Báo cáo khoa học: Amino acids Thr56 and Thr58 are not essential for elongation factor 2 function in yeast potx

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Amino acids Thr56 and Thr58 are not essential forelongation factor 2 function in yeastGalyna Bartish1,2, Hossein Moradi1,2and Odd Nyga˚rd11 School of Life Sciences, So¨derto¨rns ho¨gskola, Huddinge, Sweden2 Department of Cell Biology, Arrhenius Laboratories, Stockholm University, SwedenProtein synthesis is one of the most complicated andenergy consuming cellular processes. Approximately150 different proteins are required to facilitate the vari-ous processes involved in the translation process [1].Elongation factor 2 (eEF2) is one of the key partici-pants in the protein synthesis elongation cycle. eEF2 isa 95 kDa GTP-binding protein that binds to pretrans-location ribosomes [2]. The role of the factor, and itseubacterial homologue, elongation factor G (EFG), isto promote GTP-dependent translocation of the ribo-some along the mRNA under simultaneous transfer ofpeptidyl-tRNA and deacylated tRNA to the ribosomalP- and E-sites, respectively. This process is presumedto involve conformational changes in the ribosome aswell as in the factor itself [2–4].Yeast eEF2 is a protein of 842 amino acids [5]. Theprotein is evolutionary conserved and the amino acidsequence is 66% identical and 85% homologous to thesequence of human eEF2 [5]. eEF2 is an essential proteincoded for by two genes, EFT1 and EFT2 [5]. The cellularlevel of eEF2 is strictly regulated [6] and cell viabilityrequires that at least one of the two genes is functional.Keywordselongation factor 2; functionalcomplementation; osmostress;phosphorylation; yeastCorrespondenceO. Nyga˚rd, School of Life Sciences,So¨derto¨rns ho¨gskola, S-141 89 Huddinge,SwedenFax: +46 8608 4510Tel: +46 8608 4701E-mail: odd.nygard@sh.se(Received 10 January 2007, revised 27 June2007, accepted 17 August 2007)doi:10.1111/j.1742-4658.2007.06054.xYeast elongation factor 2 is an essential protein that contains two highlyconserved threonine residues, T56 and T58, that could potentially be phos-phorylated by the Rck2 kinase in response to environmental stress. Theimportance of residues T56 and T58 for elongation factor 2 function inyeast was studied using site directed mutagenesis and functional comple-mentation. Mutations T56D, T56G, T56K, T56N and T56V resulted innonfunctional elongation factor 2 whereas mutated factor carrying pointmutations T56M, T56C, T56S, T58S and T58V was functional. Expressionof mutants T56C, T56S and T58S was associated with reduced growth rate.The double mutants T56M ⁄ T58W and T56M ⁄ T58V were also functionalbut the latter mutant caused increased cell death and considerably reducedgrowth rate. The results suggest that the physiological role of T56 and T58as phosphorylation targets is of little importance in yeast under standardgrowth conditions. Yeast cells expressing mutants T56C and T56S were lessable to cope with environmental stress induced by increased growth tem-peratures. Similarly, cells expressing mutants T56M and T56M ⁄ T58W wereless capable of adapting to increased osmolarity whereas cells expressingmutant T58V behaved normally. All mutants tested were retained theirability to bind to ribosomes in vivo. However, mutants T56D, T56G andT56K were under-represented on the ribosome, suggesting that these non-functional forms of elongation factor 2 were less capable of competing withwild-type elongation factor 2 in ribosome binding. The presence of non-functional but ribosome binding forms of elongation factor 2 did not affectthe growth rate of yeast cells also expressing wild-type elongation factor 2.AbbreviationsCaMPKIII, Ca2+and calmodulin-dependent protein kinase III; eEF2, eukaryotic elongation factor 2; EFG, elongation factor G; MAP, mitogen-activated protein; SC, synthetic complete; 5-FOA, 5-fluoroortic acid.FEBS Journal 274 (2007) 5285–5297 ª 2007 The Authors Journal compilation ª 2007 FEBS 5285eEF2 is subjected to post-translational modifica-tions. The C-terminal part of the protein contains ahistidine residue (H699 in yeast) that is converted todiphthamide, a unique amino acid only found in eEF2[7]. The N-terminal part of eEF2 contains two highlyconserved threonine residues (T56 and T58 in yeast)that can be phosphorylated. The primary phosphoryla-tion target is T56 but phosphorylation at the secondthreonine has also been observed [8,9]. Phosphoryla-tion decreases the affinity of eEF2 for pretranslocationribosomes, thereby preventing the factor from stimu-lating translocation [10–12]. The observation thatthreonines T56 and T58 are highly conserved in eEF2[5,13] has led to the suggestion that threonine phos-phorylation may play a general role in regulating theactivity of eEF2 in eukaryotes.In mammals, an altered phosphorylation status ofeEF2 has been connected to different physiological sit-uations and severe diseases [14]. Mammalian eEF2 isphosphorylated by a specific Ca2+and calmodulin-dependent protein kinase (CaMPKIII) [15,16]. Theactivity of the eEF2 kinase is regulated by the mito-gen-activated protein (MAP) kinase and mTOR-signal-ling pathways [17]. These signalling pathways activatethe eEF2 kinase in response to mitogens and otherstimuli that increase the cellular energy demand [18–21].Unicellular eukaryotes such as yeast appear to lackCaMPKIII [22]. However, yeast eEF2 can serve assubstrate for mammalian CaMPKIII [23]. Donovanand Bodley [23] noted that yeast eEF2 was phosphory-lated in vivo by an endogenous kinase present in theyeast cells. Furthermore, peptide mapping suggestedthat both phosphorylation by the endogenous and themammalian kinases occurred at the same site in yeasteEF2 [23]. The endogenous yeast kinase was identifiedby Teige et al. [24] as the Rck2 kinase, a Ser ⁄ Thr pro-tein kinase homologous to mammalian calmodulin-dependent kinases. Like the mammalian eEF2 kinase,Rck2 activity is regulated via phosphorylation. Activa-tion of the Rck2 kinase is mediated by the MAPkinase Hog1 in response to osmostress [24], an envi-ronmental stress condition known to reduce the rate ofprotein synthesis in fission yeast [25].Site directed mutagenesis has frequently been usedto analyse the function of specific amino acids in bac-terial EFG [26–29]. To date, there are only a fewreports in which this technique has been used toacquire information on the importance of specificamino acids and amino acid motifs for eEF2 function[6,13,30,31]. In the present study, we have used sitedirected mutagenesis to analyse the importance ofthreonines T56 and T58 for cell viability in yeast.ResultsYeast eEF2 has two putative phosphorylation sites,threonines T56 and T58. We have used site directedmutagenesis to analyse the role of these two aminoacids for viability of yeast cells. A total of 13 eEF2mutants were created. All except three contained singleamino acid substitutions. The constructs were insertedin the expression vector pCBG1202 (Table 1) underthe control of the GAL1 promoter. The expressionplasmid contains a 3¢-located sequence coding for aninframe V5 epitope that could be used for immunode-tection of the plasmid-encoded protein. All constructswere sequenced to confirm the presence of the intro-duced mutations and to assure that the correct readingframe was maintained.To ascertain that the cloned constructs wereexpressed, cells from the haploid yeast strain YOR133wwere transformed with the expression vector pCBG1202containing the various constructs. YOR133w cellsretain one of the two EFT genes normally coding forthe essential protein eEF2. Viability of the cells wastherefore independent of the functional properties ofthe plasmid-encoded eEF2. Control cells were trans-formed with the identical plasmid containing thesequence coding for V5-tagged wild-type eEF2 (GA2cells Table 1).As eEF2 exert its function on the ribosome, func-tional complementation studies require that the tagattached to the C-terminus of the plasmid-encodedeEF2 do not interfere with the ribosomal bindingproperties of the factor. As shown in Fig. 1A, thetagged wild-type protein was able to bind to ribo-somes. Thus, the C-terminal tag did not prevent ribo-somal binding. Furthermore, all mutant forms of eEF2used in the present study were also capable of bindingto the ribosome (Fig. 1A).A closer examination of the total expression levelsof the mutant forms of eEF2 suggests that all mutantswere expressed to the same level as tagged wild-typeeEF2 with two exceptions (Fig. 1B). The detectablelevels of the double mutant T56V ⁄ T58V and the singlemutant T56D was 75% and 50% of the wild-type lev-els, respectively. Because all constructs are identical,except for the introduced point mutations, transcrip-tion levels should be equal. It is therefore possible thatthe lower intracellular levels of these mutant forms ofeEF2 reflect increased degradation. The expression lev-els of tagged wild-type eEF2 from plasmid pCBG1202in GB2 cells (Table 1) was used as a reference for max-imum expression levels and ribosomal binding oftagged eEF2 analysed in the absence of competingeEF2 coded for by the yeast genome. As shown inRole of Thr56 and Thr58 for eEF2 function in yeast G. Bartish et al.5286 FEBS Journal 274 (2007) 5285–5297 ª 2007 The Authors Journal compilation ª 2007 FEBSFig. 1B, the expression level was almost twice thatobserved in cells also expressing genomic eEF2. Theamount of plasmid-encoded eEF2 bound to ribosomeswas also approximately double that seen in GA2(Fig. 1B). Most of the mutant forms of eEF2 were ableto bind as efficient to ribosomes as wild-type eEF2.Table 1. Strains and plasmids used in the present study. Euroscarf (Frankfurt, Germany).Strains and plasmids SourceYOR133w (Mat a; his3D1; leu2D0 met15D0; ura3D0; yor133w::kanMX4) EuroscarfYDR385w (Mat a; his3D1; leu2D0; lys2D0; ura3D0; ydr385w::kanMX4) EuroscarfGA1 (YOR133w; pYES2.1 ⁄ URA3 ⁄ EF2) This studyGA2 (YOR133w; pCBG1202 ⁄ HIS3 ⁄ EF2) This studyGB1 (YOR133w; ydr385wDLEU2; pYES2.1 ⁄ URA3 ⁄ EF2) This studyGB2 (YOR133w; ydr385wDLEU2; pCBG1202 ⁄ HIS3 ⁄ EF2) This studyT56C as GB1 but (pCBG1202 ⁄ HIS3 ⁄ EF2T56C) This studyT56M as GB1 but (pCBG1202 ⁄ HIS3 ⁄ EF2T56M) This studyT56S as GB1 but (pCBG1202 ⁄ HIS3 ⁄ EF2T56S) This studyT58S as GB1 but (pCBG1202 ⁄ HIS3 ⁄ EF2T58S) This studyT58V as GB1 but (pCBG1202 ⁄ HIS3 ⁄ EF2T58 V) This studyT56M ⁄ T58V as GB1 but (pCBG1202 ⁄ HIS3 ⁄ EF2T56M ⁄ T58V) This studyT56M ⁄ T58W as GB1 but (pCBG1202 ⁄ HIS3 ⁄ EF2T56V ⁄ T58W) This studyOne Shot TOP10 cells (F- mcrA D(mrr-hsdRMS-mcrBC) /80lacZDM15 DlacX74recA1 araD139 D(araleu) 7697 galU galK rpsL (StrR) endA1 nupG)InvitrogenDB3.1(F–gyrA462 endA1 D(sr1-recA) mcrB mrr hsdS20(rB-, mB-) supE44 ara-14galK2 lacY1 proA2 rpsL20(SmR) xyl-5 Dleu mtl1)InvitrogenpYES2.1 (PGAL1,2l, GAL1, URA3); InvitrogenpYES3 ⁄ CT (PGAL1,2l, GAL1, TRP1) InvitrogenpDONR221 InvitrogenpCBG1202 (PGAL1,2l, GAL1, HIS3, RFC) This studyABFig. 1. Galactose induced expression levels and ribosome association of plasmid-encoded mutant and wild-type eEF2. Plasmid pCBG1202containing mutant forms of eEF2 was inserted into Yor133w cells. GA2 and GB2 cells expressing tagged wild-type eEF2 from the sameplasmid was used as control (Table 1). Expression of the plasmid-encoded eEF2 was induced by incubating the transformed cells at 30 °Cinthe presence of galactose. The induced cells were harvested and an aliquot of the total cell lysate was withdrawn before isolation of ribo-somes. 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 dotblots were quantified using computer-assisted densitometry.G. Bartish et al. Role of Thr56 and Thr58 for eEF2 function in yeastFEBS Journal 274 (2007) 5285–5297 ª 2007 The Authors Journal compilation ª 2007 FEBS 5287The exceptions were mutants T56D, T56G and T56K.These mutant forms of eEF2 were under-representedon the ribosome even after compensation for variationin total cellular levels of plasmid-encoded factor, sug-gesting that the mutation may have interfered with theribosome-binding properties of eEF2. The low expres-sion level of the double mutant T56V ⁄ T58V was notmanifested in lower levels of ribosome-bound eEF2(Fig. 1B). Instead, this mutant appears to bind well toribosomes. This is in agreement with the lack of effecton ribosome binding seen with the single mutantsT56V and T58V.The ability of the eEF2 mutants to functionallycomplement wild-type eEF2 was analysed by trans-forming GB1 cells (Table 1) with expression vectorpCBG1202 coding for mutant forms of eEF2. TheGB1 strain lacks both genomic genes normally codingfor eEF2. These cells are viable due to the presence ofan URA3-plasmid, pYES2.1, containing the gene cod-ing for wild-type eEF2 (Table 1). The transformedGB1 cells were allowed to grow on the appropriateselective medium. Colonies from each transformationwere isolated and plated onto solid media containing5-fluoroortic acid (5-FOA) for counter selection. Asshown in Fig. 2, seven of the mutant eEF2 constructswere able to support cell viability.One colony from each functional construct was fur-ther characterized by growth on selective media. Theoriginal GB1 strain was only able to grow on platescontaining histidine (supplementary Fig. S1) whereasthe colonies in which the pYES2.1 plasmid wasreplaced by the HIS3-plasmid pCBG1202 containingthe gene coding for wild-type (GB2-cells) or mutantbut functional forms of eEF2 were only able to growin the presence of uracil (supplementary Fig. S1).Sequencing of plasmid pCBG1202 confirmed that thesurviving eEF2 constructs contained the amino acidsubstitutions originally inserted in the eEF2 sequenceby PCR. The results from the functional complementa-tion assay show that the threonine at position 56 couldbe replaced by cystein, methionine and serine (Fig. 2).Mutants containing asparagine, aspartic acid, glycine,lysine or valine were nonfunctional (Fig. 2). Clonesexpressing eEF2 in which the adjacent threonine T58was replaced by amino acids serine or valine were via-ble (Fig. 2).One possibility was that threonine T56 could bereplaced by an amino acid that could not serve asphosphate acceptor (i.e. cystein or methionine) as longas the second putative phosphorylation site T58 wasleft intact. To investigate this possibility, we con-structed double mutants in which both threonines werereplaced by amino acids that could not be phosphory-lated. As shown in Fig. 2, eEF2 containing the doublemutants T56M ⁄ T58V and T56M ⁄ T58W could replacewild-type eEF2 in yeast whereas the constructT56V ⁄ T58V was nonfunctional.During the experiment, we noted that some of theclones expressing functionally active but mutant formsof eEF2 appeared to grow slower than yeast expressingwild-type eEF2 from an otherwise identical plasmid.The data presented in Fig. 3A,D show that thedoubling time for yeast cells expressing mutantT56M ⁄ T58V was increased by approximately 75%compared to that of yeast cells expressing the taggedwild-type protein. For mutants T56C, T56S and T58S,the doubling time was increased by 15–25% whereasyeast cells expressing the double mutant T56M ⁄ T58Wgrew slightly faster than the control cells at 30 °C(Fig. 3D). Expression of the double mutantT56M ⁄ T58V resulted in a marked reduction in thenumber of viable cells whereas mutants T56C, T56Sand T58S only caused a slight increase in cell death(Table 2).Fig. 2. Ability of mutant forms of eEF2 to functionally complementyeast cells lacking genomic copies of the eEF2 genes. Yeast GB1cells were transformed with plasmid pCBG1202 carrying wild-type(wt, positive control) or mutant forms of the eEF2 gene. Cellstransformed with empty plasmid pCBG1202 were used as negativecontrol. The transformed cells were grown in SC ⁄ Gal-Ura-Leu-Hismedium until the D600 nmreached approximately 1. Aliquots (5 lL)of the cell cultures (undiluted and diluted 1 : 5) were spotted ontoSC ⁄ Gal-Ura-Leu-His plates (left panel) or onto SC ⁄ Gal-Leu-Hisplates containing 5-FOA (right panel). The plates were incubated for4 days at 30 °C.Role of Thr56 and Thr58 for eEF2 function in yeast G. Bartish et al.5288 FEBS Journal 274 (2007) 5285–5297 ª 2007 The Authors Journal compilation ª 2007 FEBSA comparison of the growth rate at various temper-atures using solid medium showed that all mutantsexcept T58V grew slightly slower than the control at25 °C (Fig. 3F). Cells expressing the T56M ⁄ T58Vmutant failed to grow at this low temperature. At37 °C, cells expressing mutants T56M, T58S, T58VADBECFFig. 3. 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 os-mostress (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 nm¼ 0.2 with SC ⁄ Gal-His medium (B,E) or with SC ⁄ Gal-His-Leu medium with-out (A,F) or with 0.4M NaCl (C). The cells were allowed to grow at 30 °C under vigorous shaking. The attenuance of the yeast cultures wasmeasured at 600 nm at the intervals indicated (A–C) and the growth rates calculated (D,E). Temperature-dependent growth of yeast cellexpressing 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 3 days at the temperatures indicated.G. Bartish et al. Role of Thr56 and Thr58 for eEF2 function in yeastFEBS Journal 274 (2007) 5285–5297 ª 2007 The Authors Journal compilation ª 2007 FEBS 5289and T56M ⁄ T58W grew at nearly the same rate as cellsexpressing wild-type eEF2 whereas the growth rate ofcells expressing mutants T56C, T56S and T56M ⁄ T58Vwas more severely affected at 37 °C than at 30 °C(Fig. 3F). For mutants T56C and T56M ⁄ T58V, thereduced growth was partly accounted for by a markedincrease in the proportion of nonviable cells (notshown).The nonfunctional eEF2 constructs were able tobind to the ribosome in the presence of wild-type eEF2coded for by the remaining eEF2 gene in the yeaststrain YOR133w (Fig. 1). It was therefore possiblethat these mutants could interfere with the function ofwild-type eEF2 thereby reducing the rate of proteinsynthesis and the growth rate of the transformed cells.As shown in Fig. 3B, the doubling rate was only mar-ginally affected by the presence of a nonfunctionaleEF2. Thus, there was no negative effect on thegrowth rate caused by the presence of the nonfunc-tional ribosome-binding forms of eEF2.Phosphorylation of eEF2 in yeast cells can be trig-gered by osmostress [24]. To investigate how yeast cellsexpressing eEF2 lacking the putative phosphorylationtargets T56 and ⁄ or T58 responded to osmostress, con-trol cells and cells containing the mutants T56M,T58V and T56M ⁄ T58W were grown in the presence of0.4 m NaCl. As shown in Fig. 4C,D, mild osmostresshad a slight negative effect on the growth rate of GB2cells. A limited effect was also seen on the growth rateof cells expressing the mutant T58V, suggesting thatthe mutation had little or no effect on the response toincreased osmolarity. By contrast, yeast cells express-ing mutants T56M and T56M ⁄ T58W responded by areduction in the growth rate by approximately 35%and 45%, respectively (Fig. 3C,D). The fraction ofdead cells was increased to approximately the sameextent in cells expressing mutant eEF2 compared tothat in cells expressing plasmid-encoded wild-typeeEF2 (not shown).DiscussionThe ability of eEF2 to promote translocation in mam-mals is regulated by phosphorylation at T56 and ⁄ orT58 [8–10,12,32,33]. Phosphorylation is catalysedby an eEF2-specific Ca2+and calmodulin-dependentkinase. Threonines Thr56 and Thr58 are highly con-served in eEF2 from several different organisms(Fig. 4) [5,13]. Phosphorylation at the homologousthreonines has therefore been assumed to play a gen-eral role in the regulation of the rate of elongation ineukaryotes. Yeast cells contain a Ser ⁄ Thr proteinkinase called Rck2 that shows homology to the mam-malian CaM-dependent eEF2-kinase [24]. The Rck2kinase phosphorylates eEF2 in vitro. The activity ofthe kinase is increased under environmental stress con-ditions such as increased osmolarity [24]. The activa-tion is associated with elevated intracellular levels ofphospho-eEF2 and reduced protein synthesis [24,25].The actual phosphorylation site(s) on eEF2 have notbeen identified but previous studies suggest that thetarget amino acids are identical to those phosphory-lated by mammalian CaMPKIII in vitro (i.e. T56and ⁄ or T58) [23].Functional complementation under standardgrowth conditions and under environmentalstressOur analysis of the role of the two threonines foreEF2 function in yeast cells showed that the threonineat position 56 could be replaced with serine as well aswith cystein and methionine. Cells expressing mutantsT56C and T56S grew markedly slower than cellsexpressing tagged wild-type eEF2 while mutant T56Mhad less effect on the growth rate. The decreasedgrowth observed with T56C and T56S was partlyaccounted for by a slight increase in cell mortality.Amino acid substitutions were also allowed at posi-tion 58. Cells expressing mutants T58S and T58V grewslower than control cells expressing the tagged wild-type eEF2 and showed a slight decrease in the numberof viable cells. Thus, T58 could be replaced by valinewhereas the T56V mutation resulted in a nonfunctionaleEF2. Consequently, double mutant T56V ⁄ T58V wasalso nonfunctional. The observation that the func-tional properties of double mutant T56M ⁄ T58V wasseverely impaired was surprising because both mutantshad little effect on eEF2 function, when occurring assingle mutants. Expression of the mutant had negativeTable 2. Determination of the fraction of viable cells expressingfunctional but mutant forms of eEF2. Doubling time calculated aftercompensation for variations in the percentage of viable cells. Theoriginally observed doubling time was taken from Fig. 3D.Strain Viable cells (%)Estimated doublingtime (min)GB2 89 287T56C 80 319T56M 89 301T56S 83 342T58S 85 323T58V 81 284T56M ⁄ T58V 65 370T56M ⁄ T58W 88 275Role of Thr56 and Thr58 for eEF2 function in yeast G. Bartish et al.5290 FEBS Journal 274 (2007) 5285–5297 ª 2007 The Authors Journal compilation ª 2007 FEBSeffects on the proportion of viable cells and on thedoubling time. Double mutant T56M ⁄ T58W was fullyfunctional and the growth rate of cells expressing thisvariant of eEF2 was almost indistinguishable from thatof cells expressing the control factor. In this mutant,the sequence TDT was replaced by the homologousmotif MDW found in EFG from Escherichia coli.Interestingly none of the organisms in the alignmentshown in Fig. 5 have tryptophan at the position corre-sponding to T58 in yeast. The results from the muta-tion experiments suggest that threonines T56 and T58are not essential for the viability of yeast cells undernormal growth conditions. Both threonines could bereplaced by amino acids that cannot be phosphory-lated by serine ⁄ threonine protein kinases such as theRck2 kinase [24]. Phosphorylation of T56 and ⁄ or T58therefore appears not to play an essential role in regu-lating the rate of protein synthesis in yeast under stan-dard growth conditions.Under conditions of increased osmolarity, the yeastcells rapidly reduce protein production [25]. In Saccha-romyces cerevisiae, the HOG MAP kinase pathway isactivated under condition of increased extra cellularosmolarity [34]. Activation of Hog1 is essential for sur-vival of yeast cells at high osmolarity. Hog1 activatesRck2, which in turn phosphorylates eEF2 [24]. Mildosmostress reduced the growth rate of yeast cellsexpressing plasmid-encoded wild-type eEF2 (Fig. 4).By contrast to what might have been expected, replace-ment of the two threonies with amino acids that couldnot serve as phosphorylation targets did not preventthe osmostress-dependent reduction in growth rate.Cells expressing the T58V mutant behaved similar tocells expressing wild-type eEF2 whereas the growthrate of cells expressing mutants T56M andT56M ⁄ T58W was even more reduced than thatobserved in the presence of wild-type eEF2. The effecton the growth rate observed with the double mutantwas probably caused by the amino acid replacement atposition 56 because the effect on the growth rate wassimilar to that observed with the single mutationT56M. The data suggest that phosphorylation at posi-tion 56 and ⁄ or 58 is not critical for the cellularresponse to increased extra cellular osmolarity.Stress induced by increasing the growth temperature(37 °C) resulted in reduced growth rates for cellsexpressing wild-type as well as mutant forms of eEF2.Fig. 4. Comparison of the amino acid context surrounding the puta-tive phosphorylation site in eEF2 from various fungi, plants andmetazoans. The position of threonines T56 and T58 (yeast number-ing) are indicated by arrows. Amino acid sequences from (acces-sion numbers in parenthesis) Saccharomyces cerevisiae(NP_014776), Saccharomyces castellii (AAO32487), Saccharomyceskluyveri (AAO32562), Glugea plecoglossi (BAA11470), Ashbya gos-sypii (AAS53513), Candida albicans (CAA70857), Schizosaccharomy-ces pombe (CAB58373), Neurospora crassa (AAK49353), Gibberellazeae (XP_389750), Aspergillus nidulans (XP_663934), Aspergillus fu-migatus (XP_755686), Cryptococcus neoformans (AAW43242), Ent-amoeba 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), Cryptosporidi-um parvum (AAC46607), Plasmodium falciparum (BAA97565),Tetrahymena thermophila (AAN04122), Drosophila pseudoobscura(EAL32818), Drosophila melanogaster (P13060), Aedes aegypti(AAK01430), Spodoptera exigua (AAL83698), Caenorhabditis ele-gans (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 theposition of T56 and T58.G. Bartish et al. Role of Thr56 and Thr58 for eEF2 function in yeastFEBS Journal 274 (2007) 5285–5297 ª 2007 The Authors Journal compilation ª 2007 FEBS 5291However, cells expressing mutants T56C, T56S andT56M ⁄ T58V were clearly more affected than cellsexpressing the other functional mutants. The increasedgrowth temperature also influenced the number of via-ble cells. For most mutants, the effect was similar tothat seen in cells expressing plasmid-encoded wild-typeeEF2. The exception was cells expressing the doublemutant T56M ⁄ T58V. These cells showed markedlyincreased cell death. The observation that mutantsT56M, T58V and T56M ⁄ T58W did not alter the abil-ity of the yeast cells to respond to temperature stresssuggests that the ability to phosphorylate eEF2 at T56and ⁄ or T58 is not crucial for regulating translation inresponse to temperature stress.One possibility is that environmental stress inducesphosphorylation at an alternative site in eEF2. Arecent large-scale characterization of nuclear phospho-proteins in HeLa cells showed the presence of eEF2phosphorylated at Ser485 (yeast numbering) located inthe so-called hinge region of the factor [35]. The pres-ence of serine-phosphorylated eEF2 has also beendemonstrated in vitro upon activation of a yeast kinasehomologous to the type II Ca2+and calmodulin-dependent kinases [36].It has been speculated that the general role of eEF2phosphorylation may not be a massive shut-down ofprotein synthesis but rather a mechanism to promotetranslation of specific mRNAs that have difficulties incompeting with more translation efficient mRNAs byslowing down the elongation rate [19,37]. The situationwould be analogous with that observed on translationafter administration of limited concentrations of cyclo-heximide [38–42]. Our results cannot rule out the pos-sibility that a limited reduction of the elongation ratethrough phosphorylation at T56 is necessary to pro-mote translation of mRNAs needed under specificstress situations.An alignment of the amino acid sequences from avariety of eukaryotic organisms showed that Thr56often is replaced by methionine in fungal eEF2whereas Thr58 is much more conserved (Fig. 4). Itshould be noted that none of the listed eEF2 sequenceshave amino acids S or V in position 58 and none ofthe sequences have C or S in position 56. The lattercould be explained by the slower growth rate of yeastcells expressing eEF2 carrying these mutations. Theslower growth of the T58S mutant could also be anevolutionary disadvantage and hence explain the lackof serine at position 58, even if the resulting protein isfunctional. However, the absence of valine at posi-tion 58 is notable because replacement of T58 withvaline had limited effect on eEF2 function as deter-mined by the effect of the mutation on the growth rateunder both normal growth conditions and conditionsof increased environmental stress.The T56M mutation had little if any effect on thegrowth rate of yeast cells under standard laboratorygrowth conditions. However, yeast cells expressing theT56M mutant (or the double mutant T56M ⁄ T58W)have considerable difficulties in coping with environ-mental stress situations as demonstrated by the effectof increased osmolarity. Thus, the better ability toadapt to environmental stress may have constituted astrong evolutionary pressure in favour of threonine atposition 56 in eEF2.Properties of the eEF2 mutantseEF2 is a GTP-binding protein that interacts with pre-translocation ribosomes and promotes ribosomal trans-location along the mRNA under GTP-hydrolysis [2].All mutant forms of eEF2 described here (i.e. even themutants that were unable to functionally complementwild-type eEF2) were able to bind to ribosomes in cellsalso expressing wild-type eEF2 from one of theremaining eEF2 coding genes. Because binding ofeEF2 to the ribosome is dependent on the preforma-tion of a guanosine nucleotide-factor complex [43], theobservation suggests that the mutant forms of the fac-tor were also able to interact with guanosine nucleo-tides. Three of the nonfunctional mutants, T56D,T56G and T56K, were under-represented on the ribo-some even after adjusting for variations in the intra-cellular concentrations of plasmid-encoded eEF2,indicating that these mutations had a negative effecton the ability of the factor to bind to ribosomes. Theother nonfunctional mutants had approximately thesame ability to bind to ribosomes as the plasmid-encoded wild-type eEF2, suggesting that the mutationsinterfere with the ability of the factor to sustain elon-gation rather than with the ability to associate withribosomes.The function of eEF2 in translocation requires recip-rocation between two conformational states associatedwith the phosphorylation status of the bound guanosinenucleotide [2]. The two putative phosphorylation sitesare located in the so-called switch I region (also knownas the effector-domain) [44,45], a flexible region knownto be involved in the dynamic properties of elongationfactors [46]. Due to its flexible nature, the peptidesequence containing threonines T56 and T58 is missingin the crystal structure of yeast eEF2 [3]. It is thereforedifficult to estimate the structural effects caused by theintroduced point mutations. The observed phenotypiceffects of the analysed eEF2 mutants, an inability tofunctionally complement wild-type eEF2 and theRole of Thr56 and Thr58 for eEF2 function in yeast G. Bartish et al.5292 FEBS Journal 274 (2007) 5285–5297 ª 2007 The Authors Journal compilation ª 2007 FEBSreduced growth rates obtained after expression of func-tional eEF2 mutants may be related to a loss of thedynamic properties of the factor. Such a loss could leadto a reduced ability of the factor to participate in theelongation cycle, resulting in a reduced growth rate.It has previously been shown that the level of eEF2in yeast cells is tightly regulated [6]. The regulationinvolves a post-transcriptional mechanism that keepsthe cellular level of eEF2 constant. Thus, over-expres-sion of mutated eEF2 in cells also expressing plasmidencoded wild-type eEF2 result in decreased levels ofthe wild-type protein as the proportion of mutated fac-tor increases. As a consequence, nonfunctionalmutants of eEF2 (e.g. point mutations at V488 andH699) cause a dominant negative phenotype whenexpressed in cells also expressing wild-type eEF2 [6].The nonfunctional eEF2 mutants described in the pres-ent study were capable of binding to the ribosome andcould therefore have been expected to interfere withthe function of wild-type eEF2 even if expression ofthese mutants would have no effect on the intracellularlevel of wild-type eEF2. However, the nonfunctionalmutants did not interfere with the growth rate of yeastcells also expressing wild-type eEF2. Only the expres-sion of mutant T56V had a slight effect on the growthrate. Thus, no dominant negative effect of the non-functional mutants could be observed. In the presentstudy, the nonfunctional mutants were expressed inyeast cells retaining one of the two genes normallycoding for eEF2 in wild-type cells. It is possible thatthese cells have a sub-optimal content of wild-typeeEF2. If this is the case, the presence of nonfunctionaleEF2 in the ribosomal fraction without noticeableeffects on the growth rate may reflect an increasedpopulation of ‘hungry’ pretranslocation ribosomeswaiting to interact with a functional eEF2.Experimental proceduresChemicalsBP clonase enzyme mix, LR clonase enzymes mix, Readingframe cassette C, DNA polymerase (Klenow fragment) andanti-V5-HRP serum were obtained from Invitrogen (Carls-bad, CA, USA). Restriction nucleases AatII, ClaI, BamHIand XhoI were obtained from Roche (Mannheim, Germany).Alkaline phosphatase, Ready-To-Go ligation kit, ECL wes-tern blotting detection kit was obtained from AmershamPharmacia Biotech Inc. (Uppsala, Sweden). 5-FOA was pro-vided by Larodan Fine Chemicals (Malmo, Sweden). Ampi-cillin, kanamycin, chloramphenicol and synthetic dropoutmedium supplement lacking histidine, leucine, tryptophanand uracil were obtained from Sigma (St Louis, MO, USA).Taq DNA polymerase, Pfu DNA polymerase, RNasine wereobtained from SDS Promega (Madison, WI, USA). Yeastnitrogen base without amino acids and agar were from BD(Franklin Lakes, NJ, USA). Ammonium sulphate and aminoacids were from Merck (Darmstadt, Germany).Strains, plasmids and primersThe strains and plasmids used are listed in Table 1. PlasmidpFA6a-HIS3MX6 was kindly provided by C. Sjo¨gren(Department of Cell and Molecular Biology, KarolinskaInstitutet, Stockholm, Sweden). All primers were synthe-sized by CyberGene AB (Huddinge, Sweden). The primersused are listed in supplementary Tables S1–S2.Growth mediaEscherichia coli cells were grown in LB containing the properantibiotics. Yeast strains were grown on synthetic completemedium, SC, containing 0.67% (weight by volume) bacto-yeast nitrogen base without amino acids, 0.14% (w ⁄ v) yeastsynthetic drop-out medium without histidine, leucine, trypto-phan and uracil, 0.5% (w ⁄ v) ammonium sulphate). Themedium was supplemented with uracil (20 lgÆmL)1) and theappropriate amino acids: histidine (20 lgÆmL)1) and leucine(60 lgÆmL)1) as indicated. Galactose (2% weight by volume)was added as carbon source unless noted.For counter selection, we used SC-Leu-His media supple-mented with 5-FOA (1 gÆL)1) and uracil (50 lgÆmL)1).Solid growth media contained 2% (w ⁄ v) agar.Construction of a conditional null strainFor cloning of the yeast eEF2 gene, total yeast DNA wasprepared form strain YDR385w as described by Hoffmanand Winston [47]. The gene for eEF2 was amplified byPCR using primers eEF2F and eEF2R (supplementaryTable S1). The 2.5 kb PCR-product was introduced intothe TOPO vector pYES2.1 and the resulting plasmid wastransformed into strain YOR133w carrying only one of thetwo genomic alleles for eEF2. The transformed cells wereplated onto SC-Ura and a positive colony (YOR133w;pYES2.1 ⁄ URA3 ⁄ eEF2) was selected. This strain is referredto as GA1 (Table 1).For deletion of the remaining genomic copy of the eEF2gene, the LEU2 gene was amplified from plasmid pAT3using primers Leu2F and Leu2R (supplementary Table S1).These two primers contained 20 nucleotides that matchedthe 5¢- and 3¢-sequence of LEU2, and 40 nucleotides with asequence identical to the 5¢- and 3¢-sequences flanking thegenomic eEF-2 in strain YOR133w. The purified PCR frag-ment was introduced into the GA1 cells and the genomiceEF2 coding sequence replaced by the LEU2 gene viahomologous recombination. The transformed cells wereG. Bartish et al. Role of Thr56 and Thr58 for eEF2 function in yeastFEBS Journal 274 (2007) 5285–5297 ª 2007 The Authors Journal compilation ª 2007 FEBS 5293plated on SC-Ura-Leu for selection of positive colonies.After 4 days at 30 °C, positive colonies were selected andthe replacement of the genomic copy of eEF2 by LEU2 wasconfirmed by PCR and sequencing. The resulting yeaststrain is referred to as GB1 (Table 1).Construction of plasmid for counter selectionVector pYES3 ⁄ CT was digested with restriction enzymesAatII and ClaI to remove the TRP1 gene. The digestionproducts were separated by agarose gel-electrophoresis andthe vector without TRP1 was isolated. The HIS3 gene wasamplified from plasmid pFA6a-HIS3MX6 using primersHisF, HisR, HisFL and HisRL (supplementary Table S1).The latter primer set was used to create overhangs, whichwere complementary to the overhangs generated after AatIIand ClaI digestion of the vector. The PCR productsobtained using the two sets of primers were pooled, heatedto 95 °C for 10 min and allowed to gradually anneal bystepwise lowering of the temperature. The annealing mix-ture was ligated into the digested pYES3 ⁄ CT plasmid. Theligated plasmid was transformed into TOP10 cells. Thepresence of the HIS3 gene in the plasmid was confirmed byPCR analysis.The new vector, pYES3 ⁄ CT⁄ HIS3, was used for construc-tion of a destination vector suitable for use in the Gatewaytechnology cloning system [48–51]. For this purpose, the vec-tor was digested with restriction enzymes BamHI and XhoIand treated with the Klenow polymerase fragment followedby treatment with alkaline phosphatase. Reading frame cas-sette C.1 was ligated with the digested vector and the result-ing plasmid was transformed into DB3.1a E. coli cells, whichwere plated onto LB plates containing chloramphenicol.Positive colonies were selected and the presence of the read-ing frame cassette in the new destination plasmid referred toas pCBG1202 was confirmed by restriction analysis.Site directed mutagenesisAll mutants were generated using the mega-primer methoddescribed by Brons-Poulsen et al. [52]. Primer GateEF2Fwas used in combination with one of the reverse primerscarrying the point mutation to produce a short PCR frag-ment (supplementary Table S2). This fragment was used asa mega-primer together with primer GateEF2R (supple-mentary Table S2) for amplifying the full-length gene. ThePCR products were inserted into the donor vectorpDONR221 using BP clonase. The presence of the muta-tion was confirmed by sequence analysis. Mutant eEF2genes were transferred to the vector pCBG1202 by recombi-nation using LR clonase. The destination vector was trans-formed into strain YOR133w for confirming geneexpression, and into strain GB1 for functional analysis byplasmid shuffling. A copy of the wild-type eEF2 geneobtained by PCR amplification using primers GateEF2Fand GateEF2R was cloned into the pCBG1202 vector asdescribed above. This plasmid served as control.Cell transformationBacterial transformations were performed according tostandard methods [53]. Yeast cells were transformed usingthe lithium acetate method, as described by Soni et al.[54].Detection of eEF-2 expression by immunoblottingYeast strain YOR133w containing plasmid pCBG1202 witha wild-type or mutated eEF2 gene was grown overnight at30 °C in 5 mL of SC-His medium containing 2% (w ⁄ v) glu-cose. The cells were collected by centrifugation, washed andresuspended in 30 mL of SC-His medium with galactose.After induction during approximately 20 h at 30 °C, thecells were harvested, washed in 20 mm Hepes-KOH(pH 7.4), 2 mm Mg(CH3COO)2, 100 mm KCl and 1 mmdithiothreitol, and suspended in the same buffer containing1mm PMSF and 4000 U RNasine. The cell suspension wasmixed with glass beads and the yeast cells lysed asdescribed [55]. The crushed cells were centrifuged for 5 minat 5000 g with a Haereus Biofuge (Berlin, Germany). Analiquot of the supernatants were withdrawn for analysis ofthe total level plasmid-encoded eEF2. The remaining super-natants were transferred to new tubes and centrifuged foranother 15 min at 15000 g. The supernatants were used forpreparation of ribosomes. Deoxycholate and Triton X-100were added at a final concentration of 1% (w ⁄ v) each. Thesupernatants (1 mL), were layered onto 2 mL sucrose cush-ions containing 0.75 m sucrose in 75 mm KCl, 20 mmTris ⁄ HCl, pH 7.6, 2 mm Mg(CH3COO)2and 15 mm 2-mer-captoethanol. The material was centrifuged in aTLA100.3 rotor (Beckman Instruments, Palo Alto, CA,USA) for 150 min, at 198 000 g and 4 °C. The ribosomalpellets were dissolved in 0.25 m sucrose, 25 mm KCl,30 mm Hepes-KOH (pH 7.6), 2 mm Mg(CH3COO)2and1mm dithiothreitol. Dissolved ribosomes and the post-ribo-somal supernatants were stored in aliquots at )80 °C untilused.For detection of total cellular levels of plasmid-encodedeEF2 crude cell lysates, 40 lg protein in 2 lL, were spot-ted on nitrocellulose membranes. For estimation of theribosomal binding capacity of plasmid-encoded eEF2 iso-lated ribosomes, 40 lg ribosomes in 2 lL, were spotted onnitrocellulose membranes. The dried membranes were forimmunoblotting. The ribosome bound eEF2, 50 lgofribosomes, was also analysed by SDS gel electrophoresison 10% (w ⁄ v) polyacrylamid gels [56]. The separated pro-teins were transferred to a nitrocellulose membrane, andthe membrane was incubated with anti-V5-HRP serum.Bound antibodies were detected using the ECL westernblotting detection kit.Role of Thr56 and Thr58 for eEF2 function in yeast G. Bartish et al.5294 FEBS Journal 274 (2007) 5285–5297 ª 2007 The Authors Journal compilation ª 2007 FEBS[...]... Council FEBS Journal 27 4 (20 07) 528 5– 529 7 ª 20 07 The Authors Journal 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 Ejiri S (20 02) Moonlighting functions of polypeptide elongation factor 1: from actin bundling to zinc finger protein R1-associated... to the effector domain in elongation factor G Proteins 37, 29 3–3 02 529 6 27 Kolesnikov AV & Gudkov AT (20 03) Mutational analysis of the functional role of the loop region in the elongation factor G fourth domain in the ribosomal translocation Mol Biol (Mosk) 37, 719– 725 28 Savelsbergh A, Matassova NB, Rodnina MV & Wintermeyer W (20 00) Role of domains 4 and 5 in elongation factor G functions on the ribosome... 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 pCBG 120 2 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... Livingston DM & Bodley JW (19 92) Saccharomyces cerevisiae elongation factor 2 Genetic cloning, characterization of expression, and G-domain modeling J Biol Chem 26 7, 1190–1197 6 Ortiz PA & Kinzy TG (20 05) Dominant-negative mutant phenotypes and the regulation of translation elongation factor 2 levels in yeast Nucleic Acids Res 33, 5740–5748 7 Robinson EA, Henriksen O & Maxwell ES (1974) Elongation factor. .. sites in elongation factor 2 FEBS Lett 27 5, 20 9 21 2 10 Ryazanov AG, Shestakova EA & Natapov PG (1988) Phosphorylation of elongation factor 2 by EF -2 kinase affects rate of translation Nature 334, 170–173 11 Redpath NT & Proud CG (1989) The tumour promoter okadaic acid inhibits reticulocyte-lysate protein synthesis by increasing the net phosphorylation of elongation factor 2 Biochem J 26 2, 69–75 ˚ 12 Carlberg... peptidechain elongation in mammalian cells Eur J Biochem 26 9, 5360–5368 Wang X, Li W, Williams M, Terada N, Alessi DR & Proud CG (20 01) Regulation of elongation factor 2 kinase by p90 (RSK1) and p70, S6 kinase EMBO J 20 , 4370–4379 ˚ Nilsson A & Nygard O (1995) Phosphorylation of eukaryotic elongation factor 2 in differentiating and proliferating HL-60 cells Biochim Biophys Acta 126 8, 26 3 26 8 Chen Y,... (20 01) Mechanisms for increased levels of phosphorylation of elongation factor2 during hibernation in ground squirrels Biochemistry 40, 11565–11570 Rose AJ, Broholm C, Kiillerich K, Finn SG, Proud CG, Rider MH, Richter EA & Kiens B (20 05) Exercise rapidly increases eukaryotic elongation factor 2 phosphorylation in skeletal muscle of men J Physiol 569, 22 3 22 8 Drennan D & Ryazanov AG (20 04) Alpha-kinases:... family and comparison with conventional protein kinases Prog Biophys Mol Biol 85, 1– 32 Donovan MG & Bodley JW (1991) Saccharomyces cerevisiae elongation factor 2 is phosphorylated by an endogenous kinase FEBS Lett 29 1, 303–306 Teige M, Scheikl E, Reiser V, Ruis H & Ammerer G (20 01) Rck2, a member of the calmodulin-protein kinase family, links protein synthesis to high osmolarity MAP kinase signaling in. .. [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 pCBG 120 2 vector and the same vector containing wild-type eEF2 were... (1998) Elongation factor 2 as a novel target for selective inhibition of fungal protein synthesis J Biol Chem 27 3, 3148–3151 32 Ryazanov AG & Davydova EK (1989) Mechanism of elongation factor 2 (EF -2) inactivation upon phosphorylation Phosphorylated EF -2 is unable to catalyze translocation FEBS Lett 25 1, 187–190 33 Redpath NT, Price NT, Severinov KV & Proud CG (1993) Regulation of elongation factor- 2 by . Amino acids Thr56 and Thr58 are not essential for elongation factor 2 function in yeast Galyna Bartish1 ,2 , Hossein Moradi1 ,2 and Odd Nyga˚rd11. yeast genome. As shown in Role of Thr56 and Thr58 for eEF2 function in yeast G. Bartish et al. 528 6 FEBS Journal 27 4 (20 07) 528 5– 529 7 ª 20 07 The Authors Journal
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