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J Biol Chem, Vol. 273, Issue 39, 25041-25044, September 25, 1998
From the Research Department, Glaxo Wellcome, S. A., Severo Ochoa
2, 28760 Tres Cantos, Spain
Sordarin derivatives are remarkably selective
inhibitors of fungal protein synthesis. Available evidence points to a
binding site for these inhibitors on elongation factor 2, but high
affinity binding requires the presence of ribosomes. The gene mutated
in one of the two isolated complementation groups of
Saccharomyces cerevisiae mutants resistant to the sordarin
derivative GM193663 has now been identified. It is RPP0,
encoding the essential protein of the large ribosomal subunit stalk
rpP0. Resistant mutants are found to retain most of the binding
capacity for the drug, indicating that mutations in rpP0 endow the
ribosome with the capacity to perform translation elongation in the
presence of the inhibitor. Other proteins of the ribosomal stalk
influence the expression of resistance, pointing to a wealth of
interactions between stalk components and elongation factors. The
involvement of multiple elements of the translation machinery in the
mode of action of sordarin antifungals may explain the large
selectivity of these compounds, even though the individual target
components are highly conserved proteins.
The ribosomal stalk is a defined morphological feature most
evident in prokaryotic ribosomes, where it is composed of the L7/L12
tetramer, L10 and L11 proteins, and a highly conserved domain of the 23 S rRNA termed the "GTPase center." The stalk plays a prominent role
in the elongation phase of ribosomal protein synthesis (see Refs. 1 and
2 for a review).
Equivalent structures have been visualized on eukaryotic ribosomes (3)
and functional homologies with the bacterial components have been
described (see Ref. 4 for a review). The recent nomenclature proposal
for Saccharomyces cerevisiae ribosomal proteins (5) will be
followed throughout this manuscript. In this organism, proteins
rpP1 The molecular detail of the interplay between stalk components and
elongation factors is unknown. Recent morphological evidence (13)
indicates that elongation factors make multiple contacts with stalk
components, and a detailed footprinting analysis of EF-G on rRNA place
the factor at the base of the stalk (14). Resolution is however not
high enough to ascribe roles to individual molecules.
Semisynthetic derivatives of the natural product sordarin (15, 16) have
proven to be highly potent inhibitors of eukaryotic protein synthesis,
with remarkable selectivity for the fungal translation machinery (17,
18). Sordarin inhibitors seem to bind to elongation factor
21,2
(18), and
point mutations on the factor make cells resistant to the
inhibitors1 (18), but high affinity binding requires the
presence of ribosomes,2 and resistant mutants were
detected, which did not map on EF2.1
This work describes the gene mutated in a second complementation
group of S. cerevisiae mutants resistant to GM193663, a
potent sordarin derivative. The gene encodes the ribosomal stalk
protein rpP0. It is further shown that other stalk components are also involved in the translation elongation function inhibited by GM193663, substantiating the ribosomal requirement for binding of the inhibitor to EF2 and pointing at interactions between the factor and specific stalk components.
Strains and Growth Conditions--
All the strains used in
this study are derivatives of: S. cerevisiae SEY6210
(Mat
Ribosomal P-protein Stalk Function Is Targeted by Sordarin
Antifungals*
ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References
, rpP1
, rpP2, and rpP2 take the place of the bacterial
L7/L12 tetramer. rpP0 corresponds functionally to bacterial L10 and
rpL12 to bacterial L11 (6-10). rpP0 is thought to play a central role
in stalk architecture, interacting with rpL12 and 26 S rRNA at its
amino-terminal half and with the other four P-proteins at its
carboxyl-terminal end (4, 11). None of the P1/P2 proteins are
absolutely essential for growth, although in the absence of at least
one member of each class (P1 and P2), vegetative growth is strongly
retarded (12).
EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References
ura3-52 leu2-3, 112 his3
200 trp1901 lys2-81
suc29), its ade2-101 derivative SEY6211 (S. Emr),
S. cerevisiae 373 (Mat a ade2-101) (A. Jimenez),
S. cerevisiae W303 (Mat a leu2-3, 112 trp1-1 his
3-11, 15 ade 2-1 can 1-100) and S. cerevisiae
W303dGP0 (W303 strain with the cassette pGAL1-rpP0-URA3 replacing the endogenous RPP0 locus) (19). For genetic
mapping of the FPR2-15 mutation, crosses were made between
strain W303dGP0 and W303 derivatives D46 (rpp1b::TRP1
rpp2a::URA3) and D57 (rpp1a::LEU2 rpp2b::HIS3). Heterologous rpP0 proteins were expressed
in the W303dGP0 strain growing in glucose, to repress the chromosomal RPP0 gene, using the centromeric plasmid pFL39-HIS and the
endogenous S. cerevisiae promoter. All W303 derivatives and
pFL constructs were obtained from the laboratory of J. P. G. Ballesta (12, 19).
In Vitro Activity and Binding Assays-- Growth inhibitory activity of GM193663 was determined on liquid medium by the antibiotic 2-fold serial dilution technique; 100 µl of YPD were inoculated with 105 colony-forming units/well. IC50 was defined as the minimal concentration of compound that inhibited 50% of the control growth after 24 h of incubation at 30 °C. For strains living with heterologous rpP0 proteins, A600 was measured after 48 h of incubation because of their slower growth. Cycloheximide (Sigma) and GM193663 stocks were prepared in Me2SO.
Cell-free S50 extracts were prepared from cells grown in YPD medium to an A600 = 1.5. Washed cells were broken in lysis buffer (30 mM Hepes/KOH, 100 mM potassium acetate, 12.5 mM magnesium acetate, 2 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 8.5% D-mannitol, pH = 7.4) by vortexing with glass beads for 10 1-min periods, with intervening 1-min cooling in ice. Unbroken cells and debris were pelleted at 4,000 × g for 10 min, and the S50 supernatant was obtained by centrifugation at 50,000 × g for 30 min. [3H]Sordarin was labeled at 180 GBq/mmol in the aldehyde group by the Isotope Chemistry Group at Glaxo Wellcome (Stevenage, United Kingdom). Binding assays were performed with 0.5 mg of protein from S50 extracts and 0.1 µg of [3H]sordarin (36 kBq, 20 nmol), in 80 mM Hepes/KOH, 1 mM dithiothreitol, 10 mM magnesium acetate, in a final volume of 500 µl. This amount of sordarin was enough to have at least 90% of the input compound free at equilibrium. After 30 min of incubation at room temperature, 400 µl from each sample were applied to PD-10 Sephadex-G 25 M columns (Amersham Pharmacia Biotech), presoaked in lysis buffer without D-mannitol. [3H]Sordarin bound to excluded macromolecules was measured by scintillation counting. All incubations were done in duplicate tubes and the results averaged. Measurements from paired tubes never differed by more than 20%. Counts obtained in parallel incubations containing a 100-fold excess of cold sordarin were subtracted from all data points. This nonspecific binding was always less than 10% of the total binding detected.Molecular Mapping of rpP0 Mutations-- Localization of mutations that conferred resistance to GM193663 within the rpP0 open reading frame was carried out by the single strand conformation polymorphism technique (22). Briefly, standard PCRs were performed using cloned rpP0 from all mutant and wild type strains as template. Three overlapping fragments covering the entire open reading frame were amplified and electrophoresed under denaturing conditions. PCR fragments showing an altered mobility compared with their corresponding wild types were sequenced using an Applied Biosystems PRISM 310 genetic analyzer.
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RESULTS |
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Abstract
Introduction
Procedures
Results
Discussion
References
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Mutations in Ribosomal Protein P0 Confer Resistance against Sordarin Antifungals-- It has been communicated previously that genetic analysis of spontaneous mutants resistant to the sordarin antifungal GM193663, an inhibitor of translation elongation, identified two different genetic complementation groups, denominated FPR1 and FPR2.1 The major one, FPR1, grouped mutations in EFT2, encoding translation elongation factor 2. FPR2 was composed of just four mutants and, contrary to expectations, did not arise from EFT1, the second EF2-coding gene in S. cerevisiae. Growth and resistance characteristics of the mutants in rich medium are presented in Table I. All mutants displayed cross-resistance on plates to other members of the sordarin family, but not to the protein synthesis inhibitors cycloheximide, hygromycin, or verrucarin A, showing that sordarins act by a mechanism different from the one used by those other inhibitors.
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, rpP1, rpP2, and rpP2 were obtained from the
laboratory of J. P. G. Ballesta (12) and crossed to
GM193663R mutant FPR2-15, which displayed the
most dominant resistance phenotype. Tetrad analysis did not detect a
genetic linkage between the resistance locus and the P-proteins, except
in the case of rpP0, where a tight genetic linkage between the
resistance mutation and RPP0, the rpP0 locus, was found. All
36 tetrads dissected displayed the parental ditype phenotype. This
result strongly suggested that the resistance and RPP0 loci
were very close to each other or were the same one.
RPP0 was amplified and cloned from the four mutants in group
2 as well as from their corresponding parental strains. The cloned genes were transformed into a sensitive haploid strain, and the transformants were tested for their sensitivity to GM193663. Strains transformed with an RPP0 gene from a resistant strain became
resistant themselves in all cases, confirming that the gene mutated in
the second complementation group was RPP0. Thus, mutations
in protein rpP0, an essential protein of the large ribosomal subunit,
can confer a level of resistance to sordarins higher than the one afforded by mutations in EF2, the proposed binding protein for sordarins (Table II). This could be the
first such occurrence described, and it strongly suggests that rpP0 and
EF2 interact functionally.
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Resistance Mutations Cluster Together in the rpP0 Sequence-- Chromosomal RPP0 genes from the four mutants were cloned by gap repair and analyzed by single-stranded conformational polymorphism (see "Experimental Procedures"). Mobility changes were found in the central one-third of all alleles. The region was sequenced on both strands, and amino acid changes were detected in all mutants (Table I, Fig. 1). To confirm that the observed changes were not introduced by the PCR, genomic fragments from the four mutants were recovered by the plasmid gap repair technique (24). A wild type RPP0 gene was gapped by removing all the open reading frame except the last 86 amino acids and then transformed into the resistant mutants. Plasmids were recovered from stable transformants, sequenced, and re-transformed into a wild type strain. All recovered plasmids had the expected mutation and conferred resistance to GM193663, establishing that the observed amino acid changes (Table I) were responsible for the resistance to the inhibitor. Unexpectedly, spontaneous mutant FPR2-17 had four nucleotides changed, leading to two consecutive substitutions in the amino acid sequence of the protein.
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Mutations in rpP0 Do Not Abolish Binding of the Inhibitor to Macromolecules-- Sordarin is a close analogue of GM193663 (Fig. 2) and both compounds display cross-resistance and compete for binding to macromolecules.2 Fig. 2 shows [3H]sordarin binding to macromolecules in the rpP0 mutants. Substantial binding was found in all mutant strains, showing that growth, and therefore protein synthesis, can take place in the presence of nearly wild type levels of inhibitor binding in some strains. Data from an EF2 mutant have been included for comparison, and it can be seen that in this case resistance is accompanied by the total loss of sordarin binding capacity, as described previously1 (Fig. 2). The measured binding is to the ribosomal fraction of the extracts plus associated cytoplasmic factors, because the post-ribosomal supernatant showed negligible binding (data not shown). Therefore, the mechanism of resistance mediated by the rpP0 protein is different from the simple loss of affinity for the drug, and it may be related to the molecular details of the interaction between EF2 and the ribosomal stalk.
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Other Proteins of the Ribosomal Stalk Modulate Resistance Conferred by rpP0-- Given the known or inferred interactions between the acidic P-proteins and rpP0 (see Ref. 4 for a review), it was of interest to know if lack of the former affected resistance due to rpP0, which would substantiate the relevance of the interactions between P-proteins in translation elongation.
All four nonessential P-proteins were interrupted singly and in pairs in the FPR2-15 mutant background. It was found that deleting either rpP1 or rpP2 reduced resistance levels more than
10-fold. Deletion of either rpP1 or rpP2 did not have a significant effect. Deleting both rpP1 and rpP2 had an additive effect, whereas deleting the other pair did not affect resistance (Table II). The effect of the protein composition of the ribosomal stalk on the sensitivity toward GM193663 is observed in the mutant RPP0 background only. The same deletions do not affect the
sensitivity of a wild type strain (data not shown). These results
confirm that the four P-proteins are not functionally equivalent, with rpP1 and rpP2 establishing a type of interaction with rpP0, and
probably EF2, different from the one maintained by rpP1 or rpP2.
The influence of the stalk on the sensitivity toward sordarin
antifungals seems to be specific for this type of translational inhibitors, because deleting the acidic P-proteins, either on a wild
type strain or in the FPR2-15 mutant, did not change their sensitivity toward a different elongation inhibitor, cycloheximide. However, GM193663 resistance mediated by a mutant EF2 was also diminished by deleting rpP1 and to a lesser extent by deleting rpP2 (Table II). This is in agreement with the findings presented above and further indicates that the soluble factor establishes functional interactions with the ribosomal stalk, and not only with
rpP0, but also with rpP1 and perhaps with rpP2 as well.
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DISCUSSION |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Sordarins are a new family of antifungal compounds and, together with tenuazonic acid (25), the only protein synthesis inhibitors reported to show selectivity between different eukaryotes (17, 18). They have been described to target and bind to elongation factor 21,2 (18), but it was also immediately apparent that the inhibitor's mode of action involved additional cellular components, because resistant mutants were obtained outside EF21, and high affinity binding of the drug to macromolecules required the presence of ribosomes,2 suggesting that the binding site on EF2 is fully formed only after the factor interacts with a ribosome. In this work, experiments are described showing that rpP0, an essential component of the ribosomal large subunit stalk, plays a large role in determining the sensitivity to sordarin antifungal GM193663, as single point mutations in rpP0, and substitution of heterologous rpP0 proteins for the endogenous one, reduce measurably the sensitivity of yeast toward these compounds (Tables II and III). The results also indicate that there is a functional interaction between rpP0 and EF2, because mutations in any one of them render cells insensitive to the same translational inhibitor. This fits well with evidence indicating that elongation factors are probably recruited to the ribosome through interactions with the stalk (7, 9, 13, 26).
The mechanism of resistance afforded by mutations in rpP0 is
intriguing, and its elucidation may shed light into the molecular details of ribosomal translocation. The binding capacity for the inhibitor has been retained to a large extent by the resistant mutants
(Fig. 2). If the hypothesis that sordarin antifungals impede
conformational changes in EF2 is correct1 (18), then
changes in rpP0 could somehow force or facilitate the appropriate
bending of the EF2 molecule. According to the data presented here, in
order for rpP0 to be able to "help" EF2 when blocked by GM193663,
the protein must be part of a ribosomal stalk in which rpP1 and
rpP2 are present. Stalk structures deficient in either of those
proteins substantially reduce the capacity of mutant rpP0 to overcome
the presence of the inhibitor. Interestingly, rpP1 and rpP2 are
also the P-proteins whose deletion has the largest effect on growth
rate (12). rpP1 may be able to establish some interaction with EF2
by itself, because lack of this ribosomal protein also reduces
significantly resistance levels afforded by mutations in EF2 (Table
II). There seems to be also an effect of the rpP2 deletion on
EF2-mediated resistance, although the reduction in the IC50
of the inhibitor is smaller in this case. All this hints at a wealth of
functional interactions between elongation factors, or at least EF2,
and components of the ribosomal P-protein stalk. Pairwise physical
interactions between all these proteins are currently being analyzed by
genetic and biochemical means.
The screen for GM193663R mutants is by no means saturated. Ongoing work at several laboratories will possibly detect additional gene products involved in the mode of action of sordarin antifungals, which may in turn uncover new functional interactions, or confirm suspected ones, between components of the translation apparatus.
In yeast there are described ribosomal protein mutations involved in resistance to cycloheximide, cryptopleurine, trichodermin, and now sordarin analogues (27-31). Sordarin antifungals may however be unique in targeting the functional interaction between a soluble translation factor and ribosomal proteins, in such a manner as to allow both types of proteins to influence the cell's sensitivity to the inhibitor. The contribution of multiple elements to sensitivity may explain the large selectivity of these compounds, despite the high sequence conservation between the individual components of the eukaryotic translation apparatus. Sordarin antifungals are very selective drugs with a good therapeutic potential, and they are also turning out to be useful tools for the analysis of the elongation cycle during protein translation.
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ACKNOWLEDGEMENTS |
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We are indebted to members of the group of J. P. G. Ballesta, especially to M. A. Rodríguez-Gabriel, for numerous discussions and for allowing us access to their strains and unpublished information. We also thank our colleagues in the Isotope Chemistry Group (Glaxo Wellcome, Stevenage, UK) for providing tritiated sordarin. The frequent help of all our colleagues in the New Targets and Biochemistry groups of Glaxo Wellcome (Spain) is also gratefully acknowledged.
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FOOTNOTES |
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* 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.
To whom correspondence should be addressed. Tel.: 34-91-807-0606;
Fax: 34-91-807-0550; E-mail: jfg32652{at}glaxowellcome.co.uk.
The abbreviation used is: PCR(s), polymerase chain reaction(s).
1 Capa, L., Mendoza, A., Lavandera, J. L., Gómez de las Heras, F., and García-Bustos, J. F. (1998) Antimicrob. Agents Chemother., in press.
2 Domínguez, J. M., and Martín, J. J. (1998) Antimicrob. Agents Chemother., in press.
4 M. A. Rodríguez-Gabriel, M. Remacha, and J. P. G. Ballesta, submitted for publication.
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REFERENCES |
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