Ribosomal P0 protein domain involved in selectivity of antifungal sordarin derivatives.

The ribosomal stalk protein P0 is involved in the susceptibility to the antifungal sordarin derivatives, as reported for a number of Saccharomyces cerevisiae resistant mutants. Mammals and some lower eukaryotes are naturally resistant to these compounds. It is shown here that expression in S. cerevisiae of the ribosomal protein P0 from Homo sapiens and from other sordarin-resistant organisms results in a decrease in the sensitivity of the cells to an agent of this class. To further characterize the P0 region responsible for inducing sordarin resistance, a series of protein chimeras containing complementary regions of the human and yeast P0 proteins were constructed and expressed in yeast. The chimeras complement the absence of the native yeast P0 except in chimeras containing the human P0 carboxyl-terminal domain. Resistance to sordarins was found to be associated with the presence of an HsP0 amino acid sequence comprising P118 to F138, which unexpectedly led to higher resistance than the presence of the complete human P0. A comparison of the corresponding region in P0 from yeast and sordarin-insensitive organisms, followed by site-directed mutagenesis, indicates that residues in positions 119, 124, and 126 have an important role in determining resistance to sordarins. Moreover, since sordarins block the eukaryotic elongation factor 2 (EF2) function, the P0 region affecting sordarin susceptibility must correspond to EF2-interacting domains of the ribosomal stalk protein, which affects the drug-binding site in the elongation factor.

Characterization of interaction sites in the Saccharomyces cerevisiae ribosomal stalk components.

The interactions among the yeast stalk components (P0, P1alpha, P1beta, P2alpha and P2beta) and with EF-2 have been explored using immunoprecipitation, affinity chromatography and the two-hybrid system. No stable association was detected between acidic proteins of the same type. In contrast, P1alpha and P1beta were found to interact with P2beta and P2alpha respectively. An interaction of P0 with P1 proteins, but not with P2 proteins, was also detected. This interaction is strongly increased with the P0 carboxyl end, which is able to form a pentameric complex with the four acidic proteins. The P1/P2 binding site has been located between residues 212 and 262 using different C-terminal P0 fragments. Immunoprecipitation shows the association of EF-2 with protein P0. However, the interaction is stronger with the P1/P2 proteins than with P0 in the two-hybrid assay. This interaction improves using the 100-amino-acid-long C-end of P0 and is even higher with the last 50 amino acids. The data indicate a specific association of P1alpha with P2beta and of P1beta with P2alpha rather than the dimerization of the acidic proteins found in prokaryotes. In addition, they suggest that stalk assembly begins by the interaction of the P1 proteins with P0. Moreover, as functional interactions of the complete P0 were found to increase using protein fragments, the data suggest that some active sites are exposed in the ribosome as a result of conformational changes that take place during stalk assembly and function.

Characterization of sparsomycin resistance in Streptomyces sparsogenes.

The antitumor antibiotic sparsomycin, produced by Streptomyces sparsogenes, is a universal translation inhibitor that blocks the peptide bond formation in ribosomes from all species. Sparsomycin-resistant strains were selected by transforming the sensitive Streptomyces lividans with an S. sparsogenes library. Resistance was linked to the presence of a plasmid containing an S. sparsogenes 5.9-kbp DNA insert. A restriction analysis of the insert traced down the resistance to a 3.6-kbp DNA fragment, which was sequenced. The analysis of the fragment nucleotide sequence together with the previous restriction data associate the resistance to srd, an open reading frame of 1,800 nucleotides. Ribosomes from S. sparsogenes and the S. lividans-resistant strains are equally sensitive to the inhibitor and bind the drug with similar affinity. Moreover, the drug was not modified by the resistant strains. However, resistant cells accumulated less antibiotic than the sensitive ones. In addition, membrane fractions from the resistant strains showed a higher capacity for binding the drug. The results indicate that resistance in the producer strain is not connected to either ribosome modification or drug inactivation, but it might be related to an alteration in the sparsomycin permeability barrier.

Asymmetric interactions between the acidic P1 and P2 proteins in the Saccharomyces cerevisiae ribosomal stalk.

The Saccharomyces cerevisiae ribosomal stalk is made of five components, the 32-kDa P0 and four 12-kDa acidic proteins, P1alpha, P1beta, P2alpha, and P2beta. The P0 carboxyl-terminal domain is involved in the interaction with the acidic proteins and resembles their structure. Protein chimeras were constructed in which the last 112 amino acids of P0 were replaced by the sequence of each acidic protein, yielding four fusion proteins, P0-1alpha, P0-1beta, P0-2alpha, and P0-2beta. The chimeras were expressed in P0 conditional null mutant strains in which wild-type P0 is not present. In S. cerevisiae D4567, which is totally deprived of acidic proteins, the four fusion proteins can replace the wild-type P0 with little effect on cell growth. In other genetic backgrounds, the chimeras either reduce or increase cell growth because of their effect on the ribosomal stalk composition. An analysis of the stalk proteins showed that each P0 chimera is able to strongly interact with only one acidic protein. The following associations were found: P0-1alpha.P2beta, P0-1beta.P2alpha, P0-2alpha.P1beta, and P0-2beta.P1alpha. These results indicate that the four acidic proteins do not form dimers in the yeast ribosomal stalk but interact with each other forming two specific associations, P1alpha.P2beta and P1beta.P2alpha, which have different structural and functional roles.

Acidic ribosomal P proteins are phosphorylated in Trypanosoma cruzi.

Trypanosoma cruzi ribosomes from epimastigote forms were purified as determined by electron microscopy and isoelectrofocusing was used to analyse this purified ribosome fraction. Silver stained gels revealed that acidic proteins are present in at least 10 different isoforms, in accord with previous cloning studies. To detect phosphorylation, in vitro phosphorylation assays using the recombinant protein TcP2beta-mbp were carried out. The results showed that T. cruzi cytosolic fraction possesses protein kinase activity able to phosphorylate the recombinant protein. Purified ribosomes contain protein kinases that could also phosphorylate the recombinant protein TcP2beta-mbp. Labelling parasites with [(32)Pi] in a phosphate free medium demonstrated that ribosome proteins, recognised with a specific mouse antiserum against recombinant TcP2beta proteins, are phosphorylated in vivo. All these results suggest that in vivo phosphorylation of ribosome TcP2beta proteins are mediated by protein kinase(s) not yet identified.

Conformational changes induced in the Saccharomyces cerevisiae GTPase-associated rRNA by ribosomal stalk components and a translocation inhibitor.

The yeast ribosomal GTPase associated center is made of parts of the 26S rRNA domains II and VI, and a number of proteins including P0, P1alpha, P1beta, P2alpha, P2beta and L12. Mapping of the rRNA neighborhood of the proteins was performed by footprinting in ribosomes from yeast strains lacking different GTPase components. The absence of protein P0 dramatically increases the sensitivity of the defective ribosome to degradation hampering the RNA footprinting. In ribosomes lacking the P1/P2 complex, protection of a number of nucleotides is detected around positions 840, 880, 1100, 1220-1280 and 1350 in domain II as well as in several positions in the domain VI alpha-sarcin region. The protection pattern resembles the one reported for the interaction of elongation factors in bacterial systems. The results exclude a direct interaction of these proteins with the rRNA and are compatible with an increase in the ribosome affinity for EF-2 in the absence of the acidic P proteins. Interestingly, a sordarin derivative inhibitor of EF-2 causes an opposite effect, increasing the reactivity in positions protected by the absence of P1/P2. Similarly, a deficiency in protein L12 exposes nucleotides G1235, G1242, A1262, A1269, A1270 and A1272 to chemical modification, thus situating the protein binding site in the most conserved part of the 26S rRNA, equivalent to the bacterial protein L11 binding site.

Phosphorylation and N-terminal region of yeast ribosomal protein P1 mediate its degradation, which is prevented by protein P2.

The stalk proteins P1 and P2, which are fundamental for ribosome activity, are the only ribosomal components for which there is a cytoplasmic pool. Accumulation of these two proteins is differentially regulated in Saccharomyces cerevisiae by degradation. In the absence of P2, the amount of P1 is drastically reduced; in contrast, P2 proteins are not affected by a deficiency in P1. However, association with P2 protects P1 proteins. The half-life of P1 is a few minutes, while that of P2 is several hours. The proteasome is not involved in the degradation of P1 proteins. The different sensitivity to degradation of these two proteins is associated with two structural features: phosphorylation and N-terminus structure. A phosphorylation site at the C-terminus is required for P1 proteolysis. P2 proteins, despite being phosphorylated, are protected by their N-terminal peptide. An exchange of the first five amino acids between the two types of protein makes P1 resistant and P2 sensitive to degradation.

Assembly of Saccharomyces cerevisiae ribosomal stalk: binding of P1 proteins is required for the interaction of P2 proteins.

The yeast ribosomal stalk is formed by a protein pentamer made of the 38 kDa P0 and four 12 kDa acidic P1/P2. The interaction of recombinant acidic proteins P1 alpha and P2 beta with ribosomes from Saccharomyces cerevisiae D4567, lacking all the 12 kDa stalk components, has been used to study the in vitro assembly of this important ribosomal structure. Stimulation of the ribosome activity was obtained by incubating simultaneously the particles with both proteins, which were nonphosphorylated initially and remained unmodified afterward. The N-terminus state, free or blocked, did not affect either the binding or reactivating activity of both proteins. Independent incubation with each protein did not affect the activity of the particles, however, protein P2 beta alone was unable to bind the ribosome whereas P1 alpha could. The binding of P1 alpha alone is a saturable process in acidic-protein-deficient ribosomes and does not take place in complete wild-type particles. Binding of P1 proteins in the absence of P2 proteins takes also place in vivo, when protein P1 beta is overexpressed in S. cerevisiae. In contrast, protein P2 beta is not detected in the ribosome in the P1-deficient D67 strain despite being accumulated in the cytoplasm. The results confirm that neither phosphorylation nor N-terminal blocking of the 12 kDa acidic proteins is required for the assembly and function of the yeast stalk. More importantly, and regardless of the involvement of other elements, they indicate that stalk assembling is a coordinated process, in which P1 proteins would provide a ribosomal anchorage to P2 proteins, and P2 components would confer functionality to the complex.

Structural differences between Saccharomyces cerevisiae ribosomal stalk proteins P1 and P2 support their functional diversity.

The eukaryotic acidic P1 and P2 proteins modulate the activity of the ribosomal stalk but playing distinct roles. The aim of this work was to analyze the structural features that are behind their different function. A structural characterization of Saccharomyces cerevisaie P1 alpha and P2 beta proteins was performed by circular dichroism, nuclear magnetic resonance, fluorescence spectroscopy, thermal denaturation, and protease sensitivity. The results confirm the low structure present in both proteins but reveal clear differences between them. P1 alpha shows a virtually unordered secondary structure with a residual helical content that disappears below 30 degrees C and a clear tendency to acquire secondary structure at low pH and in the presence of trifluoroethanol. In agreement with this higher disorder P1 alpha has a fully solvent-accessible tryptophan residue and, in contrast to P2 beta, is highly sensitive to protease degradation. An interaction between both proteins was observed, which induces an increase in the global secondary structure content of both proteins. Moreover, mixing of both proteins causes a shift of the P1 alpha tryptophan 40 signal, pointing to an involvement of this region in the interaction. This evidence directly proves an interaction between P1 alpha and P2 beta before ribosome binding and suggests a functional complementation between them. On a whole, the results provide structural support for the different functional roles played by the proteins of the two groups showing, at the same time, that relatively small structural differences between the two stalk acidic protein types can result in significant functional changes.

Three-dimensional cryo-electron microscopy localization of EF2 in the Saccharomyces cerevisiae 80S ribosome at 17.5 A resolution.

Using a sordarin derivative, an antifungal drug, it was possible to determine the structure of a eukaryotic ribosome small middle dotEF2 complex at 17.5 A resolution by three-dimensional (3D) cryo-electron microscopy. EF2 is directly visible in the 3D map and the overall arrangement of the complex from Saccharomyces cerevisiae corresponds to that previously seen in Escherichia coli. However, pronounced differences were found in two prominent regions. First, in the yeast system the interaction between the elongation factor and the stalk region of the large subunit is much more extensive. Secondly, domain IV of EF2 contains additional mass that appears to interact with the head of the 40S subunit and the region of the main bridge of the 60S subunit. The shape and position of domain IV of EF2 suggest that it might interact directly with P-site-bound tRNA.

Cloning and characterization of the ribosomal protein CcP0 of the medfly Ceratitis capitata.

The gene of the ribosomal protein CcP0, the third member of the ribosomal P-protein family of the medfly Ceratitis capitata, was identified by genomic and cDNA sequence analysis. It codes for a polypeptide of 317 amino acids and its predicted amino acid sequence shows great similarity to the P0 proteins of other eukaryotic organisms. The CcP0 gene was expressed in Escherichia coli and the 34-kDa recombinant protein was identical to the P0 protein of purified medfly ribosomes. Both proteins reacted positively with a specific monoclonal antibody against the highly conserved C terminus of eukaryotic ribosomal P proteins. Interestingly, the medfly CcP0 seems to be the only P0 protein of higher eukaryotic organisms with basic character (pI 8.5), as shown by electrofocusing of purified ribosomes.

Ribosomal stalk protein phosphorylating activities in Saccharomyces cerevisiae.

With ribosomal P protein as a substrate, five peaks of protein kinase activity are eluted after chromatography of a Saccharomyces cerevisiae cellular extract on DEAE-cellulose. Two of them correspond to CK-II and the other three have been called RAP-1, RAP-II, and RAP-III. RAP-I was previously characterized. RAP-III is present in a very small amount, which hindered its purification. RAP-II was further purified on phosphocellulose, heparin-Sepharose, and P protein-Sepharose, studied in detail, and compared with other acidic protein kinases, including RAP-I, CK-II, and PK60. RAP-II is shown by SDS-PAGE and centrifugation on glycerol linear density gradients to have a molecular mass of around 62 kDa and it is immunologically different from RAP-I and PK60. RAP-II phosphorylates the P proteins in the last serine residue at the highly conserved carboxyl terminal domain as other P-protein kinases. The ribosome-bound stalk P proteins are not equally phosphorylated by the different kinases. Thus, RAP-II and PK60 mainly phosphorylate P1beta and P2alpha whereas RAP-I and CK-II modify all of them. A comparative study of the K(m) and V(max) of the phosphorylation reaction by the different kinases using individual purified acidic proteins suggests changes in the substrate susceptibility upon binding to the ribosome. All the data available reveal clear differences in the characteristics of the various P protein kinases and suggest that the cell may use them to differentially modify the stalk depending, perhaps, on metabolic requirements.

The ribosomal P-proteins of the medfly Ceratitis capitata form a heterogeneous stalk structure interacting with the endogenous P-proteins, in conditional P0-null strains of the yeast Saccharomyces cerevisiae.

The genes encoding the ribosomal P-proteins CcP0, CcP1 and CcP2 of Ceratitis capitata were expressed in the conditional P0-null strains W303dGP0 and D67dGP0 of Saccharomyces cerevisiae, the ribosomes of which contain either standard amounts or are totally deprived of the P1/P2 proteins, respectively. The presence of the CcP0 protein restored cell viability but reduced the growth rate. In the W303CcP0 strain, all four acidic yeast proteins were found on the ribosomes, but in notably less quantity, while a preferable binding of the YP1alpha/YP2betapair was established. In the absence of the endogenous P1/P2 proteins in the D67CcP0 strain, the complementation capacity of the CcP0 protein was considerably reduced. The simultaneous expression of the three medfly genes resulted in alterations of the stalk composition: both the CcP1 and CcP2 proteins were found on the particles substituting the YP1alphaand YP2alpha proteins, respectively, but their presence did not alter the growth rate, except in the case of the YP1alpha/betadefective strain, where a helping effect on the binding of the YP2alphaand YP2betaproteins on the ribo-somes was confirmed. Therefore, the medfly ribosomal P-proteins complement the yeast P-protein deficient strains forming an heterogeneous ribosomal stalk, which, however, is not functionally equivalent to the endogenous one.

The RNA interacting domain but not the protein interacting domain is highly conserved in ribosomal protein P0.

Protein P0 interacts with proteins P1alpha, P1beta, P2alpha, and P2beta, and forms the Saccharomyces cerevisiae ribosomal stalk. The capacity of RPP0 genes from Aspergillus fumigatus, Dictyostelium discoideum, Rattus norvegicus, Homo sapiens, and Leishmania infantum to complement the absence of the homologous gene has been tested. In S. cerevisiae W303dGP0, a strain containing standard amounts of the four P1/P2 protein types, all heterologous genes were functional except the one from L. infantum, some of them inducing an osmosensitive phenotype at 37 degrees C. The polymerizing activity and the elongation factor-dependent functions but not the peptide bond formation capacity is affected in the heterologous P0 containing ribosomes. The heterologous P0 proteins bind to the yeast ribosomes but the composition of the ribosomal stalk is altered. Only proteins P1alpha and P2beta are found in ribosomes carrying the A. fumigatus, R. norvegicus, and H. sapiens proteins. When the heterologous genes are expressed in a conditional null-P0 mutant whose ribosomes are totally deprived of P1/P2 proteins, none of the heterologous P0 proteins complemented the conditional phenotype. In contrast, chimeric P0 proteins made of different amino-terminal fragments from mammalian origin and the complementary carboxyl-terminal fragments from yeast allow W303dGP0 and D67dGP0 growth at restrictive conditions. These results indicate that while the P0 protein RNA-binding domain is functionally conserved in eukaryotes, the regions involved in protein-protein interactions with either the other stalk proteins or the elongation factors have notably evolved.

Structure and function of the stalk, a putative regulatory element of the yeast ribosome. Role of stalk protein phosphorylation.

The ribosomal stalk is involved directly in the interaction of the elongation factors with the ribosome during protein synthesis. The stalk is formed by a complex of five proteins, four small acidic polypeptides and a larger protein which directly interacts with the rRNA at the GTPase center. In eukaryotes, the acidic components correspond to the 12 kDa P1 and P2 proteins, and the RNA binding component is protein P0. All these proteins are found to be phosphorylated in eukaryotic organisms. Previous in vitro data suggested this modification was involved in the activity of this structure. To confirm this possibility a mutational study has shown that phosphorylation takes place at a serine residue close to the carboxyl end of proteins P1, P2 and P0. This serine is part of a consensus casein kinase II phosphorylation site. However, by using a yeast strain carrying a temperature sensitive mutant, it has been shown that CKII is probably not the only enzyme responsible for this modification. Three new protein kinases, RAPI, RAPII and RAPIII, have been purified and compared with CKII and PK60, a previously reported enzyme that phosphorylates the stalk proteins. Differences among the five enzymes have been studied. It has also been found that some typical effectors of the PKC kinase stimulate the in vitro phosphorylation of the stalk proteins. All the data available suggest that phosphorylation, although it is not involved in the interaction of the acidic proteins with the ribosome, affects ribosome activity and might participate in some ribosome regulatory mechanism.

Phosphorylation of the yeast ribosomal stalk. Functional effects and enzymes involved in the process.

The ribosomal stalk is directly involved in the interaction of the elongation factors with the ribosome during protein synthesis. The stalk is formed by a complex of five proteins, four small acidic polypeptides and a larger protein which directly interacts with the rRNA at the GTPase center. In eukaryotes the acidic components correspond to the 12-kDa P1 and P2 proteins, and the RNA binding component is the P0 protein. All these proteins are found phosphorylated in eukaryotic organisms, and previous in vitro data suggested this modification was involved in the activity of this structure. Results from mutational studies have shown that phosphorylation takes place at a serine residue close to the carboxy end of the P proteins. Modification of this serine residue does not affect the formation of the stalk and the activity of the ribosome in standard conditions but induces an osmoregulation-related phenotype at 37 degrees C. The phosphorylatable serine is part of a consensus casein kinase II phosphorylation site. However, although CKII seems to be responsible for part of the stalk phosphorylation in vivo, it is probably not the only enzyme in the cell able to perform this modification. Five protein kinases, RAPI, RAPII and RAPIII, in addition to the previously reported CKII and PK60 kinases, are able to phosphorylate the stalk proteins. A comparison of the five enzymes shows differences among them that suggest some specificity regarding the phosphorylation of the four yeast acidic proteins. It has been found that some typical effectors of the PKC kinase stimulate the in vitro phosphorylation of the stalk proteins. All the data suggest that although phosphorylation is not involved in the interaction of the acidic P proteins with the ribosome, it can affect the ribosome activity and might participate in a possible ribosome regulatory mechanism.

Disruption of six Saccharomyces cerevisiae novel genes and phenotypic analysis of the deletants.

As a part of the EUROFAN programme, six open reading frames from Saccharomyces cerevisiae (YNL083w, YNL086w, YNL087w, YNL097c, YDL100c and YOR086c) were disrupted in two genetic backgrounds, FY1679 and W303. Individual deletions in diploid strains and tetrad analysis of heterozygous deletants revealed that none of them is essential. Basic phenotypic analysis did not reveal any significant difference between the parental and mutant strains. Although YNL087w and YOR086c are 55% identical, the double disruptant also behaves the same as the parental cells. Ydl100p seems to be involved in metal detoxification, the phenotype of the null mutants being enhanced when the assays are performed at 37 degrees C.

Deletion of 24 open reading frames from chromosome XI from Saccharomyces cerevisiae and phenotypic analysis of the deletants.

As a part of the EUROFAN program, 24 open reading frames from Saccharomyces cerevisiae (YKR010c to YKR013w, YKR015c to YKR025w, YKR081c to YKR083c, YKR087c to YKR091w and YKR096w) were disrupted in two genetic backgrounds, FY1679 and W303. Systematic deletions and phenotypic analysis were performed following a hierarchical strategy, the so-called 'mass murder'. Of the 24 genes thus deleted, four are essential, whereas the deletion of 17 did not reveal any significant difference between the parental and mutant strains. Deletions of the remaining three show some growth phenotype; ykr024c mutants grow slowly under any conditions, ykr019c mutants grow slower in a rich medium and ykr082w mutants are temperature sensitive, being unable to germinate at 30 degrees C and above.

Isolation and expression of the genes encoding the acidic ribosomal phosphoproteins P1 and P2 of the medfly Ceratitis capitata.

The genes of the acidic ribosomal proteins P1 and P2 (CcP1 and CcP2) of the medfly Ceratitis capitata were isolated from a genomic library using homologue DNA probes prepared by PCR. Sequencing and characterization of the two genes revealed strong similarities of the encoded amino acid sequence to the homologous proteins of Drosophila melanogaster and other eukaryotic species. The predicted amino acid sequences of the CcP1 and CcP2 proteins shared an almost identical carboxyl terminal sequence of 10 amino acids common to most known acidic ribosomal proteins. The CcP2 gene lacked intervening sequences in contrast to the CcP1 gene, which was interrupted by an intron of 188 nucleotides. Both genes were cloned in expression pT7 vectors and were expressed in Esherichia coli. The 17- and 15-kDa recombinant proteins reacted with a monoclonal antibody specific to the highly conserved carboxyl terminus of eukaryotic acidic ribosomal proteins, confirming their equivalence to these ribosomal components. Both recombinant proteins were electrophoretically identical to acidic proteins extracted from purified ribosomes of C. capitata.