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INTRODUCTION |
Eukaryotic elongation factor 2 (eEF2)1 and its prokaryotic
counterpart, elongation factor G (EF-G), promote the translocation of
the ribosome along messenger RNA during the elongation phase of protein
synthesis. Hydrolysis of GTP to GDP drives translocation and is
associated with a presumed conformational change in eEF2. Sordarin (1)
and its analogs are fungal-specific translation inhibitors (2, 3) that
bind to the eEF2-ribosome-GDP complex in Saccharomyces
cerevisiae, stabilizing the post-translocational GDP form in a
manner similar to that of fusidic acid (3). However, in contrast to
fusidic acid, which binds both EF-G and eEF2 and is a general
translocation inhibitor, sordarin inhibits translation only in
susceptible fungi, deriving its unique specificity from the source of
eEF2 (3-5). The observation that eEF2 is the major determinant of
sordarin specificity was confirmed by the identification of 15 unique
sordarin-resistant alleles of EFT1 and EFT2 that encode eEF2 in S. cerevisiae. In our original
characterization of 21 sordarin-resistant mutants, five mutations were
not linked to the EFT1 or EFT2 genes. In this
work, we show that these five mutations map to the essential ribosomal
protein L10e.
The ribosome, although not contributing significantly to the fungal
specificity of sordarin, is a critical partner in forming the
stabilized post-translocational complex (3). Detection of a complex
between fungal eEF2 and a labeled sordarin analog is strongly dependent
upon the presence of ribosomes. L10, the prokaryotic counterpart of
S. cerevisiae L10e, has been localized to the base of the
stalk structure conserved in all large ribosomal subunits (6-8). The
eukaryotic ribosomal stalk proteins L10e, L12eIA, L12eIIA, L12eIB, and
L12eIIB in S. cerevisiae (9, 10) comprise a pentameric
structure that is similar to the L10 and L7/L12 complex in
Escherichia coli ribosomes. The conservation of the stalk
structure has been visualized in recent cryoimages of both 70S (11), in
which the binding position of the EF-G-GDP-fusidic acid complex is
observed in detail, and 80S (12) ribosomes. The prokaryotic L7/L12
ribosomal proteins have been studied extensively by many physical and
biological techniques, but much less is known about the structure and
function of prokaryotic or eukaryotic L10. L10 is among the proteins
reported to be cross-linked to eEF2 in 80S ribosomes by bifunctional
reagents (13). In yeast, mutational analysis of the L10e ribosomal
protein gene has shown that only L10e is essential, and that the
carboxyl-terminal 132 amino acids of the L10e protein are required for
viability. However, the L12e proteins that comprise the L10e/L12e
pentameric complex are not essential (14). Several findings suggest
that there are associations between the stalk proteins and elongation
factors. Mutations in L7/L12 perturb both EF-Tu and EF-G functions in
E. coli (15). Chemical cross-links have been observed
between EF-Tu and EF-G and the L7/L12 complex (reviewed in Ref. 16). Of
particular relevance to the present case, cross-links are observed
between the EF-G and L7/L12 proteins in the presence of the
nonhydrolyzable GTP analog GMPPCP, but not in the presence of GDP and
fusidic acid (17, 18). L7/L12 proteins in the EF-G-fusidic
acid-ribosome complex are resistant to trypsin proteolysis (19). Our
current results add to the body of information implicating L10e and
eEF2 interactions to be important in translocation. These studies
define a role for a small region of L10e in mediating inhibition by a new class of natural product with unprecedented selectivity for fungal
protein synthesis and provide evidence for a functional interaction
between L10e and eEF2.
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EXPERIMENTAL PROCEDURES |
Yeast Strains, Plasmids, and Compounds--
Sordarin was
isolated essentially as described from Sordaria arenosa (1),
and preparation of the sordarin analog L-793,422 has been described
previously (3, 20). The sordarin-resistant strains sRb1, sRb2, sRb5,
sRb13, and sRb14 were generated essentially as described previously
(3), except that mutant strains that demonstrated sordarin resistance
unlinked to EFT1 or EFT2 were selected for
characterization in this study. The wild-type yeast strain YPH54 used
for genetic analysis and the plasmid YCplac111 used for subcloning the
S. cerevisiae L12e and L10e genes have both been described
previously (3).
Cloning of L10e and L12e Genes--
The ribosomal protein genes
L10e, L12eIA, L12eIB, L12eIIA, and L12eIIB were cloned by polymerase
chain reaction (PCR) using synthetic oligonucleotides (Table I) that
were designed based on published sequences (10). These oligonucleotides
also contain the 5' BglII restriction enzyme recognition
sequence 5'-AGATCT-3' after the sequence 5'-GCGCGC-3' for subcloning
purposes. S. cerevisiae genomic DNA from wild-type and
sordarin-resistant strains was prepared by standard procedures and
purified with a QIAamp Tissue Kit (Qiagen) according to the
manufacturer's specifications. PCR products were amplified from
genomic DNA with the synthetic oligonucleotides listed in Table I,
using Klentaq polymerase () according to
the manufacturer's recommendations. The PCR products were purified over Qiaquick columns (Qiagen), and putative PCR-generated L10e and
L12e ribosomal protein genes were identified by restriction analysis.
Each PCR product was digested with BglII, followed by ligation into the BamHI site of YCplac111. Ligation mixes
were transformed into the E. coli strain DH5
(Life
Technologies Inc.). E. coli transformants were pooled,
inoculated into LB broth containing 100 µg/ml ampicillin, and grown
overnight at 37 °C. Plasmid DNA was prepared, and the constructs
YCpL10e, YCpL12eIA, YCpL12eIB, YCpL12eIIA, and YCpL12eIIB were verified
by restriction analysis. Plasmid DNA pools of each construct described
above were used to transform the S. cerevisiae
eft
1/eft
2 strain YEFD12 h (21) to leucine prototrophy.
Colonies from the individual transformations were pooled, and 1 × 106 cells were plated on SC medium containing 2% agar and
5 µg/ml sordarin. Cultures were incubated at 29 °C for
approximately 4 days until colonies appeared. The pooled transformants
were also tested in liquid culture essentially as described previously
(3). In this assay, cells were inoculated in SC medium depleted of leucine, followed by incubation at 29 °C in a humidified chamber for
approximately 8 days. Transformants exhibiting
plasmid-dependent sordarin resistance were clonally
purified, followed by plasmid rescue and transformation into E. coli.
Molecular Mapping of eEF2 Mutations--
Plasmid DNA from
transformants with the resistance phenotype was sequenced with an ABI
Prism 373 DNA Sequencer according to the manufacturer's
recommendations (Perkin-Elmer Applied Biosystems). Sequences were
analyzed using Sequencher DNA analysis software (Gene Codes Corp.). The
products of three additional independent PCR reactions were sequenced
to rule out the possibility of errors due to polymerase infidelity.
Cell Extracts--
Washed cells grown to logarithmic phase
(A600, 1-1.5) in YPAD medium at 30 °C were
disrupted, and post-ribosomal extracts were prepared essentially as
described by Skogerson (22). Wild-type eEF2 was purified to a final
specific activity of 2.5 pmol/µg in the diphtheria-catalyzed ADP
ribosylation assay. Ribosomes were prepared using standard procedures
(23), and their concentration was estimated using the figure of 18.6 pmol/A260 unit. Subunits were separated by
sucrose gradient sedimentation (23). Ribosomal proteins were extracted
using acetic acid precipitation and acetone extraction, and core
particles remaining after the removal of the acidic L12e proteins were
prepared by treating intact ribosomes with 50% ethanol and either 0.4 or 1.0 M KCl (24). SDS-polyacrylamide gel electrophoresis
chromatography and Coomassie staining were used to monitor the release
of protein of the expected size (about 13 kDa).
Binding--
[3H]L-793,422 was prepared at 20 mCi/mg (8000 mCi/mmol) at a concentration of 0.004 mg/ml by the Drug
Metabolism Department at Merck Research Laboratories. The gel
filtration method used previously (3) was replaced by a filter binding
assay. Filter choice, presoaking, and washing regimens were carefully
optimized. GF/C filters (Millipore) were presoaked in 0.15%
polyethyleneimine plus 0.25% Triton X-100. Binding was performed in a
1-ml volume (or greater, to check the validity of Kd
measurements) of 50 mM Tris-HCl, pH 7.4, 10 mM
MgCl2, 25 µM guanosine
5'-3-O-(thio)triphosphate, and 10 mM
-mercaptoethanol in glass tubes for 30 min at 22 °C. Chilled
0.5% Triton X-100 (3 ml) was added, and the tube contents were
immediately filtered for approximately 3 s under a vacuum maintained at a constant pressure of 300 mm Hg. Two additional 3 ml
washes were performed, and the filter was removed for counting in
scintillation fluid. Background binding of about 70 dpm was obtained
with [3H]L-793,422 at a concentration of 2 nM. Parallel ribosylation determinations with and without
ribosomes on GF/C filters using filtration conditions similar to the
assay for [3H]L-793,422 binding showed that the presence
of ribosomal particles makes no difference to the trapping efficiency
of eEF2 by GF/C filters. Maximal binding (Bmax)
was reached at 0.15 pmol of [3H]L-793,422 bound with 10 µg of S30 extract or at 0.39 pmol of [3H]L-793,422 with
0.46 pmol of purified eEF2 plus 1.5 pmol of salt-washed ribosomes. The
apparent Kd values were deduced from Scatchard transformations of the binding data.
GTP Binding--
The gel filtration assay used previously (3)
was also replaced by a filter binding assay. Nitrocellulose filters
(0.45 µm; 25-mm diameter; BA85; Schleicher & Schuell) were presoaked in 50 mM Tris-HCl, pH 7.5, 10 mM
MgCl2, 100 mM NH4Cl, and 2 mM
-mercaptoethanol. The binding reaction (200 µl) was
performed in the same buffer plus 0.92 pmol of purified eEF2, 3 pmol of salt-washed ribosomes, 0.1 µCi of
[guanosine-8-3H]GTP (12 Ci/mmol), and
increasing amounts of sordarin or analogs. After 30 min at 22 °C, 1 ml of ice-cold buffer was added, and the contents were filtered as
described above, followed by a final 1 ml wash. Under these conditions,
approximately 0.1 mol of nucleotide was trapped per mole of eEF2 in the
absence of any compound.
GTPase Assay--
The eEF2-dependent ribosomal
GTPase activity was measured at 22 °C for wild-type ribosomes and
each mutant using 0.46 pmol of wild-type eEF2 and 0.8 pmol of ribosomes
per 20-µl reaction at 20 mM Tris, pH 7.4, 5 mM Mg(OAc)2, 20 mM
NH4Cl, 2 mM dithiothreitol, and 500 µM [
-32P]GTP (200 dpm/pmol). Duplicate
1-µl samples were removed at the indicated times and quenched, and
GDP release was analyzed as described previously (25).
Translation Assays--
Polymerization using S30 extracts was
performed at 22 °C essentially as described by Hussain and Leibowitz
(26) with poly(U) (Calbiochem) at 160 µg/ml as a template during the
linear period of incorporation at 22 °C using
[3H]phenylalanine (4000 dpm/pmol) as a precursor.
Polymerization reactions were performed with either intact ribosomes or
those reconstituted from separated subunits and contained 0.05 A260 unit of ribosomes and 10 µg of S100
protein as a source of all soluble factors. In these assays,
incorporation of [3H]phenylalanine was limited by the
level of ribosomes.
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RESULTS AND DISCUSSION |
A Class of Sordarin-resistant Mutations Not Linked to eEF2--
We
have previously described S. cerevisiae mutants containing
unique amino acid substitutions in eEF2 that confer resistance to
sordarin (3). These mutants were obtained by spontaneous mutagenesis of
eft1
/eft2
deletion strains harboring an episomal copy
of either EFT1 or EFT2, genes that encode eEF2.
Linkage of sordarin resistance to eEF2 was confirmed by demonstrating
plasmid-dependent sordarin resistance in sensitive strains.
In addition to the eEF2 mutants, five additional sordarin-resistant
mutants (sRb1, sRb2, sRb5, sRb13, and sRb14) were identified that
harbored wild-type episomal EFT1 or EFT2. All of
these strains had sordarin IC50s for growth inhibition in
SC medium of 10-30 µg/ml (Fig. 1). No additional phenotypes were observed for these mutants in standard laboratory media, with the exception of a growth defect in strain sRb2
(data not shown).

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Fig. 1.
Non-eEF2 (sRb) mutants are partially
resistant to sordarin. Growth inhibition assays were performed in
a microtiter format in which cells were inoculated to an
A600 of 0.02 in SC medium containing sordarin
serially diluted 2-fold from 0.2 to 100 µg/ml, followed by incubation
at 29 °C for approximately 24 h. , sRb1; , sRb2; ×,
sRb5; , sRb13; , sRb14; , wild-type (YEFD12h).
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To characterize the genetic elements that mediate sordarin resistance,
diploids were generated from crosses of the sRb strains and wild-type
strain YPH54. In growth inhibition assays, sRb/YPH54 diploids exhibited
a codominant phenotype at low concentrations of sordarin. Tetrad
analysis of all diploids showed that sordarin resistance segregated
2s:2r, indicating that the resistance phenotype
results from mutations in a single chromosomal gene (data not shown).
Complementation testing was performed on sRb/sRb diploid strains to
determine the number of genes represented. Approximately the same level of resistance to sordarin was observed among the sRb/sRb diploids, suggesting that all the mutant strains belong to the same
complementation group. This result was confirmed by tetrad analysis of
the sRb/sRb mutant strains, indicating that the sRb mutation is defined
by a single nuclear gene (data not shown).
Resistance in Vitro Is Associated with the Large Ribosomal
Subunit--
S30 extracts prepared from each of the sRb mutants showed
a marked reduction in sordarin sensitivity in in vitro
translation assays using poly(U) as a template. This is illustrated for
the sRb5 mutant in Fig. 2A.
The other four mutants displayed essentially identical inhibition
curves (data not shown), which shifted about 6-fold from the control
value at low sordarin concentrations and showed no further inhibition
at levels up to 5 µg/ml. This biphasic response could be due to the
functional redundancy of a ribosomal protein(s) or to the limitations
of the assay.

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Fig. 2.
Resistance to sordarin in translation assays
is conferred by the large ribosomal subunit. In vitro
translation assays were performed as described under "Experimental
Procedures." A, , S30 extracts derived from wild-type
yeast; , sRb5. 100% incorporation for wild-type and mutant was 75 and 68 pmol, respectively. B, reconstitution from S100
extracts and ribosomes. , wild-type S100 plus wild-type ribosomes
(11 pmol); , sRb5 S100 plus sRb5 ribosomes (13.3 pmol); ,
wild-type S100 plus sRb5 ribosomes (12.5 pmol); , sRb5 S100 plus
wild-type ribosomes (11.8 pmol). C, reconstitution after
ribosomal subunit separation. In each case, wild-type S100 was used as
the source of elongation factors. , wild-type large subunit plus
wild-type small subunit (5.6 pmol); , sRb5 large subunit plus sRb5
small subunit (6.7 pmol); , wild-type large subunit plus sRb5 small
subunit (7.2 pmol); , sRb5 large subunit plus wild-type small
subunit (7.8 pmol). In each case, the bracketed value is 100%
incorporation for the 10-min reaction.
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Fractionation of the S30 extracts into salt-washed ribosomes and S100,
followed by reconstitution, did not alter the sordarin inhibition curve
for the sRb mutants. However, experiments using S100 extracts and
ribosomes from either the sordarin-sensitive parental strain or the sRb
mutants allowed for the identification of the resistance determinant as
the ribosome in all cases (sRb5 is shown in Fig. 2B).
Ribosomes prepared from strain sRb5 were further separated into large
and small subunits, and, as shown in Fig. 2C, resistance was
found to be associated with the large ribosomal subunit via in
vitro translation experiments. Reconstitution studies between
subunits of wild-type strains and those of the other sRb strains gave
similar results (data not shown).
Ribosomal Protein L10e Confers Sordarin
Resistance--
Identification of the large ribosomal subunit as the
determinant of sordarin resistance, in addition to the biochemical and genetic associations of the stalk proteins with EF-G or eEF2 (11, 13,
15-19), focused our efforts on the ribosomal stalk proteins. Because
both fusidic acid and sordarin stabilize the eEF2-GDP-ribosome complex,
we hypothesized that proteins in the stalk were candidate biochemical
targets of sordarin. In S. cerevisiae ribosomes, the stalk
is composed of five proteins: L10e and a tetramer of L12eIA, L12eIIA,
L12eIB, and L12eIIB. These proteins are the eukaryotic counterparts of
the L10 and L7/L12 proteins in E. coli. S. cerevisiae L10e- and L12e-specific oligonucleotides were
synthesized (Table I) and used in PCR
amplification of genomic DNA from the sordarin-resistant strain sRb5.
Putative L10e and L12e ribosomal protein genes were verified,
subcloned, and transformed into sordarin-sensitive yeast. Colonies from
individual transformations were pooled and plated on SC agar medium
containing sordarin or tested in growth inhibition assays. After
incubation for 4 days at 29 °C, colonies appeared only on plates
with transformed cells containing episomal L10e from sRb5 (data not
shown). Representative data from a growth inhibition assay are shown in
Fig. 3A. Based on these
results, L10e clones from the four remaining sRb strains were generated and examined in a similar manner. As shown in Fig. 3B, L10e
from all strains conferred sordarin resistance, although less for sRb2 than for the others, as was seen for the original isolated mutants (Fig. 1).
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Table I
Sequence of synthetic oligonucleotides used for PCR amplification of
the S. cerevisiae L10e and L12e ribosomal protein genes
Sequences are written 5' to 3'; sense (+) and antisense ( ) strands
are indicated. Lower case letters represent sequences used for cloning
the PCR products; upper case letters represent the partial DNA sequence
from the L10e and L12e ribosomal protein genes (10) that were used for
PCR amplification.
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Fig. 3.
L10e cloned from the sRb mutant strains
confers sordarin resistance. Growth inhibition assays with strains
harboring YCpL10e, YCpL12eIA, YCpL12eIB, YCpL12eIIA, and YCpL12eIIB.
A, the L10e and L12e ribosomal protein genes from wild-type
and the sordarin-resistant mutant sRb5 strain were cloned and expressed
in S. cerevisiae as described under "Experimental
Procedures." , YCpL12eIA; , YCpL12eIB; ×, YCpL12eIIA; ,
YCpL12eIIB; , YCpL10e cloned from YEFD12h; , YCpL10e cloned from
sRb5; , the YCp111 yeast shuttle vector. L12e ribosomal protein
genes were cloned from strain sRb5. B, L10e from all sRb
mutants was cloned and expressed in S. cerevisiae. ,
YCpL10e from sRb1; , YCpL10e from sRb2; ×, YCpL10e from sRb5; ,
YCpL10e from sRb13; , YCpL10e from sRb14; , YCp111. Growth
inhibition assays were performed as described previously (3), except
that SC medium and incubation to 48 h were used.
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Plasmid DNA from the L10e transformed strains was recovered and
sequenced. Several nucleotides that differ from the sequence published
by Hunter Newton et al. (Ref. 10; GenBankTM
accession number M26506) were identified (Fig.
4). To determine the specific change
responsible for the resistant phenotype, the L10e gene from the
parental strain YEFD12h was amplified by PCR and sequenced. A
comparison of the published sequence with our parental strain showed
that L10e in YEFD12h contains three nucleotide changes that result in
three conservative amino acid substitutions: I4V, V27I, and T281S.
Based on this result, we were able to identify the L10e amino acid
substitutions that confer sordarin resistance (Fig. 4). All of the
amino acid substitutions resulted from single nucleotide changes, with
the exception of sRb14 (S134
), which results from the deletion of
three nucleotides. All of the changes conferring resistance were found
to be clustered within 10 residues located approximately in the middle
of the L10e polypeptide.

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Fig. 4.
Peptide sequence of L10e and the amino acid
substitutions that confer sordarin resistance. The ribosomal
protein gene L10e was cloned from the wild-type strain YEFD12h and the
sordarin-resistant mutants as described under
"Experimental Procedures." Changes in L10e that confer
the resistance phenotype map to four amino acid substitutions and one
amino acid deletion clustered in a conserved 10-amino acid region of
L10e (sRb1-Q137P, sRb2-T143I, sRb5-Q137K, sRb13-T143A, and
sRb14-S134 ). All amino acid substitutions result from single
nucleotide changes, with the exception of S134 , which results from
the deletion of three nucleotides. The amino acid sequence of L10e from
the wild-type parental strain YEFD12h contains the conservative
substitutions I4V, V27I, and T281S that differ from
GenBankTM accession number M26506.
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L-793,422 Binding Studies--
Fungal specificity by the sordarin
class of compounds is conferred by the source of eEF2. However, for a
stable interaction to be detected with [3H]L-793,422,
ribosomes must be present (Table II).
Titration of the ribosomes with a fixed amount of eEF2 showed that an
approximately 3-fold excess of ribosomes was required to give
saturation binding. Using this level, [3H]L-793,422
binding reached saturation at about 0.85 pmol/pmol eEF2, with an
apparent Kd of 2.5 nM. No substantial
differences were seen for [3H]L-793,422 binding
Bmax or the apparent Kd when
studies were conducted with mutant ribosomes (sRb1, sRb2, or sRb5) and wild-type eEF2. Also, the level of ribosomes required for saturation did not deviate significantly from the wild-type value for any of the
mutants (data not shown).
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Table II
Binding of [3H]L-793,422 to ribosomal components
Binding experiments were performed as described under "Experimental
Procedures" using 0.46 pmol of eEF2, 1.5 pmol of whole ribosomes or
individual subunits, 25 µM guanosine
5'-3-O-(thio)triphosphate, and 2 nM
[3H]L-793,422. Core ribosomes lack the acidic L12e proteins
and were prepared by treating intact ribosomes with 50% ethanol and
either 0.4 or 1.0 M KCl. Data are given for wild-type
ribosomal components. Reconstitution was performed for separated
subunits of sRB5 ribosomes with essentially identical results.
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The minimal requirements for the ribosomal contribution to binding were
assessed by dissociating ribosomes into components (Table II). The L12e
stalk was removed from ribosomes, i.e. core ribosomes,
without significant binding loss. However, the separation of subunits
caused a marked decrease in their ability to stimulate eEF2-associated
binding. Residual binding in the presence of individual subunits was
consistent with the approximately 10% cross-contamination of large
subunits by light as estimated by Western blotting with L10e antiserum.
Reconstitution of the separated subunits restored binding. Solubilized
protein from complete ribosomes or large subunits did not stimulate the
very low level of binding exhibited by eEF2 alone. Wild-type L10e
purified by reverse phase chromatography also failed to stimulate eEF2
binding of sordarin (data not shown). Thus, the ribosomal component of
binding requires both subunits and possibly the interface between them
and cannot be supplied by soluble ribosomal proteins.
Sordarin as a Translocation Inhibitor--
To examine
translocation more directly, we assessed the stability of the
nucleotide-eEF2-ribosome complex for each of the ribosomal mutants,
using [guanosine-8-3H]GTP and a rapid
filtration assay. The amount of labeled nucleotide trapped in the
presence of increasing levels of sordarin is shown in Fig.
5A for wild-type eEF2 plus
wild-type or mutant sRb5 ribosomes. Without the addition of sordarin,
approximately 0.1 mol of nucleotide is trapped per mole of eEF2.
Sordarin increased this to a maximum of 0.28 (± 0.007) mol for
wild-type ribosomes (with an IC50 of about 25 ng/ml), 0.23 (± 0.011) mol for sRb1 ribosomes, 0.18 (± 0.007) mol for sRb2
ribosomes, 0.20 (± 0.010) mol for sRb5 ribosomes, 0.22 (± 0.009) mol
for sRb13 ribosomes, and 0.16 (± 0.005) mol for sRb14 ribosomes. Thus,
the eEF2 complexes containing mutant ribosomes all showed a 20-40%
reduction in sordarin-conferred stabilization when compared with
wild-type ribosomal complex, consistent with their approximately equal
resistance to sordarin for in vitro translation.

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Fig. 5.
A, mutant ribosomes show reduced
sordarin-conferred stabilization of the eEF2-nucleotide complex.
Binding of [guanosine-8-3H]GTP was performed
with 0.92 pmol of wild-type eEF2 and 3 pmol of salt-washed ribosomes
from wild-type ( ) or sRB5 ( ) on nitrocellulose filters as
described under "Experimental Procedures." B, mutant
ribosomes show reduced stimulation of eEF2 GTPase activity. A time
course of GDP release is shown as the number of moles released per mole
of eEF2 for wild-type ribosomes (circles) and sRb1 ribosomes
(squares) in the absence (open symbols) and
presence (filled symbols) of 1 µg/ml sordarin. The value
without eEF2 (reaching approximately 0.2 mol at 10 min for both
wild-type and sRb1 ribosomes) was subtracted in each case. GTPase
activity for wild-type ribosomes in the presence of 2 mM
fusidic acid is shown (×). Data shown are the averaged results of a
single experiment performed in duplicate reactions with duplicate
samples.
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Turnover rates in translocation are reflected in the ribosomal
EF2-dependent GTPase activity. Fusidic acid at low
millimolar levels strongly inhibits the ribosomal-dependent
GTPase activity of yeast eEF2 (27). Sordarin, although exerting its
effect with much higher affinity, has less effect on GTPase activity.
Sordarin concentrations of
100 ng/ml maximally inhibited the GTPase
activity of wild-type ribosomes and eEF2 by about 60%. Similar
experiments with wild-type eEF2 and each of the ribosomal mutants
showed similar titration curves (data not shown), but the inhibition of
GTPase activity was reduced 30-40% relative to wild-type eEF2 and
ribosomes. Fig. 5B shows a time course for sRb1 compared
with wild-type ribosomes at a single saturating sordarin concentration
of 1 µg/ml. Approximately the same reduction was obtained whether the
mutant GTPase activity was lower than the wild-type (0.8 pmol/pmol
eEF2/min), as for sRb1 (0.4 pmol/pmol/min eEF2), or higher (1.1 units
for sRb2 and 1.2 units for sRb5). Consistent with these elevated GTPase
rates, ribosomes sRb2 and sRb5 both showed somewhat elevated
translation rates with wild-type eEF2 in the absence of sordarin under
the established assay conditions (data for sRb5 may be seen in the legend of Fig. 2). Overall, the magnitude of the reduction in GTPase
activity exhibited by the mutants resembles that seen for the
nucleotide complex. Because we have demonstrated that the ribosomal
mutants are not impaired in sordarin binding, the reduced sensitivity
seen in GTPase and complex stability must represent a functional
alteration in translocation for the mutants. The amino acid
substitutions that result in the sordarin-resistant phenotype cluster
to a 10-amino acid region approximately in the middle of the L10e
polypeptide and suggest that this region may be important in
conformational flexibility during translocation.
In our initial study, we emphasized the qualitative similarity between
the action of sordarin and fusidic acid. Sordarin stabilizes the
eEF2-ribosome-GDP complex in a manner similar to that of fusidic acid
(Ref. 3 and this work). Quite compellingly, there is some cross-resistance between sordarin and fusidic acid resistance in eEF2
alleles; furthermore, sordarin-resistant mutations cluster in a pattern
similar to that demonstrated for fusidic acid resistance alleles in
S. typhimurium EF-G (28). The present data demonstrate the
quantitative differences in the effects of sordarin and fusidic acid
and suggest that upon closer analysis, their precise modes of action
will most certainly differ.
Our results have implications for the physical and functional
relationship between L10e and eEF2. Others have shown that L10 is
exposed on the surface of 70S ribosomes in E. coli (29), and
chemical cross-links have been observed between rat L10 and eEF2.
Recently, a three-dimensional cryoelectron microscopy map of the
ribosome-EF-G-GDP-fusidic acid complex has been obtained (11). In this
structure, EF-G makes contacts with both the 30S and 50S subunits.
Moreover, domains I and V of EF-G make contacts with the base of the
L7/L12 stalk complex. Many reports have implicated the stalk complex in
factor binding and translocation, and conformational changes in the
stalk and EF-G are thought to drive the translocation process (6, 16,
29). The highly mobile nature of the stalk is well established, and a
recent report proposes that conformational changes in the
amino-terminal domains of L7/L12 are coupled with polypeptide synthesis
(30). In the cryoelectron microscopy map of the
ribosome-EF-G-GDP-fusidic acid complex, the stalk appears as a
well-defined structure, presumably frozen in a stable conformation by
fusidic acid. Sordarin and fusidic both inhibit translocation by
stabilizing the ribosome-eEF2-GDP complex, although the precise details
of this stabilization undoubtedly differ. Our results showed that
whereas L10e mutant ribosomes are not detectably altered in their
ability to bind [3H]L-793,422, the ability of sordarin to
stabilize the eEF2-ribosome-nucleotide complex in these mutants is
impaired. Similar results were obtained with some of the partially
resistant eEF2 mutants. Many of these mutations in eEF2 overlap with
regions in EF-G that give rise to fusidic acid resistance (3, 28).
Interestingly, these regions in EF-G contact the ribosome in the
cryoelectron microscopy map, including those that contact the base of
the stalk complex. Our genetic and biochemical data, taken together
with previous results from L10e cross-linking experiments and the
cryoelectron microscopy map of the ribosome-EF-G-GDP-fusidic acid
complex, support a model in which L10e makes direct contact with eEF2
and also plays a role in conformational changes during translocation. The L10e mutants described here provide new opportunities to examine the functional relationships between L10e and eEF2 in S. cerevisiae.