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

Amino acids Thr56 and Thr58 are not essential for
elongation factor 2 function in yeast
Galyna Bartish
1,2
, Hossein Moradi
1,2
and Odd Nyga
˚
rd
1
1 School of Life Sciences, So
¨
derto
¨
rns ho
¨
gskola, Huddinge, Sweden
2 Department of Cell Biology, Arrhenius Laboratories, Stockholm University, Sweden
Protein synthesis is one of the most complicated and
energy consuming cellular processes. Approximately
150 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 is
a 95 kDa GTP-binding protein that binds to pretrans-
location ribosomes [2]. The role of the factor, and its
eubacterial homologue, elongation factor G (EFG), is
to promote GTP-dependent translocation of the ribo-
some 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.
Keywords
elongation factor 2; functional
complementation; osmostress;
phosphorylation; yeast
Correspondence
O. Nyga
˚
rd, School of Life Sciences,
So
¨
derto
¨
rns ho
¨
gskola, S-141 89 Huddinge,
Sweden
Fax: +46 8608 4510
Tel: +46 8608 4701
E-mail:
(Received 10 January 2007, revised 27 June
2007, accepted 17 August 2007)

doi:10.1111/j.1742-4658.2007.06054.x
Yeast elongation factor 2 is an essential protein that contains two highly
conserved threonine residues, T56 and T58, that could potentially be phos-
phorylated by the Rck2 kinase in response to environmental stress. The
importance of residues T56 and T58 for elongation factor 2 function in
yeast was studied using site directed mutagenesis and functional comple-
mentation. Mutations T56D, T56G, T56K, T56N and T56V resulted in
nonfunctional elongation factor 2 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 tem-
peratures. 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 non-
functional forms of elongation factor 2 were less capable of competing with
wild-type elongation factor 2 in ribosome binding. The presence of non-
functional but ribosome binding forms of elongation factor 2 did not affect
the growth rate of yeast cells also expressing wild-type elongation factor 2.
Abbreviations
CaMPKIII, Ca
2+
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 5285
eEF2 is subjected to post-translational modifica-
tions. 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 phosphoryla-
tion target is T56 but phosphorylation at the second
threonine has also been observed [8,9]. Phosphoryla-
tion decreases the affinity of eEF2 for pretranslocation
ribosomes, thereby preventing the factor from stimu-
lating translocation [10–12]. The observation that
threonines 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 the
activity of eEF2 in eukaryotes.
In mammals, an altered phosphorylation status of
eEF2 has been connected to different physiological sit-
uations and severe diseases [14]. Mammalian eEF2 is
phosphorylated by a specific Ca
2+
and calmodulin-
dependent protein kinase (CaMPKIII) [15,16]. The
activity of the eEF2 kinase is regulated by the mito-
gen-activated protein (MAP) kinase and mTOR-signal-
ling 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 phosphory-
lated 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 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 MAP
kinase Hog1 in response to osmostress [24], an envi-
ronmental 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 bac-
terial 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.
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 immunode-
tection of the plasmid-encoded protein. All constructs
were sequenced to confirm the presence of the intro-
duced 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 trans-
formed 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, func-
tional complementation studies require that the tag
attached to the C-terminus of the plasmid-encoded
eEF2 do 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 ribo-
somes. Thus, the C-terminal tag did not prevent ribo-
somal 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 lev-
els, respectively. Because all constructs are identical,
except for the introduced point mutations, transcrip-
tion levels should be equal. It is therefore possible that
the lower intracellular levels of these mutant forms of
eEF2 reflect increased degradation. The expression lev-
els of tagged wild-type eEF2 from plasmid pCBG1202
in GB2 cells (Table 1) was used as a reference for max-
imum expression levels and ribosomal binding of
tagged eEF2 analysed in the absence of competing
eEF2 coded for by the yeast genome. As shown in
Role 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 FEBS
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.
Table 1. Strains and plasmids used in the present study. Euroscarf (Frankfurt, Germany).
Strains and plasmids Source
YOR133w (Mat a; his3D1; leu2D0 met15D0; ura3D0; yor133w::kanMX4) Euroscarf
YDR385w (Mat a; his3D1; leu2D0; lys2D0; ura3D0; ydr385w::kanMX4) Euroscarf

GA1 (YOR133w; pYES2.1 ⁄ URA3 ⁄ EF2) This study
GA2 (YOR133w; pCBG1202 ⁄ HIS3 ⁄ EF2) This study
GB1 (YOR133w; ydr385wDLEU2; pYES2.1 ⁄ URA3 ⁄ EF2) This study
GB2 (YOR133w; ydr385wDLEU2; pCBG1202 ⁄ HIS3 ⁄ EF2) This study
T56C as GB1 but (pCBG1202 ⁄ HIS3 ⁄ EF2T56C) This study
T56M as GB1 but (pCBG1202 ⁄ HIS3 ⁄ EF2T56
M) This study
T56S as GB1 but (pCBG1202 ⁄ HIS3 ⁄ EF2T56S) This study
T58S as GB1 but (pCBG1202 ⁄ HIS3 ⁄ EF2T58S) This study
T58V as GB1 but (pCBG1202 ⁄ HIS3 ⁄ EF2T58 V) This study
T56M ⁄ T58V as GB1 but (pCBG1202 ⁄ HIS3 ⁄ EF2T56
M ⁄ T58V) This study
T56M ⁄ T58W as GB1 but (pCBG1202 ⁄ HIS3 ⁄ EF2T56V ⁄ T58W) This study
One Shot TOP10 cells (F- mcrA D(mrr-hsdRMS-mcrBC) /80lacZDM15 DlacX74
recA1 araD139 D(araleu) 7697 galU galK rpsL (StrR) endA1 nupG)
Invitrogen
DB3.1(F
–
gyrA462 endA1 D(sr1-recA) mcrB mrr hsdS20(r
B
-, m
B
-) supE44 ara-14
galK2 lacY1 proA2 rpsL20(Sm
R
) xyl-5 Dleu mtl1)
Invitrogen
pYES2.1 (P
GAL1
,2l, GAL1, URA3); Invitrogen

pYES3 ⁄ CT (P
GAL1
,2l, GAL1, TRP1) Invitrogen
pDONR221 Invitrogen
pCBG1202 (P
GAL1
,2l, GAL1, HIS3, RFC) This study
AB
Fig. 1. 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 °Cin
the 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 dot
blots were quantified using computer-assisted densitometry.
G. Bartish et al. Role of Thr56 and Thr58 for eEF2 function in yeast
FEBS Journal 274 (2007) 5285–5297 ª 2007 The Authors Journal compilation ª 2007 FEBS 5287
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, sug-
gesting that the mutation may have interfered with the
ribosome-binding properties of eEF2. The low expres-
sion 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 trans-
forming 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 cod-
ing 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 fur-
ther 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 complementa-
tion 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 via-
ble (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 con-
structed double mutants in which both threonines were
replaced by amino acids that could not be phosphory-
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).
Fig. 2. 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 D
600 nm
reached approximately 1. Aliquots (5 lL)
of the cell cultures (undiluted and diluted 1 : 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
4 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 FEBS
A comparison of the growth rate at various temper-
atures 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
AD
BE
CF
Fig. 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 D
600 nm

¼ 0.2 with SC ⁄ Gal-His medium (B,E) or with SC ⁄ Gal-His-Leu medium with-
out (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 3 days at the temperatures indicated.
G. Bartish et al. Role of Thr56 and Thr58 for eEF2 function in yeast
FEBS Journal 274 (2007) 5285–5297 ª 2007 The Authors Journal compilation ª 2007 FEBS 5289
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 mar-
ginally affected by the presence of a nonfunctional
eEF2. Thus, there was no negative effect on the
growth 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 cells

expressing eEF2 lacking the putative phosphorylation
targets 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 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 express-
ing 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
that in cells expressing plasmid-encoded wild-type
eEF2 (not shown).
Discussion
The ability of eEF2 to promote translocation in mam-
mals is regulated by phosphorylation at T56 and ⁄ or
T58 [8–10,12,32,33]. Phosphorylation is catalysed
by an eEF2-specific Ca
2+
and calmodulin-dependent
kinase. Threonines Thr56 and Thr58 are highly con-
served in eEF2 from several different organisms
(Fig. 4) [5,13]. Phosphorylation at the homologous
threonines has therefore been assumed to play a gen-
eral 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 mam-
malian CaM-dependent eEF2-kinase [24]. The Rck2
kinase phosphorylates eEF2 in vitro. The activity of
the kinase is increased under environmental stress con-
ditions such as increased osmolarity [24]. The activa-
tion 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 phosphory-
lated 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 posi-
tion 58. Cells expressing mutants T58S and T58V grew
slower than control cells expressing the tagged wild-
type 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 func-
tional 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
Table 2. 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 89 287
T56C 80 319
T56M 89 301
T56S 83 342
T58S 85 323
T58V 81 284
T56M ⁄ T58V 65 370
T56M ⁄ T58W 88 275
Role 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 FEBS
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. 5 have tryptophan at the position corre-
sponding to T58 in yeast. The results from the muta-
tion 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 phosphory-
lated 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 regu-
lating the rate of protein synthesis in yeast under stan-
dard growth conditions.
Under conditions of increased osmolarity, the yeast
cells rapidly reduce protein production [25]. In Saccha-
romyces cerevisiae, the HOG MAP kinase pathway is
activated under condition of increased extra cellular
osmolarity [34]. Activation of Hog1 is essential for sur-
vival 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, replace-
ment 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 posi-
tion 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.
Fig. 4. Comparison of the amino acid context surrounding the puta-
tive phosphorylation site in eEF2 from various fungi, plants and
metazoans. 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), Saccharomyces
kluyveri (AAO32562), Glugea plecoglossi (BAA11470), Ashbya gos-
sypii (AAS53513), Candida albicans (CAA70857), Schizosaccharomy-
ces pombe (CAB58373), Neurospora crassa (AAK49353), Gibberella
zeae (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 the
position of T56 and T58.
G. Bartish et al. Role of Thr56 and Thr58 for eEF2 function in yeast
FEBS Journal 274 (2007) 5285–5297 ª 2007 The Authors Journal compilation ª 2007 FEBS 5291
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 via-
ble 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 abil-
ity 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 phospho-
proteins 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 pres-

ence of serine-phosphorylated eEF2 has also been
demonstrated in vitro upon activation of a yeast kinase
homologous to the type II Ca
2+
and calmodulin-
dependent 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 cyclo-
heximide [38–42]. Our results cannot rule out the pos-
sibility that a limited reduction of the elongation rate
through phosphorylation at T56 is necessary to pro-
mote 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 posi-
tion 58 is notable because replacement of T58 with
valine had limited effect on eEF2 function as deter-
mined by the effect of the mutation on the growth rate
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 environ-
mental 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 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 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 preforma-
tion of a guanosine nucleotide-factor complex [43], the
observation 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 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 plasmid-
encoded wild-type eEF2, suggesting that the mutations
interfere with the ability of the factor to sustain elon-
gation rather than with the ability to associate with
ribosomes.
The function of eEF2 in translocation requires recip-
rocation 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
Role 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 FEBS
reduced growth rates obtained after expression of func-
tional 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-expres-
sion 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 fac-
tor 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 pres-
ent 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 expres-
sion of mutant T56V had a slight effect on the growth
rate. Thus, no dominant negative effect of the non-
functional 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 (Carls-
bad, CA, USA). Restriction nucleases AatII, ClaI, BamHI
and XhoI were obtained from Roche (Mannheim, Germany).
Alkaline phosphatase, Ready-To-Go ligation kit, ECL wes-
tern blotting detection kit was obtained from Amersham
Pharmacia Biotech Inc. (Uppsala, Sweden). 5-FOA was pro-
vided by Larodan Fine Chemicals (Malmo, Sweden). Ampi-
cillin, kanamycin, chloramphenicol and synthetic dropout
medium supplement lacking histidine, leucine, tryptophan
and uracil were obtained from Sigma (St Louis, MO, USA).
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 1. Plasmid
pFA6a-HIS3MX6 was kindly provided by C. Sjo
¨
gren
(Department of Cell and Molecular Biology, Karolinska
Institutet, Stockholm, Sweden). All primers were synthe-

sized 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) bacto-
yeast nitrogen base without amino acids, 0.14% (w ⁄ v) yeast
synthetic drop-out medium without histidine, leucine, trypto-
phan and uracil, 0.5% (w ⁄ v) ammonium sulphate). The
medium was supplemented with uracil (20 lgÆmL
)1
) and the
appropriate 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 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 frag-
ment was introduced into the GA1 cells and the genomic
eEF2 coding sequence replaced by the LEU2 gene via
homologous recombination. The transformed cells were
G. Bartish et al. Role of Thr56 and Thr58 for eEF2 function in yeast
FEBS Journal 274 (2007) 5285–5297 ª 2007 The Authors Journal compilation ª 2007 FEBS 5293
plated on SC-Ura-Leu for selection of positive colonies.
After 4 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 min and allowed to gradually anneal by
stepwise lowering of the temperature. The annealing mix-
ture 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 construc-
tion of a destination vector suitable for use in the Gateway
technology cloning system [48–51]. For this purpose, the vec-
tor was digested with restriction enzymes BamHI and XhoI
and treated with the Klenow polymerase fragment followed
by 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, which
were 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 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 frag-
ment (supplementary Table S2). This fragment was used as

a mega-primer together with primer GateEF2R (supple-
mentary 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 muta-
tion was confirmed by sequence analysis. Mutant eEF2
genes were transferred to the vector pCBG1202 by recombi-
nation using LR clonase. The destination vector was trans-
formed 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
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 5 mL of SC-His medium containing 2% (w ⁄ v) glu-
cose. 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), 2 mm Mg(CH
3
COO)
2

, 100 mm KCl and 1 mm
dithiothreitol, and suspended in the same buffer containing
1mm 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 5 min
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 super-
natants were transferred to new tubes and centrifuged for
another 15 min 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 2 mL sucrose cush-
ions containing 0.75 m sucrose in 75 mm KCl, 20 mm
Tris ⁄ HCl, pH 7.6, 2 mm Mg(CH
3
COO)
2
and 15 mm 2-mer-
captoethanol. The material was centrifuged in a
TLA100.3 rotor (Beckman Instruments, Palo Alto, CA,
USA) for 150 min, at 198 000 g and 4 °C. The ribosomal
pellets were dissolved in 0.25 m sucrose, 25 mm KCl,
30 mm Hepes-KOH (pH 7.6), 2 mm Mg(CH
3
COO)
2
and
1mm dithiothreitol. Dissolved ribosomes and the post-ribo-
somal 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 2 lL, were spot-
ted on nitrocellulose membranes. For estimation of the
ribosomal binding capacity of plasmid-encoded eEF2 iso-
lated ribosomes, 40 lg ribosomes in 2 lL, were spotted on
nitrocellulose membranes. The dried membranes were for
immunoblotting. The ribosome bound eEF2, 50 lgof
ribosomes, was also analysed by SDS gel electrophoresis
on 10% (w ⁄ v) polyacrylamid gels [56]. The separated pro-
teins 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.
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
Plasmid shuffling
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 analy-
sed 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.
Cell growth and viability
For growth rate analysis, wild-type and nonfunctional
mutants of eEF2 in plasmid pCBG1202 were expressed in
YOR133w cells (Table 1). The cells were allowed to grow
overnight at 30 °C in SC-His medium. Yeast cells express-
ing functional forms of eEF2 (GB2, T56C, T56M, T56S,
T58S, T58V, T56M ⁄ T58V and T56M ⁄ T58W; Table 1)
were incubated in SC-His-Leu medium over night at
30 °C. The overnight cultures were diluted to a D
600 nm
of
0.2 in the appropriate medium. The incubation was con-
tinued at 30 °C and D
600 nm
was registered at the time
intervals indicated. Alternatively, cultures of yeast cells
expressing functional forms of eEF2 were allowed to grow
to a D
600 nm
of 1. Aliquots of the cell cultures were
diluted 1 : 10 and 1 : 100 and samples (5 lL) from each
of the three cell concentrations were spotted on solid
growth media and incubated for 3 days at the tempera-
tures indicated.
The proportion of viable cells in cultures of yeast cells
expressing plasmid-encoded forms of eEF2 were determined
by colony counting and by vital staining [58–60]. The two
methods gave similar results.

Osmostress
Mild osmostress was induced by supplementing the SC-
Leu-His growth medium with 0.4 m NaCl [24].
Acknowledgements
We are grateful to Mrs Birgit Lundberg for tech-
nical assistance. We thank Dr Camilla Sjo
¨
gren for
providing us the plasmid pFA6a-HIS3MX6. This
work was supported by the Swedish Research
Council.
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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
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 corre-
sponding author for the article.
G. Bartish et al. Role of Thr56 and Thr58 for eEF2 function in yeast
FEBS Journal 274 (2007) 5285–5297 ª 2007 The Authors Journal compilation ª 2007 FEBS 5297