From the Department of Molecular Genetics,
Microbiology & Immunology, University of Medicine and Dentistry of New
Jersey Robert Wood Johnson Medical School, Piscataway, New Jersey
08854, the § Department of Biochemistry, Medical College of
Wisconsin, Milwaukee, Wisconsin 52226, and the
Laboratory
of Gene Regulation and Development, NICHD, National Institutes of
Health, Bethesda, Maryland 20892
Received for publication, September 9, 2002, and in revised form, December 9, 2002
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ABSTRACT |
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The translation elongation machinery in fungi
differs from other eukaryotes in its dependence upon eukaryotic
elongation factor 3 (eEF3). eEF3 is essential in vivo and
required for each cycle of the translation elongation process in
vitro. Models predict eEF3 affects the delivery of cognate
aminoacyl-tRNA, a function performed by eEF1A, by removing deacylated
tRNA from the ribosomal Exit site. To dissect eEF3 function and its
link to the A-site activities of eEF1A, we have identified a
temperature-sensitive allele of the YEF3 gene. The F650S
substitution, located between the two ATP binding cassettes, reduces
both ribosome-dependent and intrinsic ATPase activities.
In vivo this mutation increases sensitivity to
aminoglycosidic drugs, causes a 50% reduction of total protein
synthesis at permissive temperatures, slows run-off of polyribosomes,
and reduces binding to eEF1A. Reciprocally, excess eEF3 confers
synthetic slow growth, increased drug sensitivity, and reduced
translation in an allele specific fashion with an E122K mutation in the
GTP binding domain of eEF1A. In addition, this mutant form of
eEF1A shows reduced binding of eEF3. Thus, optimal in vivo
interactions between eEF3 and eEF1A are critical for protein synthesis.
The process of protein synthesis is mediated by soluble protein
factors, many of which are functionally similar between prokaryotic and
eukaryotic systems (1). During elongation, eukaryotic elongation factor
(eEF)1 1A or its prokaryotic
homologue EF-Tu, recruit aminoacyl-tRNA (aa-tRNA) to the A site of
ribosome. The eEF1B complex or EF-Ts, in eukaryotes and prokaryotes,
respectively, are the nucleotide exchange factors functioning to
maintain the active pools of their respective G-proteins. eEF2 in
eukaryotes or EF-G in bacteria are the GTP-driven translocases that
move the mRNA and peptidyl-tRNA following peptide bond formation.
Most protein synthesis factors are highly conserved between single cell
eukaryotes and metazoans. For example, human and Saccharomyces
cerevisiae eEF1A are 81% identical. Fungi are different in their
absolute requirement of a third factor, eEF3. eEF3 of S. cerevisiae is essential for cell-free translation systems (2, 3),
every cycle of translation elongation (4), and cell viability (5).
Apart from the YEF3 gene of S. cerevisiae (6) and
the related, nonessential HEF3 gene (7), eEF3 has been
identified and sequenced from other fungi like Candida albicans (8, 9), Cryptococcus neoformans (10), and
Pneumocystis carini (11). A structural homolog of eEF3 has
also been reported to be present in the chlorella virus CVK2
(12). Some evidence indicates there may be an eEF3-like ATPase activity
tightly associated with ribosomes in prokaryotes (13). This ATPase,
RbbA, cross-reacts with anti-eEF3 antibody and exhibits inhibition of
polyphenylalanine synthesis as well as ribosome-associated ATPase
activity in the presence of anti-eEF3 antibody.
The 1044-amino acid sequence of eEF3 reveals multiple structural motifs
spanning the protein, some possessing homology to proteins of
established functions related to translation (Fig. 1A). A
200-amino acid motif near the N terminus bears ~30% homology with
the Escherichia coli ribosomal protein S5. rpS5 binds to the
16 S rRNA and is an important constituent of the "decoding center"
(14). Recombinant peptide derived from the N terminus of eEF3 interacts
with 18 S rRNA and inhibits the ribosome-dependent eEF3
ATPase activity (15). The 51-amino acid stretch at the C terminus
comprises highly basic amino acids containing at least three
lysine-rich clusters, which are absolutely required for binding to
yeast ribosomes (16, 17). eEF3 also possesses a highly conserved
sequence found in tRNA-binding proteins, termed ELVES, residing in the
intervening region of the "A" and "B" motifs of the second ATP
binding cassette and has been hypothesized to assist the removal of
deacylated tRNA from the ribosome (14). Thus, the various sequence
motifs of eEF3 exhibit conservation to proteins known to interact with
tRNA and rRNA and may indicate similar functions for eEF3.
eEF3 possesses both intrinsic and ribosome-dependent ATPase
and GTPase activities by virtue of a repeated bipartite nucleotide binding domain characteristic of the ATP-binding superfamily of proteins possessing ATP binding cassettes (ABCs) or Walker boxes (reviewed in Ref. 18). eEF3, Gcn20p, and UvrA, unlike other members of
the ABC family of proteins, are a subfamily of soluble proteins and do
not function as membrane bound translocators, the larger part of this
family (18). Rather, eEF3 relies upon its ATP hydrolytic activity to
perhaps induce a conformational change within the ribosome,
facilitating the exit of deacylated tRNA from the E-site and subsequent
binding of charged aa-tRNA to the A-site (19). The two ABCs are
distinct, as the first has the classic 70-90-amino acid spacing
between the A and B motifs, whereas the second has a 188-amino acid
insertion. However, eEF3 does bind two molecules of ATP in a
cooperative manner (20). A protein lacking amino acids 689-793,
including the A motif of the second ATP binding cassette, however,
maintains intrinsic ATPase activity, indicating for this function both
cassettes are not essential (18). More subtle mutations in the
conserved glycines (G463V and G701V), and lysines (K469R and K707R), in
the A motif of the ATP binding cassettes
(GXXXXGKST) abolish
ribosome-dependent ATPase activity, polyphenylalanine
synthesis, and cell growth (21). However, little is known about the
function or consequences of altered ATPase activity in vivo
or in the individual steps of eEF3 function. Overall, the many distinct
sequence motifs of the eEF3 protein may perform different functions;
however, there appears to be an interaction among different domains to
govern the overall catalytic action of the eEF3 protein.
Previous studies show that, functionally, eEF3 stimulates the delivery
of only cognate aa-tRNA by eEF1A at the A site (22, 23), in a manner
that appears to be codon-dependent (24). The role of eEF3
in an allosteric interaction between the A and E sites of the ribosome
has been demonstrated (19). Further, there is evidence that eEF3 is an
E site factor controlling the removal of deacylated tRNA to facilitate
binding of the cognate (aa-tRNA·GTP·eEF1A) ternary complex to the A
site. In fact, eEF3 binds eEF1A in vitro (25), although the
functional consequences on elongation and eEF1A function remain
unknown. In the current study we report that mutation of Phe-650 to Ser
in the intervening region of the two ATP binding cassettes of S. cerevisiae eEF3 is conditionally lethal at 37 °C. The F650S
mutant strain demonstrates reduced global translation, slower run-off
of polyribosomes, reduced intrinsic and ribosome-stimulated ATPase
activities, and reduced interaction with eEF1A. The eEF3 mutant strain
of yeast does not affect +1 ribosomal frameshifting or nonsense
suppression, but has moderate effects on sensitivity to translation
inhibitors. eEF3 also exhibits alterations in both genetic and physical
interactions with a G-domain mutation in eEF1A in a highly
allele-specific fashion. The present study demonstrates a direct
allele-specific genetic interaction between the two translation
elongation factors and shows intact and functional nucleotide binding
domains of both proteins are necessary for their physical association.
Taken together these results provide in vivo evidence
supporting a functional link between these two factors.
Yeast and Bacterial Strains, Growth, and Drug Sensitivity
Assays--
S. cerevisiae strains and their genotypes used
in this study are listed in Table I.
E. coli DH5 Isolation and Cloning of the F650S Mutant Allele of S. cerevisiae
YEF3--
A screen for temperature-sensitive (Ts Translation Assays--
ATP Hydrolysis--
His6-tagged wild type and F650S
mutant eEF3 proteins were purified from the strains TKY702 and TKY707,
respectively, on a Ni2+ HiTrap chelating column (Amersham
Biosciences). Total yeast cell extracts were clarified and
loaded on the column in buffer A (50 mM KPO4,
pH 7.6, 300 mM KCl, 1 mM DTT, and 0.2 mM PMSF) with 20 mM imidazole. The protein was
eluted with buffer A plus 250 mM imidazole. The protein
peak was dialyzed into buffer B (20 mM Tris, pH 7.5, 0.1 mM EDTA, 10% glycerol, 1 mM DTT, 0.2 mM PMSF, and 100 mM KCl). ATP hydrolysis was
performed as previously described (29). Briefly, the standard assay
mixture contained 0.5 A260 units of yeast
ribosomes and 150 µM [ GST and His6 Pull-downs of eEF1A and eEF3--
Yeast
extracts for GST pull-down analysis were prepared by glass bead lysis
in TEDG buffer (10 mM Tris-HCl, pH 7.4, 2 mM
EDTA, 5 mM DTT, 50 mM KCl, 1 mM
PMSF) from eEF1A wild type (MC214) or mutant (TKY229, TKY252, and
TKY588) strains expressing either GST alone (pMA210) or GST-eEF3
(pTKB546) fusion plasmids under the GAL4 promoter and
induced by growth in C Characterization of a Novel Temperature-sensitive Allele of
YEF3--
To apply a genetic approach to understanding eEF3 function,
a screen for randomly generated Ts The F650S Substitution in eEF3 Abolishes Both
Ribosome-dependent and Intrinsic ATP Hydrolytic
Activity--
The F650S mutation is between the two ATP binding
cassettes of eEF3 (Fig. 1A). This location is of interest as
the presence of bipartite ABCs or ATPase domains in tandem is found in
a series of proteins including the ABC transporters. The cross-talk
between the two domains is critical for the ATPase function of
these proteins (reviewed in Ref. 34), and the presence of this
mutation in the intervening region of the two motifs could potentially
affect the proper alignment of these two domains. Thus, it was
predicted that the F650S mutation would interfere with hydrolytic
activity. As seen in Fig. 2, wild type
eEF3 hydrolyzes 0.82 nmol of ATP/pmol of eEF3. The presence of
ribosomes stimulates the hydrolysis 2-fold. In comparison, F650S eEF3
protein shows only background levels of hydrolysis in the presence or
absence of ribosomes. Thus, the mutation severely compromises an eEF3
activity, ATP hydrolysis, previously shown to be critical for
elongation in vitro.
A Strain Expressing the F650S eEF3 Mutant Shows Altered Sensitivity
to Translation Elongation Inhibitors but Not Reduced Translational
Fidelity--
To identify changes in protein synthesis caused by the
F650S mutation, the wild type and mutant strains were tested for
sensitivity to translation inhibitors. Altered sensitivity to
aminoglycosidic drugs such as paromomycin and hygromycin B typically
correlates with reduced translational fidelity (35, 36), whereas
cycloheximide is a general translation elongation inhibitor. At the
permissive temperature, the F650S mutant eEF3 strain showed increased
sensitivity to paromomycin and hygromycin, but not cycloheximide (Table
II). To further analyze potential effects
on fidelity, the ability of the wild type and mutant strains to
suppress two chromosomally encoded +1 frameshift mutations
(met2-1 and his4-713) was monitored by the
ability of strains to grow on medium lacking the corresponding amino
acid. Compared with the wild type, the mutant strain did not grow
better on C eEF3 Mutant Shows a Global Reduction in Translation at the
Elongation Step--
To monitor the effects of the F650S mutation on
total protein synthesis, [35S]methionine incorporation
was monitored at the permissive temperature of 30 °C. A strain
expressing the mutant eEF3 protein shows an ~50% decrease in total
translation over 60 min of growth (Fig. 3A). This is similar to the
level of reduction seen for a strain bearing the eEF1A E286K mutant
(Fig. 3B), which also dramatically affects cell growth (37,
38). To identify the step in protein synthesis affected, standard
polyribosome profiles were analyzed for the wild type and mutant eEF3
strains at the permissive temperature and following a 2-h shift to
37 °C. No significant differences between the profiles of wild type
or F650S mutant strain extracts were noted (Fig. 3C for 30 °C and
data not shown). A similar analysis of the E286K eEF1A mutant, which
also shows a significant reduction in total translation, similarly
shows no significant polyribosome profile alterations (Fig.
3D for 30 °C and data not shown). Consequently, to look
more specifically at the elongation step, identical experiments were
performed at the permissive temperature of 30 °C except that cycloheximide was omitted from all steps of the standard experimental protocol. Because cycloheximide freezes ribosomes on the mRNA, the
lack of this drug would allow ribosomes to continue elongating and
run-off. Slower run-off would be seen as enhanced retention of
polyribosomes, thus providing a better monitor of effects on elongation. The F650S mutant eEF3 strain exhibits a much higher population of polyribosomes as compared with the wild type eEF3 strain
(Fig. 3C, The F650S Substitution in eEF3 Reduces Its Physical Interaction
with eEF1A--
It has been shown previously (25) that eEF1A and eEF3
physically interact. To decipher the cause of the translation defect seen in the F650S eEF3 mutant, plasmids expressing
His6-tagged wild type and F650S eEF3 proteins were
prepared, and shown to function in place of the untagged wild type eEF3
with essentially identical growth to their untagged counterparts (data
not shown). Using a Ni2+-NTA pull-down from total cell
extracts, the native eEF1A associates with wild type His6
eEF3 (Fig. 4). When the His6
F650S eEF3 mutant is expressed as the only form of eEF3, however, it
shows essentially no binding of eEF1A (Fig. 4). Thus, these results
demonstrate that the mutation of Phe-650 to Ser in eEF3 can reduce the
physical interaction with eEF1A in vivo as well as
elongation, indicating the physiological significance of this
interaction.
Translation Elongation Factor eEF1A Shows Genetic Interactions with
eEF3--
Based on the above results and prior models linking the
functions of eEF1A and eEF3, we sought reciprocal genetic support for
this model by testing the effect of overexpression of either protein in
a cell bearing a mutant form of the other. Overexpression of eEF1A in
eEF3 wild type yeast results in reduced growth, even at permissive
temperatures (39), and consequently is unable to suppress the
Ts
To determine whether the growth defect correlates with a change in the
protein synthesis activities of the two proteins, the effect on
sensitivity to the translation inhibitor paromomycin was determined for
wild type, E122K, and N153T/D156E eEF1A strains with and without excess
eEF3. As shown in Fig. 5B, the E122K mutant shows a more
than 10-fold increase in paromomycin sensitivity in the presence of
excess eEF3, with a 50% reduction in growth achieved at 0.45 mg/ml
paromomycin compared with 5.84 mg/ml for a wild type strain. This
effect is not seen for another mutation in the GTP binding domain of
eEF1A that is unaffected by excess eEF3 (N153T/D156E, Fig.
5C). In all strains, however, the level of eEF3
overexpression was the same (Fig. 5D and data not shown). These results clearly show an allele-specific interaction between eEF1A
and eEF3 in vivo. To determine whether the F650S eEF3 mutant compromised for eEF1A binding was consequently unable to show the
synthetic growth defect, His6-tagged wild type and mutant eEF3 were overexpressed in strains expressing wild type and E122K eEF1A. Although the wild type His6-tagged eEF3, unlike
untagged eEF3, shows a slight slow growth defect in a wild type cell
(Fig. 5E), the reduced growth of the E122K eEF1A mutant
cells in the presence of excess His6-tagged eEF3 is much
more severe. Overexpression of His6-tagged F650S eEF3 was
unable to produce the synthetic slow growth phenotype in yeast with the
E122K form of eEF1A. Thus, the genetic link between eEF1A and eEF3 is
reciprocal and dependent on the ability of the two proteins to interact.
A Specific G-domain Mutant Form of eEF1A Impairs Binding to
eEF3--
To determine whether the synthetic slow growth defect of
eEF1A E122K and excess eEF3 is through an alteration in total
translation, [35S]methionine incorporation was monitored
at the permissive temperature of 30 °C for strains expressing wild
type or the E122K mutant form of eEF1A in the presence and absence of
excess eEF3. As shown in Fig.
6A, wild type yeast show a
slight increase in total translation in the presence of excess eEF3
(closed circles), consistent with the lack of
reduced growth or altered sensitivity to translation inhibitors (Fig.
5, A and B) and potentially indicating eEF3
activity or levels are limiting relative to eEF1A. A strain bearing the E122K mutation shows a 28% reduction in total translation
(open diamonds). In the presence of excess eEF3,
however, this effect is even more pronounced, a reduction of 55%
relative to wild type and 38% relative to the E122K mutant without
excess eEF3 (closed diamonds). Compared with the
E286K mutant unaffected by eEF3 overexpression but with a 32%
reduction in total translation, a modest 20% reduction in total
translation is observed with excess eEF3 in this mutant (Fig.
6B, triangles).
To understand the basis of the altered functional interaction between
the E122K mutation in eEF1A and eEF3, a series of strains expressing
wild type and E122K, N153T/D156E and E286K mutant forms of eEF1A were
transformed with plasmids expressing GST or a GST-eEF3 fusion protein.
Following pull-down of GST or GST-eEF3 from cell extracts with
glutathione beads, the amount of associated eEF1A was determined by
Western blot analysis. Wild type eEF1A is associated with GST-eEF3 and
not with GST alone (Fig. 7,
wt). Similar analysis of the E122K mutant form of eEF1A
showed an equivalent low level of association with either GST or
GST-eEF3, indicating essentially no binding of eEF3 (Fig. 7,
E122K). Two other eEF1A mutants in domain I (N153T/D156E) or
domain II (E286K) showed no significant difference in the ability to
associate with GST-eEF3 (Fig. 7). This demonstrates those eEF1A mutants
that reduce total translation (E286K) or other mutations in the GTP
binding domain (N153T/D156E) are not sufficient to alter this
association. Taken together, these results are consistent with the
interpretation that the sensitivity of the G-domain E122K eEF1A mutant
to eEF3 levels is manifest in subtle defects in protein synthesis and
the interaction between the two proteins and further indicate a
necessary balance between the activity of the two proteins.
One of the interesting motifs and functional requirements for eEF3
is the tandem ATP binding cassettes. The location of the F650S mutation
between the two cassettes may affect the interaction between these two
ATP binding sites. Work on other members of the ABC family has
indicated the requirement for two ATPase domains, as well as the
collaboration of the two domains in ATP hydrolysis (reviewed in Ref.
34). The loss of ATPase activity in this mutant form of eEF3 may
provide insight into the communication required between the two
domains. Prior studies that inactivated either of the ATP binding
domains by mutations in the highly conserved residues of the Walker A
motif, GXXXXGK, support the
essential role of ATP hydrolysis. A mutation of Gly-463 to Val appears
at least partially functional in vivo, although the analysis
was limited to slow growth on media to monitor complementation of the
wild type protein (21). A comparative analysis between this ATP binding
site mutation and the F650S mutation may provide insight into the
unique characteristics in vivo of altering the critical ATPase activity of eEF3 by completely different mechanisms.
Although the mutant form of eEF3 studied in this work does not affect
nonsense suppression or maintenance of reading frame, it was not
isolated for these characteristics. The in vitro data on the
role of eEF3 in favoring binding of cognate aa-tRNA (22) and the link
to eEF1A function support a potential role in accuracy at the A-site.
It is clear, however, that simply reducing the activity of eEF3 in
protein synthesis is not sufficient to alter fidelity as determined
here. Genetic screens designed to select mutant forms of eEF3 that
affect fidelity may shed light on the proposed role of eEF3 on this
process. A direct and highly specific role in misincorporation is still
possible. Further in vitro analysis of the F650S eEF3 and
E122K eEF1A mutants and their functional interaction during elongation
may explain whether the observed paromomycin sensitivity could point
toward altered misreading of sense codons. However, it is of note that
mutations in eEF1A that alter misincorporation also alter other forms
of fidelity (30), in particular nonsense suppression, so this high
specificity would be a novel and unanticipated finding.
This work shows that the physical interaction between eEF1A and eEF3 is
altered by mutations that compromise the activity of the two proteins,
but in a highly specific manner with regards to eEF1A. The bank of
mutations studied in this work were either selected to affect
translational fidelity (37) or designed as point mutations to affect
the affinity and specificity of GTP binding (30, 40). The E122K
mutation, the only one affected by eEF3 levels, was originally isolated
as a dominant +1 frameshift suppressor of the met2-1
reporter allele (37). The mutation, although located in the GTP binding
domain I (41), is in a fourth loop surrounding the bound nucleotide
separate from the well conserved GTP binding consensus elements such as
the NKXD motif altered in several other domain I mutations
tested. Structurally, Glu-122 does stabilizes the base packing of
Lys-156 of the NKXD consensus sequence (33). It is also not
the strongest of the dominant +1 frameshift suppressors isolated, as
the two best suppressors lie in the proposed aa-tRNA binding pocket of
domain II. The E122K mutation also shows unique characteristics as the
only form of eEF1A, including dramatic effects on programmed The effect of the combination of eEF1A E122K and excess eEF3 on
paromomycin sensitivity provides a strong in vivo link to A-site function and fidelity. The determination that the F650S mutant
fails to cause the synthetic growth defect in the E122K mutant
indicates either ATP hydrolysis or perhaps the ability to bind eEF1A
are critical for this phenotype. It is possible that, if eEF1A and eEF3
work cooperatively at the A-site, the loss of this interaction may
reduce translation. Thus, excess eEF3 in the eEF1A mutant may lead to
nonproductive interactions of the protein, perhaps with the ribosome.
Additionally, it is of interest that the effects identified link
through the nucleotide binding domains of both proteins, but outside
the consensus elements of either. The total translation rate, most
likely through the elongation cycle, is accelerated in the presence of
excess of eEF3 in the cells with wild type eEF1A, indicating the
protein may be limiting. However, the reverse is seen in E122K eEF1A
mutant cells. Thus, excess eEF3, if unable to properly or efficiently interact with eEF1A, is deleterious to the cell. The exact ratios of
eEF1A and eEF3 required for an efficient interaction, and the effect of
aa-tRNA and nucleotide on this association, remain to be determined.
It remains unclear why fungi require the function of eEF3. There is
speculation that an eEF3-like function remains in metazoans, perhaps
associated with or part of the ribosome, even though a homologous
protein has not been found (42). There is an ATPase activity associated
with metazoan ribosomes, which may perform the same function as eEF3 in
release of the deacylated tRNA from the E-site (20, 43, 44). Thus, the
critical functional interaction with eEF1A may provide a criterion for
establishing whether such a conserved function exists. Alternatively,
the physical association of eEF3 and eEF1A may be less important than
the allosteric effects of E-site release of deacylated tRNA on the
A-site. Because eEF1A is exceptionally well conserved, 81% identical
between yeast and mammals compared with 64% identity for eEF2 or 43%
identity for eEF1B
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
DISCUSSION
REFERENCES
was used for plasmid preparation. Procedures
for cell growth and genetic manipulations were according to standard
protocols (26). Yeast cells were grown in either YEPD (1% Bacto-yeast
extract, 2% peptone, 2% dextrose) or in defined synthetic complete
medium (C or C
) supplemented with 2% dextrose as the carbon source
unless noted. Yeast were transformed by the lithium acetate method
(27). Temperature sensitivity was assayed by growing strains containing
wild type (TKY597) or the mutant form of eEF3 (TKY599) to an
A600 of 1.0. Serial 10-fold dilutions (5 µl
each) were spotted on YEPD, followed by incubation at 13, 24, 30, and
37 °C for 3-7 days. Phenotypic suppression of a nonprogrammed +1
frameshift allele (met2-1 and his4-713) were
determined by spotting 10 µl of the same dilutions onto complete
medium lacking methionine or histidine, respectively, and incubating
for 5 days at 30 and 33 °C. Halo assays for sensitivity to
cycloheximide, paromomycin, and hygromycin B were as previously
described (28). Sensitivity to paromomycin in conditions where eEF3 is
overexpressed relative to wild type levels was determined in liquid
culture for at least two independent colonies of each strain grown at 30 °C in C
Ura to mid-log phase, diluted to
A600 of 0.05, and grown in triplicate in a
96-well microtiter assay plates with varying concentrations of
paromomycin. Plates were incubated with shaking at 30 °C and growth
monitored as the mean of the triplicate A600 at
22 h.
S. cerevisiae strains
)
alleles of YEF3 was performed by passage of pYEF3 through
XL1-Red E. coli (Stratagene), transformation of TKY554, and
replica plating on 5-fluoroorotic acid to monitor for slow growing
colonies at 34 °C. The mutated YEF3 plasmid was recovered
from yeast and transformed into E. coli DH5
, and isogenic
wild type and mutant eEF3 strains were constructed by transforming
pTKB594 (YEF3 TRP1) or pTKB595 (yef3 F650S
TRP1) into yeast TKY554 (29). Loss of the YEF3
URA3 plasmid was monitored by growth on 5-fluoroorotic acid,
producing strains TKY597 (wild type) and TKY599 (F650S).
-Galactosidase-based assays for
nonsense suppression and programmed
1 ribosomal frameshifting
efficiencies on the yeast L-A virus signal were performed. Nonsense
suppression assays were performed on strains TKY597 and TKY599 using a
URA3 wild type lacZ control plasmid (pUKC815tail)
or a URA3 plasmid with an in-frame UAA nonsense codon in
lacZ; (pUKC817tail (UAA)) as described previously (30). For
1 frameshifting assays, URA3-based pT125 (0 frame) and
pT124 (L-A virus
1 frame) plasmids were used (31). Total yeast
translation was monitored by in vivo
[35S]methionine incorporation as previously described
(30) using the indicated MET2 strains. Yeast polyribosome
preparation was performed as previously described (32) with the
following specifications. Yeast cultures were grown at 30 °C to
A600 of 0.8-1.0, divided, and extracted with
and without cycloheximide added to the cells and lysis buffer. Cell
extracts (40 A260) were layered on 35 ml of
7-47% sucrose gradient and centrifuged for 4 h at 27,000 rpm in
a SW28 rotor. The A254 was monitored and
recorded using a model 185 density gradient fractionator (ISCO, Inc.,
Lincoln, NE).
-32P]ATP.
Hydrolysis was allowed to proceed for 5 min at 30 °C, and Pi release determined. ATP hydrolysis levels were
calculated after subtracting the background for buffer alone.
Ura+galactose (25). Reactions containing 50 µg of total protein (determined by Bradford reagent, Bio-Rad) and 20 µl of a 50% glutathione-Sepharose 4B slurry (Sigma) in a 200-µl
final volume in KETN 150 buffer (150 mM KCl, 1 mM EDTA, 20 mM Tris, pH 8.0, 0.5% Nonidet, 1 mM PMSF) were mixed at 4 °C for 1 h. Beads were
washed three times with KETN buffer (either 150 mM KCl or
300 mM KCl), samples resolved by SDS-PAGE, and proteins
detected with a polyclonal antibody to yeast eEF1A by ECL (Amersham
Biosciences). Ni2+-NTA pull-down of eEF1A with
His6-tagged eEF3 were performed on extracts from strains
TKY702 and TKY707, expressing wild type and F650S
His6-tagged eEF3 expressed from a 2-µm TRP1
plasmids as the only form of eEF3, respectively, as described
previously (33).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
DISCUSSION
REFERENCES
alleles of the
YEF3 gene was performed. A mutation lethal at the
nonpermissive temperature of 37 °C (Fig.
1B), substituting phenylalanine 650 with serine, was identified. Additionally, there are
two substitutions, I153F and V332L, in both the wild type and mutant
plasmid-borne alleles relative to the published (6) and annotated yeast
genome sequences. These two substitutions likely reflect polymorphisms
between yeast strains and are in residues of low conservation between
fungal eEF3 sequences (10). To help determine the mechanism causing the
Ts
phenotype, the levels of wild type and F650S eEF3
proteins were analyzed following a shift to the nonpermissive
temperature of 37 °C. At 30 °C, eEF3 of the expected molecular
mass (~116 kDa) along with several degradation products were detected
in both the wild type and mutant strains (Fig. 1C). After
5 h at 37 °C, no significant change was observed in the total
eEF3 protein in either strain, indicating the tight Ts
phenotype is not a result of instability and loss of eEF3 at 37 °C
but rather a loss of function.
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Fig. 1.
The F650S mutation in yeast eEF3 results in
lethality at 37 °C. A, a schematic representation of
yeast eEF3 indicating the major motifs: an rpS5-like domain
(gray), two Walker type ATP binding motifs with the A and B
(black) signature motifs, a unique ELVES sequence
(diagonal stripe), and a basic cluster at the C
terminus (horizontal stripe). Numbers
indicate the start of each motif. The arrow shows the F650S
mutation. B, wild type (wt, TKY597) and F650S
mutant (F650S, TKY599) eEF3 strains were grown to mid
log-phase in YEPD at 30 °C, diluted to equal
A600, and spotted as 10-fold serial dilutions on
YEPD. Growth was monitored following 2-7 days at 37, 30, or 13 °C.
C, the Ts phenotype of the F650S mutation is
not the result of protein destabilization. The same strains were grown
to mid-log phase at 30 °C in YEPD, diluted to equal
A600 of 0.5 and grown at either 30 or 37 °C
for time points up to 5 h. Total protein was extracted, equal
protein amounts as determined by Bradford reagent were separated by
SDS-PAGE, and the eEF3 protein identified by Western blot analysis with
a polyclonal antibody to eEF3. Lanes 1,
3, and 5 are extracts from wild type, and
lanes 2, 4, and 6 are
extracts from the F650S mutant strains.
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Fig. 2.
The F650S mutation in yeast eEF3 results in
loss of ATPase activity. One pmol of His6-tagged wild
type (wt) or F650S mutant (F650S) eEF3 proteins were
analyzed for ATP hydrolysis. Both intrinsic ( ) as well as
yeast ribosome-stimulated (+) ATPase activities were
monitored. The nanomoles of Pi released from
[
-32P]ATP are shown after subtracting the hydrolysis
in the presence of buffer alone. The results are an average of three
experiments and the standard deviation shown.
Met or C
His media at either permissive or semipermissive
temperatures, indicating that the mutant allele does not reduce
fidelity to allow phenotypic suppression of a +1 frameshift mutation
(data not shown). These results were corroborated by several
quantitative measurements of translational fidelity. Suppression of the
1 frameshift signal from the yeast L-A virus or a UAA stop codon was
determined using lacZ-based assays of read-through. There
was no significant increase in the
-galactosidase activity levels of
either the
1 frameshift signal (6.4 ± 1% for wild type
versus 7.7 ± 2% for the mutant), or UAA stop codon
read-through (0.15 ± 0.02% for wild type versus
0.12 ± 0.04% for the mutant) reporter system. Thus, even though
paromomycin sensitivity usually correlates with altered translational
fidelity in yeast, this effect is not manifest on frameshifting or
nonsense suppression.
Sensitivity of wild type and F650S eEF3 expressing S. cerevisiae
strains to translation inhibitors
CH), indicating a retardation of the
disassembly of the ribosome and hence a slower translation elongation
rate. Similarly, analysis of the eEF1A E286K mutant strain shows a
significant increase in polyribosomes at 30 °C compared with wild
type yeast (Fig. 3D,
CH).
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Fig. 3.
The F650S mutation in eEF3 reduces
translation at the level of elongation. A and
B, total [35S]methionine incorporation in wild
type (TKY623, circles) and F650S (TKY625,
squares) eEF3 strains (A) or wild type (TKY621,
circles) and E286K (TKY622, triangles) eEF1A
strains (B) was measured by growing the culture to mid log
phase in C Met and labeled for varying times with
[35S]methionine. Incorporation is expressed as
cpm/A600 unit of cells. C and
D, polyribosome extracts of wild type (TKY597) and F650S
(TKY599) eEF3 strains (C) or wild type (MC214) and E286K
(TKY588) eEF1A strains grown at 30 °C (D) were prepared
and analyzed in the presence (+CH) and absence
(
CH) of cycloheximide by 7-47% sucrose gradients.
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Fig. 4.
The F650S mutant form of eEF3 shows reduced
interactions with eEF1A. Cell extracts were prepared from
His6-tagged wild type (TKY702) and F650S (TKY707) eEF3
strains. Equal amounts of protein were incubated with
Ni2+-NTA beads and pull-downs performed. The pellet
(P, 100% of input), unbound supernatant (S, 5%
of input), and total extract (E, 10% of input) were
separated by SDS-PAGE and analyzed by Western blotting with a
polyclonal antibody to eEF1A.
defect in eEF3 mutant cells (data not shown). The
effect of excess eEF3 was monitored on strains bearing a series of
alleles expressing mutant forms of eEF1A with alterations of the E40K,
E122K, E122Q, D130N, T142I, N153T, D156N, or N153T/D156E residues in
the GTP binding domain I and residues E286K, E295K, or E317K in domain II near the proposed aa-tRNA binding site (30, 37, 38). A construct
expressing eEF3 from its own promoter on a 2µ URA3 plasmid, YEpEF3 (29), was used in strains bearing the wild type or
mutant TEF2 alleles on TRP1 plasmids (MC214,
TKY225, TKY226, TKY229, TKY252, and TKY588). Alternatively, eEF3 with a
functional N-terminal His6 tag expressed from a 2µ
TRP1 plasmid (pTKB628) was used in strains bearing the wild
type or mutant TEF2 alleles on a URA3 plasmid
(MC213, TKY111-117). Either plasmid results in an ~10-fold increase
in eEF3 levels (Fig. 5D and
data not shown) but no or little effect of growth on a wild type strain
(Fig. 5, A (untagged) and E
(His6-tagged eEF3)). A synthetic slow growth defect in
strains overexpressing eEF3 was observed specifically for the E122K
mutant strains (TKY113 or TKY252), but no other eEF1A mutants, at all
temperatures tested (Fig. 5 (A and E) and data
not shown).
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Fig. 5.
Excess wild type eEF3 results in growth and
drug sensitivity phenotypes in an E122K eEF1A mutant strain.
A, strains expressing wild type (wt, MC214),
E286K (TKY588), and E122K (TKY252) mutant eEF1A were transformed with a
URA3 plasmid containing no eEF3 gene (pRS426, )
or YEF3 encoding eEF3 on a 2µ URA3 plasmid
(YEpEF3, +). Strains were grown to mid-log phase at 30 °C
in C
Ura, diluted to equal A600, spotted as
10-fold serial dilutions, and grown at 37, 30, 24, or 13 °C for 2-7
days. B and C, the same strains were grown to
mid-log phase at 30 °C in C
Ura, diluted to an equal
A600 of 0.05, and grown in the indicated
concentrations of paromomycin in triplicate. Sensitivity was determined
by plotting the A600 at the indicated drug
concentration after 22 h of growth at 30 °C. Panel
B shows wild type (circles) and the E122K mutant
(diamond) strains, and C shows wild type
(circles) and the N153T/D156E mutant (triangles)
strains, without (open symbols) and with
(closed symbols) YEpEF3 overexpressing eEF3.
D, Western blot analysis of eEF3 levels in the strains shown
in panel A detected with a polyclonal antibody to
eEF3. E, strains expressing wild type (wt, MC214)
and E122K (TKY113) mutant eEF1A were transformed with a 2µ
TRP1 plasmid containing no gene (pRS424,
), or
expressing His6-tagged forms of wild type (pTKB628) or
F650S (pTKB659) eEF3. Strains were grown to mid-log phase at 30 °C
in C
Trp, diluted to equal A600, spotted as
10-fold serial dilutions, and grown at 30 °C for 3 days.
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Fig. 6.
Excess eEF3 reduces translation in a strain
expressing a mutant form of eEF1A. Total
[35S]methionine incorporation was determined in wild type
(TKY621, circles) and E122K (TKY696, diamond,
A) and E286K (TKY622, triangle, B).
eEF1A strains without (open symbols) or with
(closed symbols) YEpEF3 overexpressing eEF3.
Total translation was measured by growing the cultures to mid-log phase
in C Ura
Met and labeled for varying times in
[35S]methionine. Incorporation (cpm) is expressed per
A600 unit of cells.
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Fig. 7.
The E122K mutant form of eEF1A shows a
reduced interaction with eEF3. Cell extracts were prepared from
eEF1A wild type (MC214) and E122K (TKY252), N153T/D156E (TKY229), and
E286K (TKY588) mutant strains transformed with plasmids expressing GST
or a GST-eEF3 fusion under a galactose-inducible promoter and grown in
C Ura+galactose. Equal amounts of protein were incubated with
glutathione beads and pull-downs performed. The pellet (P,
100% of input), unbound supernatant (S, 5% of input) and
total extract (E, 10% of input) were separated by SDS-PAGE
and analyzed by Western blotting with a polyclonal antibody to
eEF1A.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
DISCUSSION
REFERENCES
1
ribosomal frameshifting, an event linked to A-site occupancy (28), as
well as less dependence on catalyzed nucleotide exchange by eEF1B
(30).
, a unique eEF3 binding site is not obvious. The
unique characteristics of the F650S mutation in eEF3 now provide an
avenue toward a genetic dissection of eEF3 function in vivo,
understanding the link between the two ATPase domains, and the
identification of other factors that interact with eEF3.
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ACKNOWLEDGEMENTS |
---|
We thank Thomas Dever for keen insights into the polyribosome experiments, Gregers Andersen and members of the Kinzy laboratory for comments, Erika Pladies and Marcello Vinces for technical assistance, and the Robert Wood Johnson Medical School DNA sequencing facility.
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FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Current address: Baxter Bioscience, Glendale, CA 91203.
** Supported by National Institutes of Health Grant RO1 GM57483. To whom correspondence should be addressed: Dept. of Molecular Genetics, Microbiology & Immunology, University of Medicine and Dentistry of New Jersey Robert Wood Johnson Medical School, 675 Hoes Ln., Piscataway, NJ 08854-5635. Tel.: 732-235-5450; Fax: 732-235-5223; E-mail: kinzytg@umdnj.edu.
Published, JBC Papers in Press, December 18, 2002, DOI 10.1074/jbc.M209224200
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ABBREVIATIONS |
---|
The abbreviations used are: eEF, eukaryotic elongation factor; aa-tRNA, aminoacyl-tRNA; GST, glutathione S-transferase; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; NTA, nitrilotriacetic acid; ABC, ATP binding cassette.
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