropout medium supplement lacking histidine, leucine, tryptophan and uracil were obtained from Sigma (St Louis, MO, USA) Role of Thr56 and Thr58 for eEF2 function in yeast Taq DNA polymerase, Pfu DNA polymerase, RNasine were obtained from SDS Promega (Madison, WI, USA) Yeast nitrogen base without amino acids and agar were from BD (Franklin Lakes, NJ, USA) Ammonium sulphate and amino acids were from Merck (Darmstadt, Germany) Strains, plasmids and primers The strains and plasmids used are listed in Table Plasmid pFA6a-HIS3MX6 was kindly provided by C Sjogren ă (Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden) All primers were synthesized by CyberGene AB (Huddinge, Sweden) The primers used are listed in supplementary Tables S1–S2 Growth media Escherichia coli cells were grown in LB containing the proper antibiotics Yeast strains were grown on synthetic complete medium, SC, containing 0.67% (weight by volume) bactoyeast nitrogen base without amino acids, 0.14% (w ⁄ v) yeast synthetic drop-out medium without histidine, leucine, tryptophan and uracil, 0.5% (w ⁄ v) ammonium sulphate) The medium was supplemented with uracil (20 lgỈmL)1) and the appropriate amino acids: histidine (20 lgỈmL)1) and leucine (60 lgỈmL)1) as indicated Galactose (2% weight by volume) was added as carbon source unless noted For counter selection, we used SC-Leu-His media supplemented with 5-FOA (1 gỈL)1) and uracil (50 lgỈmL)1) Solid growth media contained 2% (w ⁄ v) agar Construction of a conditional null strain For cloning of the yeast eEF2 gene, total yeast DNA was prepared form strain YDR385w as described by Hoffman and Winston [47] The gene for eEF2 was amplified by PCR using primers eEF2F and eEF2R (supplementary Table S1) The 2.5 kb PCR-product was introduced into the TOPO vector pYES2.1 and the resulting plasmid was transformed into strain YOR133w carrying only one of the two genomic alleles for eEF2 The transformed cells were plated onto SC-Ura and a positive colony (YOR133w; pYES2.1 ⁄ URA3 ⁄ eEF2) was selected This strain is referred to as GA1 (Table 1) For deletion of the remaining genomic copy of the eEF2 gene, the LEU2 gene was amplified from plasmid pAT3 using primers Leu2F and Leu2R (supplementary Table S1) These two primers contained 20 nucleotides that matched the 5¢- and 3¢-sequence of LEU2, and 40 nucleotides with a sequence identical to the 5¢- and 3¢-sequences flanking the genomic eEF-2 in strain YOR133w The purified PCR fragment was introduced into the GA1 cells and the genomic eEF2 coding sequence replaced by the LEU2 gene via homologous recombination The transformed cells were FEBS Journal 274 (2007) 5285–5297 ª 2007 The Authors Journal compilation ª 2007 FEBS 5293 Role of Thr56 and Thr58 for eEF2 function in yeast G Bartish et al plated on SC-Ura-Leu for selection of positive colonies After days at 30 °C, positive colonies were selected and the replacement of the genomic copy of eEF2 by LEU2 was confirmed by PCR and sequencing The resulting yeast strain is referred to as GB1 (Table 1) Construction of plasmid for counter selection Vector pYES3 ⁄ CT was digested with restriction enzymes AatII and ClaI to remove the TRP1 gene The digestion products were separated by agarose gel-electrophoresis and the vector without TRP1 was isolated The HIS3 gene was amplified from plasmid pFA6a-HIS3MX6 using primers HisF, HisR, HisFL and HisRL (supplementary Table S1) The latter primer set was used to create overhangs, which were complementary to the overhangs generated after AatII and ClaI digestion of the vector The PCR products obtained using the two sets of primers were pooled, heated to 95 °C for 10 and allowed to gradually anneal by stepwise lowering of the temperature The annealing mixture was ligated into the digested pYES3 ⁄ CT plasmid The ligated plasmid was transformed into TOP10 cells The presence of the HIS3 gene in the plasmid was confirmed by PCR analysis The new vector, pYES3 ⁄ CT ⁄ HIS3, was used for construction of a destination vector suitable for use in the Gateway technology cloning system [48–51] For this purpose, the vector was digested with restriction enzymes BamHI and XhoI and treated with the Klenow polymerase fragment followed by treatment with alkaline phosphatase Reading frame cassette C.1 was ligated with the digested vector and the resulting plasmid was transformed into DB3.1a E coli cells, which were plated onto LB plates containing chloramphenicol Positive colonies were selected and the presence of the reading frame cassette in the new destination plasmid referred to as pCBG1202 was confirmed by restriction analysis Site directed mutagenesis All mutants were generated using the mega-primer method described by Brons-Poulsen et al [52] Primer GateEF2F was used in combination with one of the reverse primers carrying the point mutation to produce a short PCR fragment (supplementary Table S2) This fragment was used as a mega-primer together with primer GateEF2R (supplementary Table S2) for amplifying the full-length gene The PCR products were inserted into the donor vector pDONR221 using BP clonase The presence of the mutation was confirmed by sequence analysis Mutant eEF2 genes were transferred to the vector pCBG1202 by recombination using LR clonase The destination vector was transformed into strain YOR133w for confirming gene expression, and into strain GB1 for functional analysis by plasmid shuffling A copy of the wild-type eEF2 gene obtained by PCR amplification using primers GateEF2F 5294 and GateEF2R was cloned into the pCBG1202 vector as described above This plasmid served as control Cell transformation Bacterial transformations were performed according to standard methods [53] Yeast cells were transformed using the lithium acetate method, as described by Soni et al [54] Detection of eEF-2 expression by immunoblotting Yeast strain YOR133w containing plasmid pCBG1202 with a wild-type or mutated eEF2 gene was grown overnight at 30 °C in mL of SC-His medium containing 2% (w ⁄ v) glucose The cells were collected by centrifugation, washed and resuspended in 30 mL of SC-His medium with galactose After induction during approximately 20 h at 30 °C, the cells were harvested, washed in 20 mm Hepes-KOH (pH 7.4), mm Mg(CH3COO)2, 100 mm KCl and mm dithiothreitol, and suspended in the same buffer containing mm PMSF and 4000 U RNasine The cell suspension was mixed with glass beads and the yeast cells lysed as described [55] The crushed cells were centrifuged for at 5000 g with a Haereus Biofuge (Berlin, Germany) An aliquot of the supernatants were withdrawn for analysis of the total level plasmid-encoded eEF2 The remaining supernatants were transferred to new tubes and centrifuged for another 15 at 15000 g The supernatants were used for preparation of ribosomes Deoxycholate and Triton X-100 were added at a final concentration of 1% (w ⁄ v) each The supernatants (1 mL), were layered onto mL sucrose cushions containing 0.75 m sucrose in 75 mm KCl, 20 mm Tris ⁄ HCl, pH 7.6, mm Mg(CH3COO)2 and 15 mm 2-mercaptoethanol The material was centrifuged in a TLA100.3 rotor (Beckman Instruments, Palo Alto, CA, USA) for 150 min, at 198 000 g and °C The ribosomal pellets were dissolved in 0.25 m sucrose, 25 mm KCl, 30 mm Hepes-KOH (pH 7.6), mm Mg(CH3COO)2 and mm dithiothreitol Dissolved ribosomes and the post-ribosomal supernatants were stored in aliquots at )80 °C until used For detection of total cellular levels of plasmid-encoded eEF2 crude cell lysates, 40 lg protein in lL, were spotted on nitrocellulose membranes For estimation of the ribosomal binding capacity of plasmid-encoded eEF2 isolated ribosomes, 40 lg ribosomes in lL, were spotted on nitrocellulose membranes The dried membranes were for immunoblotting The ribosome bound eEF2, 50 lg of ribosomes, was also analysed by SDS gel electrophoresis on 10% (w ⁄ v) polyacrylamid gels [56] The separated proteins were transferred to a nitrocellulose membrane, and the membrane was incubated with anti-V5-HRP serum Bound antibodies were detected using the ECL western blotting detection kit FEBS Journal 274 (2007) 5285–5297 ª 2007 The Authors Journal compilation ª 2007 FEBS G Bartish et al Role of Thr56 and Thr58 for eEF2 function in yeast Plasmid shuffling References Strain GB1 carrying the gene for wild-type eEF2 on a URA3-plasmid was transformed with plasmid pCBG1202 containing different mutants of eEF2 gene and plated onto solid SC-Ura-Leu-His medium The appearing colonies were isolated, incubated in the same medium, and plated onto two sets of SC plates: one containing 0.1% (w ⁄ v) 5-FOA [57] and 50 lg ml uracil and one lacking Ura, Leu and His The last one served as a control of presence of both plasmids in the cell After incubation for 4–5 days at 30 °C, colonies surviving the 5-FOA treatment were analysed by sequencing and by growing on the selective media, SC-Ura-Leu and SC-Leu-His GB1 cells transformed with empty pCBG1202 vector and the same vector containing wild-type eEF2 were used as negative and positive controls, respectively Rhoads RE (1999) Signal transduction pathways that regulate eukaryotic protein synthesis J Biol Chem 274, 30337–30340 ˚ Nygard O & Nilsson L (1990) Translational dynamics: interactions 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counting methods Biotechnol Tech 13, 787–795 60 Lu YM, Miyazawa K, Yamaguchi K, Nowaki K, Iwatsuki H, Wakamatsu Y, Ichikawa N & Hashimoto T (2001) Deletion of mitochondrial ATPase inhibitor in the yeast Saccharomyces cerevisiae decreased cellular and mitochondrial ATP levels under non-nutritional conditions and induced a respiration-deficient cell-type J Biochem (Tokyo) 130, 873–878 Supplementary material The following supplementary material is available online: Fig S1 Phenotypic analysis of strains after plasmid shuffling Table S1 Primers used for construction of strains and vectors Table S2 Primers used for site directed mutagenesis This material is available as part of the online article from http://www.blackwell-synergy.com Please note: Blackwell Publishing is not responsible for the content or functionality of any supplementary materials supplied by the authors Any queries (other than missing material) should be directed to the corresponding author for the article FEBS Journal 274 (2007) 5285–5297 ª 2007 The Authors Journal compilation ª 2007 FEBS 5297 ... compilation ª 20 07 FEBS 529 5 Role of Thr56 and Thr58 for eEF2 function in yeast 14 15 16 17 18 19 20 21 22 23 24 25 26 G Bartish et al protein that is resistant to diphtheria toxin J Biol Chem 26 8, 8665–8668... Journal 27 4 (20 07) 528 5– 529 7 ª 20 07 The Authors Journal compilation ª 20 07 FEBS G Bartish et al Role of Thr56 and Thr58 for eEF2 function in yeast Fig Comparison of the amino acid context surrounding... wild-type as well as mutant forms of eEF2 FEBS Journal 27 4 (20 07) 528 5– 529 7 ª 20 07 The Authors Journal compilation ª 20 07 FEBS 529 1 Role of Thr56 and Thr58 for eEF2 function in yeast G Bartish et al